U.S. patent application number 16/722699 was filed with the patent office on 2020-05-07 for formulations of brincidofovir.
The applicant listed for this patent is Chimerix, Inc.. Invention is credited to Irma Marisa GROSSI, Mohammed Anowrul KABIR, Odin Johann NADERER.
Application Number | 20200138835 16/722699 |
Document ID | / |
Family ID | 59313330 |
Filed Date | 2020-05-07 |
View All Diagrams
United States Patent
Application |
20200138835 |
Kind Code |
A1 |
KABIR; Mohammed Anowrul ; et
al. |
May 7, 2020 |
FORMULATIONS OF BRINCIDOFOVIR
Abstract
Disclosed are formulations comprising brincidofovir. The
formulations can be intravenous formulations. The formulations can
be used in cases where a subject experiences gastrointestinal
issues in response to oral administration of brincidofovir. The
formulations can also be used in cases where oral administration of
drug is not possible due to underlying conditions or concern around
inadequate oral absorption.
Inventors: |
KABIR; Mohammed Anowrul;
(Cary, NC) ; NADERER; Odin Johann; (Chapel Hill,
NC) ; GROSSI; Irma Marisa; (Durham, NC) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Chimerix, Inc. |
Durham |
NC |
US |
|
|
Family ID: |
59313330 |
Appl. No.: |
16/722699 |
Filed: |
December 20, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15636393 |
Jun 28, 2017 |
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16722699 |
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62512825 |
May 31, 2017 |
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62507397 |
May 17, 2017 |
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62465053 |
Feb 28, 2017 |
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62446213 |
Jan 13, 2017 |
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62394665 |
Sep 14, 2016 |
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62355844 |
Jun 28, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/675 20130101;
A61K 47/26 20130101; A61K 47/18 20130101; A61P 31/22 20180101; Y02A
50/467 20180101; Y02A 50/30 20180101; A61K 9/08 20130101; A61P
31/12 20180101; A61K 9/19 20130101; A61P 31/20 20180101; A61K
47/183 20130101; A61K 9/0019 20130101; A61P 31/14 20180101 |
International
Class: |
A61K 31/675 20060101
A61K031/675; A61K 9/00 20060101 A61K009/00; A61K 9/19 20060101
A61K009/19; A61K 47/18 20060101 A61K047/18; A61K 47/26 20060101
A61K047/26; A61K 9/08 20060101 A61K009/08 |
Claims
1. A lyophilized powder comprising: about 13-19% by weight
brincidofovir; about 48%-65% by weight mannitol; and about 22-33%
by weight arginine.
2. The lyophilized powder of claim 1, comprising: about 13% by
weight brincidofovir; about 65% by weight mannitol; and about 22%
by weight arginine.
3. The lyophilized powder of claim 1, comprising: about 19% by
weight brincidofovir; about 48% by weight mannitol; and about 33%
by weight arginine.
4. The lyophilized powder of claim 1, comprising: about 10 mg
brincidofovir; about 50 mg mannitol; and about 17.4 mg
arginine.
5. The lyophilized powder of claim 1, comprising: about 10 mg
brincidofovir; about 25 mg mannitol; and about 17.4 mg
arginine.
6. A method of treating a viral infection in a subject in need
thereof, the method comprising: (a) dissolving the lyophilized
powder of claim 1 in aqueous media to produce an aqueous
pharmaceutical composition; and (b) administering to the subject an
effective amount of the aqueous pharmaceutical composition.
7. The method of claim 6, wherein the viral infection is selected
from polyomavirus, papillomavirus, herpes virus, adenovirus,
Epstein-Barr virus, cytomegalovirus, Hepatitis B virus, Hepatitis C
virus, varicella zoster virus, adenovirus, poxvirus, or a
combination thereof.
8. The method of claim 6, wherein administration to the subject
does not result in hemolysis.
9. The method of claim 6, wherein administration to the subject
does not result in gastrointestinal toxicity.
10. The method of claim 6, wherein the aqueous media is a sugar
alcohol solution, an aqueous sugar solution, Ringer's solution or
an aqueous salt solution.
11. The method of claim 10, wherein the aqueous media is an aqueous
sugar solution.
12. The method of claim 11, wherein the aqueous sugar solution is
about 5% dextrose by weight.
13. The method of claim 11, wherein the lyophilized powder is
dissolved in about 100 mL of aqueous sugar solution.
14. The method of claim 11, wherein the lyophilized powder is
dissolved in about 200 mL of aqueous sugar solution.
15. The method of claim 6, wherein the aqueous pharmaceutical
composition comprises: brincidofovir at a concentration of about
1.0 mg/mL; mannitol at a concentration of about 2.5 mg/mL to about
5 mg/mL; L-arginine at a concentration of about 1.74 mg/mL; and
dextrose at a concentration of about 50 mg/mL; wherein the pH of
the composition is about 8.0.
16. The method of claim 6, wherein the aqueous pharmaceutical
composition comprises: brincidofovir at a concentration of about
1.78 mM; mannitol at a concentration of about 13.75 mM to about
27.5 mM; L-arginine at a concentration of about 10 mM; and dextrose
at a concentration of about 287 mM; wherein the pH of the
composition is about 8.0.
17. The method of claim 6, wherein the aqueous pharmaceutical
composition comprises: brincidofovir at a concentration of about
0.5 mg/mL; mannitol at a concentration of about 1.25 mg/mL to about
2.5 mg/mL; L-arginine at a concentration of about 0.87 mg/mL; and
dextrose at a concentration of about 50 mg/mL; wherein the pH of
the composition is about 8.0.
18. The method of claim 6, wherein the aqueous pharmaceutical
composition comprises: brincidofovir at a concentration of about
0.89 mM; mannitol at a concentration of about 6.85 mM to about 13.7
mM; L-arginine at a concentration of about 5 mM; and dextrose at a
concentration of about 287 mM; wherein the pH of the composition is
about 8.0.
19. The method of claim 6, wherein the aqueous pharmaceutical
composition comprises: about 100 mg brincidofovir; about 250 mg to
about 500 mg mannitol; about 174 mg arginine; about 5 g dextrose;
and about 100 mL water; wherein the pH of the solution is about
8.0.
20. The method of claim 6, wherein the aqueous pharmaceutical
composition comprises: about 200 mg brincidofovir; about 500 mg to
about 1000 mg mannitol; about 348 mg arginine; about 10 g dextrose;
and about 200 mL water; wherein the pH of the solution is about
8.0.
21. The method of claim 6, wherein the aqueous pharmaceutical
composition comprises: about 50 mg brincidofovir; about 125 mg to
about 250 mg mannitol; about 87 mg arginine; about 5 g dextrose;
and about 100 mL water; wherein the pH of the solution is about
8.0.
22. The method of claim 6, wherein the aqueous pharmaceutical
composition comprises: about 100 mg brincidofovir; about 250 mg to
about 500 mg mannitol; about 174 mg arginine; about 10 g dextrose;
and about 200 mL water; wherein the pH of the solution is about
8.0.
23. The method of claim 6, wherein the aqueous pharmaceutical
composition is suitable for intravenous administration.
24. The pharmaceutical composition of claim 23, wherein the aqueous
pharmaceutical composition is sterile.
25. A method of preparing the lyophilized powder of claim 1,
comprising (a) preparing an aqueous solution comprising
brincidofovir at a concentration of about 10 mg/mL; mannitol at a
concentration of about 25-50 mg/mL; and L-arginine at a
concentration of about 17.4 mg/mL; wherein the pH of the
composition is about 8.0; and (b) lyophilizing the aqueous
solution.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 15/636,393, filed Jun. 28, 2017, currently pending, which
claims priority to, and benefit of: U.S. Provisional Patent
Application No. 62/355,844, filed Jun. 28, 2016; U.S. Provisional
Patent Application No. 62/394,665, filed Sep. 14, 2016; U.S.
Provisional Patent Application No. 62/446,213, filed Jan. 13, 2017;
U.S. Provisional Patent Application No. 62/465,053, filed Feb. 28,
2017; U.S. Provisional Patent Application No. 62/507,397, filed May
17, 2017; and U.S. Provisional Patent Application No. 62/512,825,
filed May 31, 2017, the contents of each of which are incorporated
by reference in their entireties.
FIELD
[0002] The present disclosure relates to pharmaceutical
compositions (e.g., lyophilized and/or aqueous compositions)
comprising brincidofovir and methods of use thereof.
BACKGROUND
[0003] Brincidofovir (BCV, CMX001) is an orally bioavailable, lipid
acyclic nucleoside phosphonate that is converted intracellularly
into the active antiviral cidofovir diphosphate (CDV-PP).
Brincidofovir has broad spectrum antiviral activity against
double-stranded DNA viruses. The structure of brincidofovir is
shown below:
##STR00001##
SUMMARY
[0004] The present disclosure presents compositions comprising
brincidofovir and methods of using the same. The compositions can
be lyophilized (e.g., as a powder) for long-term storage. The
lyophilized formulations can be reconstituted (e.g., in an aqueous
sugar solution) as biocompatible formulations for intravenous (IV)
administration (e.g., to a subject in need thereof).
[0005] In one aspect, the present disclosure provides a
pharmaceutical composition, comprising: brincidofovir; a bulking
agent; a buffer; and water; wherein the pH of the composition is
about 8.0.
[0006] In some embodiments, the bulking agent is mannitol or
sucrose. In some embodiments, the bulking agent is mannitol. In
some embodiments, the buffer is sodium phosphate, L-arginine, or
tromethamine. In some embodiments, the buffer is L-arginine. In
some embodiments, the brincidofovir is present at a concentration
of about 10.0 mg/mL. In some embodiments, the bulking agent is
present at a concentration of about 2.5-9% (w/v). In some
embodiments, the bulking agent is present at a concentration of
about 2.5% (w/v). In some embodiments, the bulking agent is present
at a concentration of about 5% (w/v). In some embodiments, the
buffer is present at a concentration of about 100-200 mM. In some
embodiments, the buffer is present at a concentration of about 100
mM. In some embodiments, the pH is adjusted using HCl and/or
NaOH.
[0007] In some embodiments, the pharmaceutical composition
comprises: brincidofovir at a concentration of about 10.0 mg/mL;
mannitol at a concentration of about 25-50 mg/mL; L-arginine at a
concentration of about 17.4 mg/mL; and water; wherein the pH of the
composition is about 8.0.
[0008] In some embodiments, the pharmaceutical composition
comprises: brincidofovir at a concentration of about 17.8 mM;
mannitol at a concentration of about 137.5-275 mM; L-arginine at a
concentration of about 100 mM; and water; wherein the pH of the
composition is about 8.0.
[0009] In some embodiments, the liquid pharmaceutical composition
is lyophilized, e.g., to remove water, forming a lyophilized
powder.
[0010] In another aspect, the present disclosure provides a
lyophilized powder comprising: about 13-23% by weight
brincidofovir; about 48%-65% by weight mannitol; and about 22-40%
by weight arginine.
[0011] In another aspect, the present disclosure provides a
lyophilized powder comprising: about 13-19% by weight
brincidofovir; about 48%-65% by weight mannitol; and about 22-33%
by weight arginine.
[0012] In some embodiments, the lyophilized powder contains about
19% by weight brincidofovir, about 48% by weight mannitol, and
about 33% by weight arginine. In some embodiments, the lyophilized
powder contains about 13% by weight brincidofovir, about 65% by
weight mannitol, and about 22% by weight arginine.
[0013] In some embodiments, the pH of the lyophilized powder is
about 8.0.
[0014] In another aspect, the present disclosure provides an
aqueous pharmaceutical composition, comprising: brincidofovir; a
bulking agent; a buffer; and a sugar alcohol solution, an aqueous
sugar solution, Ringer's solution or an aqueous salt (e.g., sodium
chloride) solution.
[0015] In some embodiments, the aqueous pharmaceutical composition
comprises: brincidofovir; mannitol; L-arginine; and dextrose.
[0016] In some embodiments, the aqueous pharmaceutical composition
comprises: brincidofovir; mannitol; L-arginine; and dextrose;
wherein the pH of the composition is about 8.0.
[0017] In some embodiments, the aqueous sugar solution is a
solution comprising about 5% dextrose by weight. In some
embodiments, the aqueous salt solution is an aqueous sodium
chloride solution. In some embodiments, the concentration of the
sodium chloride solution is 0.9% by weight. In some embodiments,
the aqueous pharmaceutical composition further comprises additional
water.
[0018] In some embodiments, the additional water is added (e.g. to
adjust tonicity, concentration, or pH of the formulation).
[0019] In some embodiments, the aqueous pharmaceutical composition
comprises: brincidofovir at a concentration of about 1.0 mg/mL;
mannitol at a concentration of about 2.5-5 mg/mL; and L-arginine at
a concentration of about 1.74 mg/mL; and dextrose at a
concentration of about 50 mg/mL. In some embodiments, the pH of the
composition is about 8.0, about 7.5, about 7.0, about 6.5, about
6.0, or below about 6.0.
[0020] In some embodiments, the aqueous pharmaceutical composition
comprises: brincidofovir at a concentration of about 1.78 mM;
mannitol at a concentration of about 13.75-27.5 mM; L-arginine at a
concentration of about 10 mM; and dextrose at a concentration of
about 287 mM. In some embodiments, the pH of the composition is
about 8.0, about 7.5, about 7.0, about 6.5, about 6.0, or below
about 6.0.
[0021] In some embodiments, the aqueous pharmaceutical composition
comprises: brincidofovir at a concentration of about 0.5 mg/mL;
mannitol at a concentration of about 1.25-2.5 mg/mL; and L-arginine
at a concentration of about 0.87 mg/mL; and dextrose at a
concentration of about 50 mg/mL. In some embodiments, the pH of the
composition is about 8.0, about 7.5, about 7.0, about 6.5, about
6.0, or below about 6.0.
[0022] In some embodiments, the aqueous pharmaceutical composition
comprises: brincidofovir at a concentration of about 0.89 mM;
mannitol at a concentration of about 6.85-13.7 mM; L-arginine at a
concentration of about 5 mM; and dextrose at a concentration of
about 287 mM. In some embodiments, the pH of the composition is
about 8.0, about 7.5, about 7.0, about 6.5, about 6.0, or below
about 6.0.
[0023] In some embodiments, the aqueous pharmaceutical composition
comprises: brincidofovir at a concentration of about 1.0 mg/mL;
mannitol at a concentration of about 2.5-5 mg/mL; and L-arginine at
a concentration of about 1.74 mg/mL; and dextrose at a
concentration of about 50 mg/mL; wherein the pH of the composition
is about 8.0.
[0024] In some embodiments, the aqueous pharmaceutical composition
comprises: brincidofovir at a concentration of about 1.78 mM;
mannitol at a concentration of about 13.75-27.5 mM; L-arginine at a
concentration of about 10 mM; and dextrose at a concentration of
about 287 mM; wherein the pH of the composition is about 8.0.
[0025] In some embodiments, the aqueous pharmaceutical composition
comprises: brincidofovir at a concentration of about 0.5 mg/mL;
mannitol at a concentration of about 1.25-2.5 mg/mL; and L-arginine
at a concentration of about 0.87 mg/mL; and dextrose at a
concentration of about 50 mg/mL; wherein the pH of the composition
is about 8.0.
[0026] In some embodiments, the aqueous pharmaceutical composition
comprises: brincidofovir at a concentration of about 0.89 mM;
mannitol at a concentration of about 6.85-13.7 mM; L-arginine at a
concentration of about 5 mM; and dextrose at a concentration of
about 287 mM; wherein the pH of the composition is about 8.0.
[0027] In some embodiments, the volume of the aqueous sugar
solution, aqueous sugar alcohol solution, Ringer's solution, or
aqueous salt solution used to dissolve a lyophilized formulation of
brincidofovir is about 100 or 200 mL. In other words, in some
embodiments, a lyophilized powder comprising brincidofovir is
dissolved in about 100 mL or about 200 mL of aqueous sugar
solution, aqueous sugar alcohol solution, Ringer's solution,
aqueous salt solution, or water. In some embodiments, the
lyophilized powder is dissolved in about 100 mL, about 110 mL,
about 120 mL, about 130 mL, about 140 mL, about 150 mL, about 160
mL, about 170 mL, about 180 mL, about 190 mL, or about 200 mL. For
example, in some embodiments, a lyophilized powder of the
disclosure can be dissolved in about 100 mL of a 5% dextrose
solution in water. In some embodiments, a lyophilized powder of the
disclosure can be dissolved in about 200 mL of a 5% dextrose
solution in water.
[0028] In some embodiments, the aqueous pharmaceutical composition
comprises: about 100 mg brincidofovir; about 250-500 mg mannitol;
about 174 mg arginine; about 5 g dextrose; and about 100 mL water.
In some embodiments, the pH of the composition is about 8.0, about
7.5, about 7.0, about 6.5, about 6.0, or below about 6.0.
[0029] In some embodiments, the aqueous pharmaceutical composition
comprises: about 200 mg brincidofovir; about 500-1000 mg mannitol;
about 348 mg arginine; about 10 g dextrose; and about 200 mL water.
In some embodiments, the pH of the composition is about 8.0, about
7.5, about 7.0, about 6.5, about 6.0, or below about 6.0.
[0030] In some embodiments, the aqueous pharmaceutical composition
comprises: about 50 mg brincidofovir; about 125-250 mg mannitol;
about 87 mg arginine; about 5 g dextrose; and about 100 mL water.
In some embodiments, the pH of the composition is about 8.0, about
7.5, about 7.0, about 6.5, about 6.0, or below about 6.0.
[0031] In some embodiments, the aqueous pharmaceutical composition
comprises: about 100 mg brincidofovir; about 250-500 mg mannitol;
about 174 mg arginine; about 10 g dextrose; and about 200 mL water.
In some embodiments, the pH of the composition is about 8.0, about
7.5, about 7.0, about 6.5, about 6.0, or below about 6.0.
[0032] In some embodiments, the aqueous pharmaceutical composition
comprises: about 100 mg brincidofovir; about 250-500 mg mannitol;
about 174 mg arginine; about 5 g dextrose; and about 100 mL water;
wherein the pH of the composition is about 8.0.
[0033] In some embodiments, the aqueous pharmaceutical composition
comprises: about 200 mg brincidofovir; about 500-1000 mg mannitol;
about 348 mg arginine; about 10 g dextrose; and about 200 mL water;
wherein the pH of the composition is about 8.0.
[0034] In some embodiments, the aqueous pharmaceutical composition
comprises: about 50 mg brincidofovir; about 125-250 mg mannitol;
about 87 mg arginine; about 5 g dextrose; and about 100 mL water;
wherein the pH of the composition is about 8.0.
[0035] In some embodiments, the aqueous pharmaceutical composition
comprises: about 100 mg brincidofovir; about 250-500 mg mannitol;
about 174 mg arginine; about 10 g dextrose; and about 200 mL water;
wherein the pH of the composition is about 8.0.
[0036] In some embodiments, one or more of the brincidofovir, the
bulking agent, and the buffer have been lyophilized before
incorporation into the aqueous pharmaceutical composition described
herein.
[0037] In some embodiments, the aqueous pharmaceutical composition
is suitable for intravenous administration. In some embodiments,
the aqueous pharmaceutical composition is sterile.
[0038] In another aspect, the present disclosure provides a
sterile, aqueous pharmaceutical composition for intravenous
administration, comprising: brincidofovir; a bulking agent; a
buffer; and dextrose.
[0039] In another aspect, the present disclosure provides a
sterile, aqueous pharmaceutical composition for intravenous
administration, comprising: brincidofovir; a bulking agent; a
buffer; and dextrose; wherein the pH of the composition is about
8.0.
[0040] In another aspect, the present disclosure provides an
aqueous pharmaceutical composition for intravenous administration,
comprising: brincidofovir at a concentration of between about 0.5
mg/mL and about 1.0 mg/mL; a bulking agent at a concentration of
between about 2.5 mg/mL and about 5 mg/mL; a buffer at a
concentration of between about 0.87 mg/mL and about 1.74 mg/mL; and
dextrose at a concentration of about 50 mg/mL. In some embodiments,
the pH of the composition is about 8.0, about 7.5, about 7.0, about
6.5, about 6.0, or below about 6.0.
[0041] In another aspect, the present disclosure provides an
aqueous pharmaceutical composition for intravenous administration,
comprising: brincidofovir at a concentration of between about 0.5
mg/mL and about 1.0 mg/mL; a bulking agent at a concentration of
between about 2.5 mg/mL and about 5 mg/mL; a buffer at a
concentration of between about 0.87 mg/mL and about 1.74 mg/mL; and
dextrose at a concentration of about 50 mg/mL; wherein the pH of
the composition is about 8.0.
[0042] In another aspect, the present disclosure provides a method
of treating a subject with a viral infection, the method
comprising: administering to the subject a pharmaceutical
composition or pharmaceutical formulation as set forth herein.
[0043] In another aspect, the present disclosure provides a method
of treating a subject with a viral infection, the method
comprising: administering to the subject an intravenous
pharmaceutical composition comprising brincidofovir; a bulking
agent; a buffer; and dextrose
[0044] In another aspect, the present disclosure provides a method
of treating a subject with a viral infection, the method
comprising: administering to the subject an intravenous
pharmaceutical composition comprising brincidofovir; a bulking
agent; a buffer; and dextrose; wherein the pH of the composition is
about 8.0.
[0045] In another aspect, the present disclosure provides a method
of treating a subject with a viral infection, the method
comprising: administering to the subject an intravenous
pharmaceutical composition comprising brincidofovir at a
concentration of between about 0.5 mg/mL and about 1.0 mg/mL; a
bulking agent at a concentration of between about 2.5 mg/mL and
about 5 mg/mL; a buffer at a concentration of between about 0.87
mg/mL and about 1.74 mg/mL; and dextrose at a concentration of
about 50 mg/mL %. In some embodiments, the pH of the composition is
about 8.0, about 7.5, about 7.0, about 6.5, about 6.0, or below
about 6.0.
[0046] In another aspect, the present disclosure provides a method
of treating a subject with a viral infection, the method
comprising: administering to the subject an intravenous
pharmaceutical composition comprising brincidofovir at a
concentration of between about 0.5 mg/mL and about 1.0 mg/mL; a
bulking agent at a concentration of between about 2.5 mg/mL and
about 5 mg/mL; a buffer at a concentration of between about 0.87
mg/mL and about 1.74 mg/mL; and dextrose at a concentration of
about 50 mg/mL %; wherein the pH of the composition is about
8.0.
[0047] In another aspect, the present disclosure provides an
aqueous pharmaceutical formulation or composition for treatment of
a viral infection, prepared by a process comprising the steps of:
dissolving, in any order, an amount of brincidofovir, a bulking
agent, and a buffer in water to form a first solution; lyophilizing
the first solution to form a lyophilized powder; and dissolving the
lyophilized powder in an aqueous sugar alcohol solution, an aqueous
sugar solution, Ringer's solution or a sodium chloride solution to
form the aqueous pharmaceutical formulation or composition.
[0048] In another aspect, the present disclosure provides the use
of an aqueous pharmaceutical composition, comprising:
brincidofovir; a bulking agent; a buffer; and an aqueous sugar
alcohol solution, an aqueous sugar solution, Ringer's solution or a
sodium chloride solution in the manufacture of a medicament for the
treatment of a viral infection.
[0049] In another aspect, the present disclosure provides the use
of an aqueous pharmaceutical composition, comprising:
brincidofovir; a bulking agent; a buffer; and an aqueous sugar
alcohol solution, an aqueous sugar solution, Ringer's solution or a
sodium chloride solution in the treatment of a viral infection.
[0050] In some embodiments of any of the above-aspects, the viral
infection to be treated is, polyomavirus, papillomavirus, herpes
virus, adenovirus, Epstein-Barr virus, cytomegalovirus, Hepatitis B
virus, Hepatitis C virus, varicella zoster virus, adenovirus,
poxvirus, or a combination thereof.
[0051] In some embodiments, administration to the subject of the
pharmaceutical compositions set forth herein does not result in
hemolysis. In some embodiments, administration to the subject of
the pharmaceutical compositions set forth herein does not result in
gastrointestinal toxicity.
[0052] In some embodiments, the pH of any of the aqueous
formulations or compositions for intravenous administration can
have a pH below about 8.0 (e.g., about 8.0, about 7.5, about 7.0,
about 6.5, about 6.0, about 5.5, about 5.0, about 4.5, about 4.0,
or below about 4.0). In some embodiments, the pH can be higher than
8.0 (e.g., about 8.5, about 9.0, about 9.5, about 10.0, or above
about 10.0).
[0053] Without wishing to be bound by theory, administration of
oral brincidofovir can result in significantly higher exposure in
the intestine compared with other organs; this higher exposure can
lead to GI toxicity after oral administration of brincidofovir. As
set forth herein, IV administration of brincidofovir can prevent
over-exposure of the gut (e.g., intestine) to brincidofovir (e.g.,
in comparison to oral administration of BCV), with improvement in
gastrointestinal (GI) tolerability and reduction in GI toxicity. In
some embodiments, IV administration of brincidofovir can deliver
comparable (e.g., compared with oral administration) drug exposure
to blood plasma and organs such as the liver, kidney, and small
intestine, even at lower doses than necessary using oral
administration.
[0054] In some embodiments, IV brincidofovir can deliver the drug
to organs (e.g., the brain) that can be difficult to reach by other
administrative routes (e.g., oral dosage). In some embodiments,
higher CNS exposure with IV brincidofovir can treat viral
infections in the brain (e.g., herpes encephalitis in newborns and
adults; HHV-6 encephalitis; JC virus/PML in transplant recipients
or patients with multiple sclerosis).
[0055] Intravenous brincidofovir can avoid GI side effects that can
be observed with oral administration of BCV and provide
opportunities for treatment of DNA viruses (e.g., in patients who
experience GI-side effects associated with oral administration of
BCV). For example, like oral administration of brincidofovir, IV
administration of brincidofovir can be used as a broad spectrum
antiviral agent with limited toxicity (e.g., substantially no heme
toxicity, and no kidney toxicity), and can be effective at
treating, e.g., adenovirus and smallpox. Additionally, IV
administration of brincidofovir can be used to treat and prevent,
for example, cytomegalovirus, adenovirus, and BK or JC virus. These
diseases can be treated, for instance, in hematopoietic cell
transplant patients.
[0056] Additional features and advantages will be apparent to one
of skill in the art and are set forth in the Detailed Description
of the Invention, below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] The skilled artisan will understand that the drawings
primarily are for illustrative purposes and are not intended to
limit the scope of the inventive subject matter described herein.
The drawings are not necessarily to scale; in some instances,
various aspects of the inventive subject matter disclosed herein
may be shown exaggerated or enlarged in the drawings to facilitate
an understanding of different features. In the drawings, like
reference characters generally refer to like features (e.g.,
functionally similar and/or structurally similar elements).
[0058] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0059] The above and further features will be more clearly
appreciated from the following detailed description when taken in
conjunction with the accompanying drawings.
[0060] FIG. 1 is a graph showing shows product temperature profiles
as recorded by product probes as set forth in Example 4.
[0061] FIG. 2 is a pair of graphs illustrating the evaluation of
shelf life in an accelerated stability study.
[0062] FIG. 3 is a graph showing the time course of blood and
plasma concentrations of radioactivity for male Sprague Dawley (SD)
and Long-Evans (LE) rats following a single 2-h intravenous
infusion (IV) of [.sup.14C]brincidofovir at a target dose of 15
mg/kg or 2 mg/kg and after a single oral (P0) dose at 15 mg/kg as
set forth in Example 9.
[0063] FIG. 4 is a plot of tissue concentration versus time for the
small intestine for [.sup.14C]brincidofovir dosed intravenously and
orally as set forth in Example 9.
[0064] FIG. 5 is a plot of tissue concentration versus time for the
kidney cortex for [.sup.14C]brincidofovir dosed intravenously and
orally as set forth in Example 9.
[0065] FIG. 6 is a plot of the plasma concentration of
brincidofovir after doses of 100 mg orally, 10 mg IV, and 25 mg
IV.
[0066] FIG. 7A is a histogram of rat intestine after oral
administration of brincidofovir as set forth in Example 10.
[0067] FIG. 7B is a histogram of rat intestine after IV
administration of brincidofovir as set forth in Example 10.
[0068] FIG. 8 is a graph showing the mean (+/-SE) alanine
aminotransferase (ALT) levels observed in a IV BCV single ascending
dose (SAD) trial.
[0069] FIG. 9 is a graph illustrating mean plasma brindidofovir
concentration as a function of time for subjects in cohorts 1-4,
and mean plasma BCV concentration as a function of time for
subjects administered BCV orally.
[0070] FIG. 10 is a graph illustrating median plasma cidofovir
concentration as a function of time following IV and Oral BCV
Doses.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0071] The present disclosure is related to pharmaceutical
compositions (e.g., lyophilized or aqueous compositions) comprising
brincidofovir and methods of use thereof. The disclosure also
relates to pharmaceutical formulations comprising a reconstituted
lyophilized pharmaceutical composition described herein. The
compositions and formulations can be for intravenous
administration. In some embodiments, IV administration of the
compositions or formulations of the present disclosure do not
result in hemolysis. The formulations can also be used in cases
where oral administration of drug is not possible or substantially
impossible, for example due to underlying conditions or because of
inadequate oral absorption.
[0072] The present disclosure relates to at least three types of
compositions of brincidofovir. First, the present disclosure
relates to bulk formulations of brincidofovir (e.g., liquid
formulations of the disclosure or pre-lyophilization formulations
of the disclosure) that can be lyophilized prior to storage. The
lyophilized formulations (e.g., powders) can be stored for extended
periods of time. Additionally, the lyophilized formulations can be
reconstituted (e.g. by dissolution in a sugar solution) prior to
(e.g., immediately prior to) administration to a patient, e.g., a
patient in need of treatment. Accordingly, the present disclosure
teaches pre-lyophilization formulations of brincidofovir,
lyophilized powders, and re-constituted formulations (e.g.,
pharmaceutical formulations) for administration.
Formulations of the Disclosure
Pre-Lyophilization Formulations
[0073] In some embodiments, brincidofovir is dissolved in water
prior to lyophilization (e.g., freeze-drying). The brincidofovir
can be co-dissolved with, for instance, a bulking agent and a
buffer. The dissolution of the components may occur separately or
concurrently, and may occur in any order.
[0074] In some embodiments, pre-lyophilization compositions of the
disclosure comprise a buffer. In some embodiments,
pre-lyophilization compositions of the disclosure comprise a
bulking agent. In some embodiments, pre-lyophilization formulations
of the disclosure comprise a buffer and a bulking agent. In some
embodiments, the concentration of brincidofovir is 10 mg/mL. In
some embodiments, the pH of the solution is adjusted with HCl
and/or NaOH.
[0075] In some embodiments, the buffer is selected from sodium
phosphate, arginine, tromethamine and pH-adjusted water. In some
embodiments, the buffer is sodium phosphate, L-arginine, or
tromethamine. In some embodiments, the buffer is Na-phosphate. In
some embodiments, the buffer is arginine. In some embodiments, the
buffer is L-arginine. In some embodiments, the buffer is
tromethamine. In some embodiments, the buffer is pH-adjusted water.
In some embodiments, the buffer is present at a concentration of
about 100 mM to about 200 mM. In some embodiments, the buffer is
present in an amount of about 25 mM, about 50 mM, about 75 mM,
about 100 mM, about 125 mM, about 150 mM, about 175 mM, or about
200 mM.
[0076] In some embodiments, the bulking agent is mannitol or
sucrose. In some embodiments, the buffer is sodium phosphate,
arginine, or tromethamine. In some embodiments, the bulking agent
is present at a concentration of about 5-9% (w/v). In some
embodiments, the bulking agent is present at a concentration of
about 2.5-9% (w/v). In some embodiments, the bulking agent is
present at a concentration of about 2.5% (w/v), about 5% (w/v), or
about 9% (w/v).
[0077] In some embodiments, the compositions of the disclosure
comprise brincidofovir at a concentration of about 10.0 mg/mL,
about 6.4 mg/mL, or about 3.2 mg/mL.
[0078] In some embodiments, the compositions of the disclosure
comprise brincidofovir at a concentration of about 10.0 mg/mL,
mannitol at a concentration of about 25-50 mg/mL, L-arginine at a
concentration of about 17.4 mg/mL and water, and have pH=8.0.
[0079] In some embodiments, the compositions of the disclosure
comprise brincidofovir at a concentration of about 17.8 mM,
mannitol at a concentration of about 137.5-275 mM, L-arginine at a
concentration of about 100 mM and water, and have pH=8.0.
[0080] In some embodiments, any of the pre-lyophilization
formulations described herein can be lyophilized to remove water
(e.g., for storage).
[0081] In some embodiments, pre-lyophilization formulations can be
used directly as pharmaceutical formulations (i.e., can be given
directly to a patient). However, in some embodiments, these
formulations are lyophilized for storage and are later
reconstituted (e.g., in 5% dextrose in water) for administration to
a patient.
[0082] In some embodiments liquid formulations of brincidofovir are
for intravenous administration after dilution into infusion
vehicles (e.g., 5% dextrose).
[0083] In some embodiments, the liquid formulations of the
disclosure (i.e., pre-lyophilization formulations of the
disclosure) are compatible with infusion vehicles and materials
(e.g., containers) used in a clinical setting. For example, the
liquid formulations of the disclosure display no significant
changes in appearance, pH or recovery of brincidofovir, or
introduction of impurities, upon contact with materials such as
sterile filters, vials, stoppers, infusion bags, or IV systems. In
some embodiments, liquid formulations of the disclosure experience
no significant change in pH, loss of brincidofovir, or introduction
of impurities upon filtration through a syringe filter.
Lyophilized Formulations
[0084] The pre-lyophilization formulations can be lyophilized to
produce lyophilized formulations (e.g., powders) of BCV, bulking
agent, and buffer. The lyophilized formulations can be stable for
extended periods of time and can be reconstituted prior to
administration to a patient. In some embodiments, the lyophilized
formulations are sterile.
[0085] In some embodiments, lyophilization comprises freezing,
annealing, and drying of the lyophilized composition. In some
embodiments, drying comprises a primary drying and a secondary
drying. In some embodiments, freezing and annealing comprises
exposing the formulation to temperatures between about 5.degree. C.
and -50.degree. C. In some embodiments primary drying is conducted
at a temperature of about 35.degree. C. In some embodiments
secondary drying is conducted at a temperature of about 20.degree.
C.
[0086] In some embodiments, freezing and/or annealing lasts for
about 16 h. In some embodiments, the primary drying phase lasts for
about 20 h. In further embodiments, the secondary drying takes
about 22 h (e.g., 21.7 h) or about 28 h (e.g., 27.7 h).
[0087] In some embodiments, liquid formulations of the disclosure
(e.g., liquid formulations comprising brincidofovir, arginine and
mannitol) do not foam during the lyophilization process, in
contrast to other formulations which suffered from foaming during
lyophilization.
Reconstituted Pharmaceutical Compositions for IV Administration
[0088] Prior to IV administration to a patient, the lyophilized
powders discussed above can be reconstituted, for example by
dissolution in an aqueous solvent such as water. In some
embodiments, the aqueous solvent is water containing a sugar
alcohol or sugar (e.g., dextrose). In some embodiments, the aqueous
solvent is 5% dextrose in water. The aqueous solvent can likewise
be sterile (e.g., like the sterile lyophilized formulation) and can
be suitable for administration to a patient in need thereof. In
some embodiments, reconstitution of the lyophilized powders of the
disclosures yields aqueous compositions of the disclosure.
[0089] As set forth below, the pharmaceutical compositions for IV
administration described herein can provide therapeutically
relevant blood plasma concentrations of brincidofovir using lower
doses of brincidofovir than those necessary when orally
administering brincidofovir. For example, IV administration of
brincidofovir using formulations of the present disclosure provided
blood plasma concentrations of brincidofovir in humans that had
previously demonstrated anti-viral potency in cytomegalovirus
prevention and adenovirus treatment. The amount of BCV used in the
IV formulations (e.g., about 10 mg or about 25 mg) was about one
tenth of the amount of BCV required to achieve similar blood-plasma
concentrations using oral dosing.
[0090] Accordingly, in some embodiments, the present disclosure
teaches the treatment of a viral infection comprising administering
a subject in need thereof an IV formulation of brincidofovir as set
forth herein. In some embodiments, the IV dose is less than the
orally administered dose of brincidofovir (e.g., about 50% of an
oral dose, about 40% of an oral dose, about 30% of an oral dose,
about 20% of an oral dose, or about 10% of an oral dose) necessary
to achieve a similar result (e.g., blood plasma concentration,
anti-viral activity, etc.). Thus, in some embodiments, it is
possible to use a smaller quantity of IV brincidofovir than used
for oral administration to achieve a similar (e.g., substantially
the same) clinical outcome. Additionally, because IV administration
results in lower quantities of brincidofovir in the intestine, in
some embodiments the present disclosure also provides for the
treatment of viral infections without resulting in gastrointestinal
toxicity. The present disclosure teaches the treatment of patients
who cannot be administered drugs orally (e.g., patients with
sensitive intestinal tracts or those unable to swallow oral
medications).
[0091] In some embodiments, the compositions of the disclosure
comprise brincidofovir at a concentration of about 1.0 mg/mL,
mannitol at a concentration of about 2.5-5 mg/mL, L-arginine at a
concentration of about 1.74 mg/mL and dextrose at a concentration
of about 50 mg/mL, and have pH=8.0.
[0092] In some embodiments, the compositions of the disclosure
comprise brincidofovir at a concentration of about 17.8 mM,
mannitol at a concentration of about 13.75-27.5 mM, L-arginine at a
concentration of about 10 mM and dextrose at a concentration of
about 287 mM, and have pH=8.0.
[0093] In some embodiments, the compositions of the disclosure
comprise brincidofovir at a concentration of about 0.5 mg/mL,
mannitol at a concentration of about 1.25-2.5 mg/mL, L-arginine at
a concentration of about 0.87 mg/mL and dextrose at a concentration
of about 50 mg/mL, and have pH=8.0.
[0094] In some embodiments, the compositions of the disclosure
comprise brincidofovir at a concentration of about 0.89 mM,
mannitol at a concentration of about 6.85-13.7 mM, L-arginine at a
concentration of about 5 mM and dextrose at a concentration of
about 287 mM, and have pH=8.0.
[0095] In some embodiments, the pharmaceutical compositions of the
disclosure further comprises a sugar. In some embodiments, the
sugar is in an aqueous sugar solution. In some embodiments, the
aqueous sugar solution is a 5% dextrose solution. In some
embodiments, the volume of the aqueous sugar solution is about 100
or 200 mL.
[0096] In some embodiments, the compositions of the disclosure
comprise brincidofovir in an amount of about 100 mg, mannitol in an
amount between about 250 mg and about 500 mg, arginine in an amount
of about 174 mg, dextrose in an amount of about 5 g, and water in
an amount of about 100 mL, and have pH=8.0.
[0097] In some embodiments, the compositions of the disclosure
comprise brincidofovir in an amount of about 200 mg, mannitol in an
amount between about 500 mg and about 1000 mg, arginine in an
amount of about 348 mg, dextrose in an amount of about 10 g, and
water in an amount of about 100 mL, and have pH=8.0.
[0098] In some embodiments, the compositions of the disclosure
comprise brincidofovir in an amount of about 50 mg, mannitol in an
amount between about 125 mg and about 250 mg, arginine in an amount
of about 87 mg, dextrose in an amount of about 5 g, and water in an
amount of about 100 mL, and have pH=8.0.
[0099] In some embodiments, the compositions of the disclosure
comprise brincidofovir in an amount of about 100 mg, mannitol in an
amount between about 250 mg and about 500 mg, arginine in an amount
of about 174 mg, dextrose in an amount of about 10 g, and water in
an amount of about 200 mL, and have pH=8.0.
[0100] In some embodiments, compositions of the disclosure comprise
brincidofovir at a concentration of about 0.1-1.0 mg/mL within
100-200 mL of a 5% dextrose solution. In some embodiments, the
compositions comprise: 10 mg brincidofovir, 100 mM arginine, and 5%
mannitol (w/v), and have pH=8.0.
[0101] In some embodiments, lyophilized compositions of the
disclosure are for IV administration by infusion after
dilution.
[0102] In some embodiments, the buffer is present at about 200 mM.
In some embodiments, the brincidofovir has been previously
lyophilized. In some embodiments, the compositions of the
disclosure are lyophilized to remove the water. In some
embodiments, the brincidofovir, the bulking agent, and the buffer
have previously been lyophilized.
[0103] In some embodiments, the present disclosure provides an
aqueous pharmaceutical composition for intravenous administration
comprising brincidofovir, mannitol, and arginine. In some
embodiments, the pH of the formulation is about 8.0. In some
embodiments, the pH of the composition is adjusted using HCl and/or
NaOH.
[0104] In some embodiments, compositions of the disclosure are
suitable for reconstitution with deionized water. In some
embodiments, compositions of the disclosure do not foam upon
reconstitution. In some embodiments, compositions of the disclosure
foam upon reconstitution and foam dissipates in less than 20
minutes, less than 10 minutes, less than 5 minutes, or less than 1
minute. In some embodiments, compositions comprising 100 mM or more
of arginine display faster dissipation of foam or bubbles upon
reconstitution than compositions comprising 50 mM or less of
arginine.
[0105] In some embodiments, lyophilized compositions of the
disclosure do not experience changes in appearance, pH or
brincidofovir recovery upon dilution with an infusion vehicle
(e.g., 5% dextrose), e.g., prior to IV administration.
[0106] In some embodiments, reconstitution of lyophilized
compositions of the disclosure does not result in a loss of
brincidofovir. In some embodiments, lyophilized formulations of the
disclosure are clear and colorless after reconstitution.
Excipients and Pharmaceutically Acceptable Carriers
[0107] In addition to brincidofovir, a buffer and a bulking agent,
the pharmaceutical compositions of the disclosure can contain
additional pharmaceutically acceptable carriers. Such
pharmaceutically acceptable carriers may include any and all
solvents, diluents, or other liquid vehicle, dispersion or
suspension aids, surface active agents, isotonic agents, thickening
or emulsifying agents, preservatives, solid binders, lubricants and
the like, as suited to the particular dosage form desired.
Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W.
Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various
carriers used in formulating pharmaceutical compositions and known
techniques for the preparation thereof. Except insofar as any
conventional carrier medium is incompatible with the active
compound (i.e., brincidofovir) such as by producing any undesirable
biological effect or otherwise interacting in a deleterious manner
with any other component(s) of the pharmaceutical composition, its
use is contemplated to be within the scope of this disclosure. Some
examples of materials which can serve as pharmaceutically
acceptable carriers include, but are not limited to, sugars such as
lactose, glucose and sucrose; starches such as corn starch and
potato starch; cellulose and its derivatives such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatine; talc; excipients such as cocoa
butter and suppository waxes; oils such as peanut oil, cottonseed
oil; safflower oil, sesame oil; olive oil; corn oil and soybean
oil; glycols; such as propylene glycol; esters such as ethyl oleate
and ethyl laurate; agar; buffering agents such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen free water;
isotonic saline; Ringer's solution; ethyl alcohol, and phosphate
buffer solutions, as well as other non-toxic compatible lubricants
such as sodium lauryl sulfate and magnesium stearate, as well as
coloring agents, releasing agents, coating agents, sweetening,
flavoring and perfuming agents, preservatives and antioxidants can
also be present in the composition, according to the judgment of
the formulator.
Methods of Use
[0108] In some embodiments, the present disclosure provides methods
of treating a subject with a viral infection, comprising
administering to the subject a composition of the disclosure. In
some embodiments, a composition of the disclosure is administered
intravenously to the subject.
[0109] In some embodiments, formulations of the present disclosure
can comprise a volume of concentrated sterile liquid solution
(e.g., for dilution) and/or lyophilized powder (e.g., for
reconstitution). In some embodiments, the formulations can be
stored for at least 12 months (e.g., at about 25.degree. C.). In
some embodiments, the formulations can be stored upside down (e.g.,
for about 24 months, or for about 36 months). In some embodiments,
the formulations can be stored in a refrigerator (e.g., for about
12 months). In some embodiments, the lyophilized powder is readily
dissolvable (e.g., for reconstitution) in a liquid. In some
embodiments, the liquid volume can be small (e.g., for parenteral
administration). In some embodiments, the formulations demonstrate
acceptable filter and tubing compatibility. In some embodiments,
the formulations are compatible with normal resuscitation fluids
including but not limited to D5W, NS, and D51/2 NS.
Patient Population
[0110] In some embodiments, administration of a composition of the
disclosure does not result in hemolysis. In some embodiments,
administration of a composition of the disclosure does not result
in gastrointestinal toxicity.
[0111] Accordingly, in some embodiments, the present disclosure
provides for the dosing of patients with brincidofovir who may
experience unwanted side effects associated with oral
administration of brincidofovir (e.g., gastro-intestinal side
effects such as diarrhea, pain, constipation and the like). For
instance, in some embodiments, IV formulations can be used for
patients with symptoms such as anemia, irritable bowel syndrome,
constipation, diarrhea, bowel pain, and the like. Additionally, in
some embodiments the present disclosure provides for sustained
and/or repeated dosing of brincidofovir to subjects in need
thereof. For instance, a patient who has undergone a stem cell
transplant can be administered IV formulations of the present
disclosure to prevent gastro-intestinal side effects associated
with oral administration (e.g., repeated oral administration) of
brincidofovir.
Viral Indications
[0112] In some embodiments, the viral infection to be treated is
selected from polyomavirus (including BK, John Cunningham virus
(JCV), Merkel cell virus (MCV), KI polyomavirus (KIV), WU
polyomavirus (WUV), Simian virus 40 (SV 40)), papillomavirus
(including human papillomavirus, cottontail rabbit papillomavirus,
equine papillomavirus and bovine papillomavirus), herpes virus
(e.g., herpes simplex virus), adenovirus, Epstein-Barr virus (EBV),
human cytomegalovirus (HCMV), Hepatitis B virus, Hepatitis C virus,
varicella zoster virus (VZV) or a combination thereof.
[0113] In some embodiments, the IV formulations can be used to
prevent clinically significant cytomegalovirus infection in at-risk
(e.g., CMV seropositive) adult and pediatric allogenic
hematopoietic stem cell transplant recipients. In some embodiments,
the present formulations can be used for treatment of adenovirus
infection in adult and pediatric immunocompromised hosts. In some
embodiments, the formulations can be used for hematopoietic stem
cell transplant or solid organ transplant patients.
Dosage Regimens
[0114] In some embodiments, the formulations (e.g., IV
formulations) of the present disclosure are administered at a dose
of between about 1 mg and 1000 mg BCV. For example, the
formulations can be administered at a dose of between about 10 mg
and 200 mg BCV. In some embodiments, the IV formulations can be
administered at about 5 mg; about 10 mg; about 15 mg; about 20 mg;
about 25 mg; about 30 mg; about 35 mg; about 40 mg; about 45 mg;
about 50 mg; about 55 mg; about 60 mg; about 65 mg; about 70 mg;
about 75 mg; about 80 mg; about 85 mg; about 90 mg; about 95 mg;
about 100 about 105 mg; about 110 mg; about 115 mg; about 120 mg;
about 125 mg; about 130 mg; about 135 mg; about 140 mg; about 145
mg; about 150 mg; about 155 mg; about 160 mg; about 165 mg; about
170 mg; about 175 mg; about 180 mg; about 185 mg; about 190 mg;
about 195 mg; or about 200 mg BCV.
[0115] In some embodiments, IV formulations of the present
disclosure can be administered to deliver between about 5 and about
50 mg; about 10 and about 50 mg; about 10 and about 40 mg; about 10
and about 30 mg; about 5 and about 25 mg; about 10 and about 25 mg;
and about 15 and about 25 mg of BCV. The formulations can be
administered to a human (e.g., an adult human).
[0116] In some embodiments, the formulations are safe and
well-tolerated when administered intravenously at doses of between
about 10 mg and 100 mg (e.g., at about 10 mg or at about 25 mg).
For example, 10 mg and 25 mg doses of brincidofovir formulations of
the present disclosure can give favorable tolerability profiles
without adverse events. In some embodiments, doses of about 10 mg
and 25 mg of the formulations described herein do not produce
gastrointestinal side effects.
[0117] In some embodiments, IV formulations taught herein can be
administered multiple times per day, or can be administered as
single-doses. For instance, IV formulations can be administered
once a day or can be administered twice a day. In some embodiments,
formulations of the disclosure are administered once a week, or
twice a week. In some embodiments, formulations of the disclosure
are administered every other day. In some embodiments, formulations
of the disclosure are administered every other week. In some
embodiments, formulations of the disclosure are administered once a
month, or twice a month. One of skill in the art will be able to
determine an appropriate dosage regimen for a patient.
[0118] The duration of administration (i.e., for each dose) of IV
brincidofovir can vary according to the needs of an individual
subject or patient in need thereof and it is within the expertise
of one skilled in the art (e.g., a clinician such as a nurse or
doctor) to determine the appropriate amount of time that a subject
is administered brincidofovir. For instance, IV administration of
brincidofovir can last about 15 minutes, about 30 minutes, about 45
minutes, about 60 minutes, about 75 minutes, about 90 minutes,
about 105 minutes, or about 120 minutes.
[0119] In some embodiments, the formulations of the disclosure can
be administered in combination with other therapeutic agents or
treatments. For example, other therapeutic agents can be cisplatin,
doxorubicin, etoposide, irinotecan, topotecan, paclitaxel,
docetaxel, the epothilones, tamoxifen, 5-fluorouracil,
methotrexate, temozolomide, cyclophosphamide, lonafarib,
tipifarnib,
4-((5-(((4-(3-chlorophenyl)-3-oxopiperazin-1-yl)methyl)-1H-imidazol-1-yl)-
methyl)benzonitrile hydrochloride,
(R)-1-((1H-imidazol-5-yl)methyl)-3-benzyl-4-(thiophen-2-ylsulfonyl)-2,3,4-
,5-tetrahydro-1H-benzo di azepine-7-carb onitrile, cetuximab,
imatinib, interferon alfa-2b, pegylated interferon alfa-2b,
aromatase combinations, gemcitabine, uracil mustard, chlormethine,
ifosfamide, melphalan, chlorambucil, pipobroman,
triethylenemelamine, triethylenethiophosphoramine, busulfan,
carmustine, lomustine, streptozocin, dacarbazine, floxuridine,
cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate,
leucovorin, oxaliplatin, pentostatine, vinblastine, vincristine,
vindesine, bleomycin, dactinomycin, daunorubicin, epirubicin,
idarubicin, mithramycin, deoxycoformycin, mitomycin-C,
L-asparaginase, teniposide 17.alpha.-ethinyl estradiol, diethyl
stilbestrol, testosterone, prednisone, fluoxymesterone,
dromostanolone propionate, testolactone, megestrol acetate,
methylprednisolone, methyltestosterone, prednisolone,
triamcinolone, chlorotrianisene, 17.alpha.-hydroxyprogesterone,
aminoglutethimide, estramustine, medroxyprogesterone acetate,
leuprolide acetate, flutamide, toremifene citrate, goserelin
acetate, carboplatin, hydroxyurea, amsacrine, procarbazine,
mitotane, mitoxantrone, levamisole, vinorelbine, anastrazole,
letrozole, capecitabine, raloxifene, droloxafine,
hexamethylmelamine, bevacizumab, trastuzumab, tositumomab,
bortezomib, ibritumomab tiuxetan, arsenic trioxide, porfimer
sodium, cetuximab, thioTEPA, altretamine, fulvestrant, exemestane,
rituximab, alemtuzumab, dexamethasone, bicalutamide, chlorambucil,
or valrubicin. The agents can be administered as part of a
treatment regime and are not necessarily administered concurrently
or on the same schedule.
Definitions
[0120] For the purposes of promoting an understanding of the
embodiments described herein, reference made to preferred
embodiments and specific language are used to describe the same.
The terminology used herein is for the purpose of describing
particular embodiments only, and is not intended to limit the scope
of the present invention. As used throughout this disclosure, the
singular forms "a," "an," and "the" include plural reference unless
the context clearly dictates otherwise. Thus, for example, a
reference to "a composition" includes a plurality of such
compositions, as well as a single composition, and a reference to
"a therapeutic agent" or "an active compound" is a reference to one
or more therapeutic and/or pharmaceutical agents (e.g.,
brincidofovir) and equivalents thereof known to those skilled in
the art. All percentages and ratios used herein, unless otherwise
indicated, are by weight.
[0121] The term "about" is used herein to mean approximately, in
the region of, roughly or around. When the term "about" is used in
conjunction with a numerical range, it modifies that range by
extending the boundaries above and below the numerical values set
forth. In general, the term "about" is used herein to modify a
numerical value above and below the stated value by a variance of
20%.
[0122] As used in the present disclosure, whether in a transitional
phrase or in the body of a claim, the terms "comprise(s)" and
"comprising" are to be interpreted as having an open-ended meaning.
That is, the terms are to be interpreted synonymously with the
phrases "having at least" or "including at least." When used in the
context of a process the term "comprising" means that the process
includes at least the recited steps, but may include additional
steps. When used in the context of a molecule, compound, or
composition, the term "comprising" means that the compound or
composition includes at least the recited features or components,
but may also include additional features or components.
[0123] A "pharmaceutical composition" is a formulation containing a
compound of the present disclosure (e.g., brincidofovir) in a form
suitable for administration to a subject. The term "pharmaceutical
composition" includes preparations suitable for administration to
mammals, e.g., humans. A pharmaceutical composition can be, e.g.,
an intravenous (IV) formulation, or an oral formulation.
[0124] The term "monotherapy" is understood to mean the use of a
single drug to treat a particular disorder or disease. Monotherapy
is different from combination therapy in that combination therapy
includes the use of at least two drugs in combination to treat a
particular disorder or disease.
[0125] As used herein, "treating," "treatment" or "treat" describes
the management and care of a patient for the purpose of combating a
disease, condition, or disorder and includes the administration of
active compound of the present disclosure (i.e., brincidofovir), to
alleviate the symptoms or complications of a disease, condition or
disorder, or to eliminate the disease, condition or disorder. The
term "treat" can also include treatment of a cell in vitro or an
animal model.
[0126] As used herein, "compounds of the disclosure" refers to both
active compounds (e.g., brincidofovir) as well as non-active
compounds (e.g., bulking agents, buffers, sweeteners, and the
like).
[0127] The active compound of the present disclosure (i.e.,
brincidofovir) may also be used to prevent a relevant disease,
condition or disorder, or used to identify suitable candidates for
such purposes. As used herein, "preventing," "prevent," or
"protecting against" describes reducing, ameliorating or
eliminating the onset of the symptoms or complications of such
disease, condition or disorder.
[0128] Brincidofovir can also be used in the prophylaxis of a
disease such as a viral infection. Prophylaxis is understood to
mean action taken to prevent a disease. In the context of the
current disclosure, prophylaxis can mean treatment with, e.g.,
brincidofovir.
[0129] As used herein, "therapeutically effective amount" means
that amount necessary to make a clinically observed improvement in
the patient. In some embodiments, the compounds of the disclosure
are formulated such that they comprise an amount that would not
cause one or more unwanted side effects. For instance, the term
"therapeutically effective amount," can refer to an amount of any
pharmaceutical agent or agents (e.g., brincidofovir) to treat,
ameliorate, or prevent an identified disease or condition (e.g., a
viral infection), or to exhibit a detectable therapeutic or
inhibitory effect. The effect can be detected by any assay method
known in the art. The precise effective amount for a subject will
depend upon the subject's body weight, size, and health; the nature
and extent of the condition; and the dosing schedule of
therapeutics selected for administration.
[0130] Therapeutically effective amounts for a given situation can
be determined by routine experimentation that is within the skill
and judgment of the clinician. In a preferred aspect, the disease
or condition to be treated is viral infection. Furthermore, an
effective amount of a pharmaceutical agent is that which provides
an objectively identifiable improvement as noted by the clinician
or other qualified observer.
[0131] As used herein, the phrase "pharmaceutically acceptable"
refers to those compounds, materials, compositions, carriers,
and/or dosage forms which are, within the scope of sound medical
judgment, suitable for use in contact with the tissues of human
beings and animals without excessive toxicity, irritation, allergic
response, or other problem or complication, commensurate with a
reasonable benefit/risk ratio.
[0132] "Pharmaceutically acceptable excipient or carrier" means an
excipient or carrier that is useful in preparing a pharmaceutical
composition that is generally safe, non-toxic and neither
biologically nor otherwise undesirable, and includes excipient that
is acceptable for veterinary use as well as human pharmaceutical
use. A "pharmaceutically acceptable excipient" as used in the
specification and claims includes both one and more than one such
excipient. Pharmaceutically acceptable excipients and carriers are
listed above.
[0133] As used herein, a "subject" is interchangeable with a
"subject in need thereof", both of which refer to a subject (e.g.,
a patient) having a disorder in which viral infection plays a part,
or a subject having an increased risk of developing viral infection
associated disease or disorder relative to the population at large.
A "subject" includes a mammal. The mammal can be e.g., a human or
appropriate non-human mammal, such as primate, mouse, rat, dog,
cat, cow, horse, goat, camel, sheep or a pig. The subject can also
be a bird or fowl. In one embodiment, the mammal is a human.
[0134] Representative "pharmaceutically acceptable salts" of
brincidofovir include, e.g., water-soluble and water-insoluble
salts, such as the acetate, amsonate
(4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzonate,
bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate,
calcium, calcium edetate, camsylate, carbonate, chloride, citrate,
clavulariate, dihydrochloride, edetate, edisylate, estolate,
esylate, fiunarate, gluceptate, gluconate, glutamate,
glycollylarsanilate, hexafluorophosphate, hexylresorcinate,
hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate,
iodide, sethionate, lactate, lactobionate, laurate, magnesium,
malate, maleate, mandelate, mesylate, methylbromide, methylnitrate,
methylsulfate, mucate, napsylate, nitrate, N-methylglucamine
ammonium salt, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate,
pamoate (1,1-methene-bis-2-hydroxy-3-naphthoate, einbonate),
pantothenate, phosphate/diphosphate, picrate, polygalacturonate,
propionate, p-toluenesulfonate, salicylate, stearate, subacetate,
succinate, sulfate, sulfosalicylate, suramate, tannate, tartrate,
teoclate, tosylate, triethiodide, and valerate salts. In some
embodiments, a pharmaceutically acceptable salt is a sodium salt, a
potassium salt, or a calcium salt.
Brincidofovir
[0135] Brincidofovir (CMX001, BCV), a lipid conjugate of cidofovir
(CDV), can be used for the prevention and treatment of infection
and disease caused by double stranded DNA viruses with high unmet
medical need. The present disclosure teaches IV formulations of
brincidofovir not only as a way to deliver BCV to patients unable
to take oral medications, but also as a way to potentially reduce
or avoid adverse GI events observed with oral dosing.
[0136] As used herein, the term "brincidofovir" is understood to
encompass the neutral compound below:
##STR00002##
[0137] as well as any pharmaceutically acceptable salts thereof. In
some of the examples below, brincidofovir is referred to as the
"test item."
Cidofovir
[0138] Cidofovir, also known as CDV, has the structure below:
##STR00003##
Brincidofovir Deamination Product
[0139] In some embodiments, brincidofovir can be deaminated under
aqueous conditions (e.g., at acidic pH) to produce the
corresponding uracil-derivative. The deaminated product is shown
below:
##STR00004##
[0140] The present disclosure relates to aqueous and lyophilized
formulations comprising brincidofovir and methods of using the
same. As used herein, a "lyophilized formulation" means a
formulation comprising an amount of solid lyophilized brincidofovir
that is dissolved in a solvent (e.g., water, or a sugar solution).
A "lyophilized formulation" can be a pharmaceutical composition of
the present disclosure.
Characteristics of Pre-Lyophilized Formulations
[0141] As set forth in is set forth in Example 4,
pre-lyophilization formulations comprising arginine as buffer and
mannitol as bulking agent yield a white uniform solid with only
minor melt-back upon lyophilization. Further, the lyophilized
product obtained from a pre-lyophilization formulation comprising
arginine as buffer and mannitol as bulking agent showed only minor
foaming upon reconstitution, in contrast to other formulations
tested. For example, formulations comprising sodium phosphate or
pH-adjusted water as a buffer showed foaming upon reconstitution
and generally required several minutes (e.g., more than 30 minutes)
before the foam dissipated. Likewise, formulations comprising
sucrose as a bulking agent also were found to exhibit foaming upon
reconstitution and generally required several minutes (e.g., more
than 30 minutes) before the foam dissipated.
[0142] Furthermore, as shown below in Example 4, mannitol-based
aqueous formulations of the disclosure were also found to yield a
more compact, uniform lyophilization cake upon lyophilization
compared with other formulations. For example, sucrose based
formulations exhibited at least partial cake collapses (and in some
cases significant cake collapse) as well as an increased melt-back
(e.g., extensive melt-back) upon lyophilization. However,
mannitol-based aqueous formulations were found to exhibit less cake
collapse and less melt-back.
[0143] Additionally, in some embodiments, phosphate-based buffers
showed pH shifts on freezing during the lyophilization process, in
contrast to formulations using arginine as a buffer. Accordingly,
the compositions set forth herein were found to have desirable
properties (e.g., greater stability and greater ease of use)
compared with other formulations tested.
Stability and Material Compatibility of Liquid Formulations
[0144] As set forth in Example 1, when brincidofovir sample stock
solutions (50 .mu.g/mL) in a 50 mM phosphate buffer at pH 6.0-8.0
containing 20% methanol were stored at 2-8.degree. C.,
precipitation was observed for of pH 7.0 and below. However,
samples prepared at a higher pH (e.g., pH=7.5 and pH=8.0) revealed
no observable change in appearance throughout the time course of
the experiment (e.g., precipitation was not observed). For samples
stored at 40.degree. C., no physical change in appearance was
observed for samples at any pH and minimal variation in
brincidofovir concentration was observed for all of the studied pH
conditions at 40.degree. C.
[0145] In the study presented in Example 1, vehicles for
brincidofovir formulations were prepared from 5.times. sodium
phosphate buffer stocks (e.g., at pH 6.0, 6.5, 7.0, 7.5, or 8.0),
various tonicifiers (e.g., 66.5 mM NaCl, 68.8 mM dextrose, 49.8 mM
MgCl.sub.2, or 35.7 mM Ca D-gluconate) and various excipients
(e.g., Cremophor.RTM., edetate disodium (EDTA), hydroxypropyl
beta-cyclodextrin (HPbCD), polyethyleneglycol (PEG) 300, ethanol,
glycerin, propylene glycol (PG), N,N-dimethylacetamide (DMA),
N-methyl-2-pyrrolidone (NMP), polysorbate 80, albumin, or benzyl
alcohol, or mixtures thereof), with a brincidofovir concentration
of 6.4 mg/mL. Without wishing to be bound by theory, tested
formulations comprising dextrose or NaCl as a tonicifier and
Cremophor.RTM. and DMA as excipients, did not precipitate over the
course of 12 days at a pH of 6.5, and tested formulations
comprising dextrose, but no excipient, did not precipitate over the
course of 12 days at a pH of 8.0, but did precipitate at lower pH
values. Similarly, and also without wishing to be bound by theory,
formulations comprising NaCl, PEG300, and polysorbate 80, and
formulations comprising NaCl, Cremophor.RTM. and ethanol, did not
precipitate at a pH of 7, but did precipitate at lower pH values of
12 days. Formulations comprising NaCl, and Cremophor.RTM. did not
precipitate at a pH of 7. Without wishing to be bound by theory,
the HPLC recovery data set forth in Example 1, suggests that under
storage condition (2-8.degree. C.) and at c(BCV)=6.4 mg/mL, some
formulations are not suited to continuously solubilize
brincidofovir in a stable fashion.
[0146] As shown in Example 2, the equilibrium solubility of
brincidofovir in aqueous infusion vehicles comprising 100 mM
phosphate, and 69 mM dextrose was found to increase at higher pH.
For example, the equilibrium solubility of brincidofovir in aqueous
infusion vehicles comprising 100 mM phosphate, and 69 mM dextrose
was determined as about 25 mg/mL at pH=6.9 and about >270 mg/mL
at pH=8.0.
[0147] As set forth in Example 5, solubility of a liquid
formulation of the disclosure in a 5% dextrose solution to yield a
liquid formulation comprising 10 mg/mL brincidofovir, 100 mM
arginine and 5% (w/v) mannitol, was 100% for at least 24 h after
dilution of the liquid formulation. Furthermore, the resulting
solution stayed clear and colorless, and no significant shifts in
pH were observed.
[0148] As set forth in Example 5 and Example 3, liquid formulations
of brincidofovir (e.g., formulations comprising 10 mg/mL
brincidofovir, 100 mM arginine and 5% (w/v) mannitol; formulations
comprising 10 mg/mL or 15 mg/mL brincidofovir and 200 nM sodium
phosphate; or formulations comprising 2 mg/mL brincidofovir, and 4
mM sodium phosphate) were tested for compatibility with infusion
vehicles as well as a series of materials from the manufacturing
and clinical stage (e.g., sterilization filters, vials and stoppers
in upright and inverted conditions, infusion bags, and infusion
systems). In some instances, the formulations were diluted with 5%
dextrose prior to testing. Without wishing to be bound by theory,
no precipitation, pH shift or significant loss in recovery of
brincidofovir were observed for any of the formulations at the end
of the studies.
Hemolytic Potential of Brincidofovir
[0149] In some embodiments, it is advantageous for drugs (e.g.,
intravenous drugs) to not cause hemolysis (i.e., not to destroy red
blood cells).
[0150] Example 7 describes the hemolytic potential of brincidofovir
using immediately collected (within 4 hrs) whole human or whole rat
blood using a cyanmethemoglobin method to evaluate hemolysis. The
study measured hemoglobin released from red blood cells in the
presence of brincidofovir to determine the extent to which
brincidofovir lysed red blood cells.
[0151] Without wishing to be bound by theory, lysis of red blood
cells can cause local reactions such as cellulitis, phlebitis and
can impact organ function. The results presented herein demonstrate
that the IV formulations of brincidofovir do not cause a hemolytic
reaction when administered intravenously.
[0152] Rat and human whole blood (stabilized with K.sub.2EDTA) were
obtained from 4 rats and 1 human donor. Each concentration (0.2,
0.5 and 1.6 mg/mL) of brincidofovir for the rat was mixed
separately in a 1:4 ratio with rat whole blood while each
concentration (0.2, 0.5 and 1.6 mg/mL) of brincidofovir for the
human was mixed separately in a 1:4 ratio with human whole blood.
All samples were then incubated for 20 minutes at 37.+-.1.degree.
C. Following incubation, the blood samples containing the BCV,
along with similarly prepared isotonic saline (negative control),
1% Saponin (positive control), 5% Dextrose (vehicle control), and
untreated whole blood control samples, were then centrifuged and
analyzed for hemolysis (supernatant hemoglobin concentration) using
a cyanmethemoglobin method. Representative BCV samples in isotonic
saline were analyzed to ensure that the BCV did not interfere with
the analysis of supernatant hemoglobin concentration.
[0153] Without wishing to be bound by theory, brincidofovir
demonstrated a hemolytic potential effect in rat whole blood that
appeared to be concentration dependent with evidence of hemolysis
observed at 1.6 mg/mL. However, brincidofovir was found to have no
hemolytic potential in human whole blood up to the highest
concentration tested corresponding to a final concentration of 0.2,
0.5 and 1.6 mg/mL.
[0154] Accordingly, in some embodiments, brincidofovir is safe for
administration to patients, including patients with hematological
diseases (e.g., anemia).
Maximum Tolerated Dose and 7-Day Dose Range-Finding (DRF) Study in
Rats
[0155] Example 8 presents a maximum-tolerated Dose and 7-day dose
range finding study (DRF) for BCV in rats. The results presented
detail an acute range finding study in the rat. In some
embodiments, this data can be used to generate information to allow
one to rationally set the doses to be administered in further
toxicology studies.
Phase 1
[0156] Phase 1 was designed to determine the Maximum Tolerated Dose
(MTD) of brincidofovir following intravenous infusion (2-hour) to
rats. Phase 1 encompassed a single escalating dose arm to assess
any acute toxicity (e.g., `toxic syndrome`) that can present in
general clinical signs generally within a 48+/- hour period of
exposure to BCV. Without wishing to be bound by theory, the
escalating dose arm can identify any overt signs or symptoms of
over-dosage.
[0157] In Phase 1 of the study, all doses were well tolerated when
administered via a single 2-hour IV infusion. Therefore, the
maximally tolerated dose of brincidofovir was not determined.
However, 15 mg/kg delivered via 2-hour IV infusion of a 0.75 mg/mL
solution of brincidofovir represented the maximum feasible dose
based on in vitro evidence of hemolysis at brincidofovir
concentrations above 0.75 mg/mL. In other words, even at the
maximum feasible dose tested, no consequential effects (e.g., on
mean body weight or weight gain) were observed in rats. Further,
there were no effects in ophthalmology, hematology, clinical
chemistry, or urinalysis parameters. Even at higher dosage levels
than can be achieved with oral administration, the rats were found
not to present with overt toxicity or death.
Phase 2
[0158] Phase 2, the repeat-dose range-finding (DRF) phase (Phase
II), was designed to assess the toxicity of brincidofovir and the
plasma toxicokinetic profiles of brincidofovir and one of its
metabolites, cidofovir. Cidofovir is known to cause nephrotoxicity
when administered intravenously to animals and humans.
Toxicokinetic parameters were evaluated following the first and
last dose administration of two dose levels of brincidofovir (1
mg/kg and 15 mg/kg) administered via intravenous (2-hour) infusion
on Days 1, 3, and 7.
[0159] In Phase 2 of the present study the animals received a short
term treatment of the high (15 mg/kg) or low (1 mg/kg) BCV IV
infusion dose (3 doses over the course of 9 days). Without wishing
to be bound by theory, this second phase of the study was used to
determine if any potential cumulative toxicity can occur with
repeat dosing. Without wishing to be bound by theory, the second
phase also included a toxicokinetic arm to determine the establish
IV toxicokinetics following single (the first dose administered)
and repeat (the last dose administered) BCV IV infusion dosing.
[0160] In Phase 2 of the study, peak concentrations for
brincidofovir were generally observed at the end of the 2-hour IV
infusion, and decreased rapidly. In general, mean brincidofovir
C.sub.max and AUC.sub.last increased approximately proportional to
the increase in dose from 1 to 15 mg/kg, though AUC was determined
on few concentration values after 1 mg/kg dosing. The brincidofovir
C.sub.max and AUC.sub.last values on Day 7 showed a trend toward
lower values on Day 7 compared to Day 1 (Day7/Day1 accumulation
ratios (AR) of 0.44 to 0.54). Further, no sex differences in
brincidofovir TK parameters were observed. Without wishing to be
bound by theory, exposure to metabolite cidofovir increased less
than proportional to the increase in brincidofovir dose, there were
no apparent sex differences in TK parameters, and no change in TK
parameters after repeat administration. Under the conditions of the
study, single dose administration of 1 or 15 mg/kg brincidofovir
via 2-hour IV infusion on Days 1, 4 and 7 were well tolerated.
Brincidofovir: A 28-Day Intravenous Infusion Toxicity Study in Rats
with a 14-Day Recovery Period
[0161] Example 10 details a repeat-dose, subchronic study with IV
BCV in rats. Without wishing to be bound by theory, this study
explores the cumulative biological effects (e.g., clinical, macro
and micro) of brincidofovir administered over a range of doses. The
effects can be qualitative, such as target organs, nature of effect
or quantitative, such as plasma or tissue levels at which effects
are observed. Without wishing to be bound by theory, the study can
define the toxicity and the potential for recovery or
progression.
[0162] As set forth in Example 10, and without wishing to be bound
by theory, none of the animals in the present study experienced
diarrhea. All of the animals gained their expected weight during
the study. There were no GI findings in intestines at terminal
necropsy. There were no in-life clinical findings at the highest
administered dose of IV brincidofovir (15 mg/kg). No transaminase
elevations were observed.
[0163] Without wishing to be bound by theory, it was observed after
oral administration of brincidofovir, there was a significant loss
of epithelium in intestinal villi. In contrast, after IV
administration of brincidofovir, there were minimal single-cell
effects noted. For example, FIG. 7A shows a histogram of histogram
of rat intestine after oral administration of brincidofovir, and
FIG. 7B shows a histogram of rat intestine after IV administration
of brincidofovir. As shown in the figures, IV administration of
brincidofovir is more well-tolerated in the intestine.
[0164] As set forth in Example 10, brincidofovir was administered
twice weekly for 28 days via 2-hour intravenous infusion to
Sprague-Dawley CD.RTM. rats (15/sex/group) at 0 (2.times. Sodium
Phosphate Buffer Solution), 1, 4 or 15 mg/kg/dose. Up to 5
animals/sex/group were held for a 14-day post-dosing recovery.
[0165] Administration of brincidofovir resulted in intermittent
(generally following infusion) clinical signs in some animals
(primarily abnormal breathing and hunched posture) in both sexes at
.gtoreq.4 mg/kg/dose and minimally decreased food consumption in
males and females at .gtoreq.1 mg/kg/dose and 15 mg/kg/dose,
respectively.
[0166] Brincidofovir-related findings were present in the male
reproductive tract (testicular germ cell depletion, luminal cell
debris in the epididymides and increased epithelial apoptosis in
the seminal vesicles) and intestinal tract (single cell necrosis)
at .gtoreq.4 mg/kg/dose and sebaceous glands (atrophy) at 15
mg/kg/dose. All brincidofovir-related findings completely recovered
with the exception of testicular changes in males at .gtoreq.4
mg/kg/dose, which without wishing to be bound by theory would not
be expected to fully recover in a 2 week period. In addition, a few
animals administered 15 mg/kg/dose had decreased bone marrow
cellularity and unilaterally or bilaterally soft and/or small
testes and epididymides at the end of recovery.
[0167] Under the conditions of the study, based on the testicular
germ cell depletion in males at .gtoreq.4 mg/kg/dose and moderate
intestinal single cell necrosis in 1 female at 15 mg/kg/dose, the
no-observed-adverse-effect level (NOAEL) for brincidofovir was
considered to be 1 mg/kg/dose in males and 4 mg/kg/dose for in
females.
[0168] Thus, in some embodiments, the study can provide support for
the initiation or continued conduct of clinical studies in humans.
In some embodiments, the study can also be used to establish doses
for longer term studies. Without wishing to be bound by theory,
this study can also guide parameters such as the number of animals
to assess when conducting longer-term studies. This study also
contained a toxicokinetic arm following the first and last
administered dose.
Mass Balance, Pharmacokinetics and Tissue Distribution by
Quantitative Whole-Body Autoradiography in Rats Following a Single
Oral or Intravenous Dose of [.sup.14C]brincidofovir
[0169] Studies were performed to characterize the tissue
distribution of total radioactivity in male Sprague Dawley (SD) and
Long-Evans (LE) rats following administration of a single
intravenous (IV, 2 h infusion) or oral (PO, gavage) dose of
[.sup.14C]brincidofovir. In addition, the rate and extent of
excretion (mass balance) and pharmacokinetics (PK) of total
radioactivity in male Sprague Dawley rats following a single
intravenous (IV, 2 h infusion) dose of [.sup.14C]brincidofovir were
examined.
[0170] [.sup.14C]Brincidofovir was administered to pigmented and
non-pigmented rats by 2-hour IV infusion or by oral gavage at a
dose of 15 mg/kg. Tissue distribution was determined by
quantitative whole body autoradiography at time points up to
35-days post-dose.
[0171] Radioactivity was well distributed with qualitative
distribution patterns of the radioactivity similar after IV or oral
administration. Quantitatively, tissue radioactive concentrations
in small intestinal tissue after IV administration were
approximately 1/10 the concentrations in small intestinal tissue
after oral administration. For most other tissues, tissue
radioactive exposure was generally higher after IV administration
than after oral gavage administration. Peak concentrations of
radioactivity in most tissues occurred at 4 to 8 h after oral
administration, or at the end of the 2-h IV infusion. Tissues with
highest concentrations of radioactivity were associated with organs
of clearance or elimination, e.g., liver, kidney and small
intestine, regardless of route of administration. The tissue to
plasma ratios (T/P) in these organs were high (>30) and for
kidney cortex and liver were similar between the IV and oral routes
of administration. Tissues with lowest concentrations of
radioactivity were brain, spinal cord, skeletal muscle, white
adipose tissue and bone. Association of radioactivity in the brain
and spinal cord was higher after IV administration (.about.20% of
plasma concentration compared to .about.5% after oral
administration). At 35 days post-dose, radioactivity was below the
limit of quantification in all tissues except for bone marrow,
lymph node, spleen and adrenal gland after IV administration; which
were different from those tissues with residual concentrations
after oral administration (kidney cortex, liver and small
intestine). No evidence of specific association with melanin
containing tissues (eye, uvea) was detected.
[0172] Compared to the oral route, IV administration of
[.sup.14C]brincidofovir results in lower small intestinal
concentrations of BCV-related radioactivity, which is consistent
with lower incidence and severity of GI findings in toxicology
studies after repeat IV BCV administration to rats.
[0173] In some embodiments, single cell necrosis in the small and
large intestine were occasionally noted following IV BCV infusion.
These minor and reversible GI findings are explained by the lower
concentrations of radioactivity seen in intestinal tissue following
IV infusion of [.sup.14C]brincidofovir. Further, the GI findings
did not result in any dose-limiting GI toxicities (i.e.,
gastropathy, enteropathy or enteritis). Accordingly, the present
disclosure provides IV formulations of BCV and methods of IV BCV
administration that can be used to mitigate BCV-associated GI
disturbances.
[0174] Distribution of radioactivity after IV
[.sup.14C]brincidofovir infusion was higher in all tissues compared
to oral BCV, except in the small intestine, where concentrations
were approximately 1/10 of oral administration.
Excretion and Distribution Summary
[0175] Excretion after IV Administration
[0176] The primary route of elimination of radioactivity after a
2-h IV infusion of [.sup.14C]brincidofovir at 15 mg/kg in male SD
rats was in urine, which accounted for an average of 51.2% of the
administered dose over the 168 h collection period. An average of
42.2% of the administered dose was recovered in feces. The total
recovery of radioactivity in urine and feces combined in Group 1
male rats averaged 93.5% of the dose over the 168 h collection
period. Most (.about.86%) of the urinary and fecal excretion of
radioactivity occurred in the first 24 h after dosing.
Distribution after IV Administration
[0177] The C.sub.max of [.sup.14C]brincidofovir total radioactivity
in plasma after a 2-h IV infusion administration to male SD rats at
15 mg/kg (Group 2) was 10.3 .mu.g equiv/mL, which occurred at a
T.sub.max of 2 h (i.e., end of infusion), and the concentration
decreased to 0.045 .mu.g equiv/mL at 72 h post-dose. The
AUC.sub.last of [.sup.14C]brincidofovir total radioactivity was
64.4 .mu.g equivh/mL, and the t.sub.1/2 was 13.0 h.
[0178] [.sup.14C]Brincidofovir-derived radioactivity was well
distributed into most tissues of albino and pigmented male rats
after a 2-h IV infusion at 15 mg/kg, and most tissues had
concentrations that were slightly higher than plasma, in
particular, excretory and alimentary canal tissues, which were much
higher than plasma (tissue AUC.sub.all:plasma ratio >1.9). The
C.sub.max of [.sup.14C]brincidofovir-derived radioactivity in most
tissues were found at 2 h post-dose (i.e., end of infusion) when
most of the tissues had concentrations that were between 1.0 and
6.0 .mu.g equiv/g.
[0179] Blood to plasma ratios generally showed that
[.sup.14C]brincidofovir-derived radioactivity was similarly
distributed after IV infusion to rats, ranging from 0.89-1.46 over
the period from 1 to 24 hours after the start of infusion. At 72
hours, blood to plasma ratios of 1.42 and 3.06 were observed after
administration of 2 mg/kg and 15 mg/kg, respectively.
[0180] The tissues of albino and pigmented male rats with the
highest concentrations (>20.0 .mu.g equiv/g) at the respective
T.sub.max after a 2-h IV infusion at 15 mg/kg were observed in
liver, kidney cortex, small intestine, kidney medulla, and urinary
bladder. The tissues with the lowest concentrations (<1.0 .mu.g
equiv/g) at their respective T.sub.max were: brain, spinal cord,
white adipose, bone, skeletal muscle, and eye lens.
[0181] The highest overall concentrations (>400 .mu.g equiv/g)
after a 2-h IV infusion at 15 mg/kg determined by QWBA were
observed in the urinary bladder contents, bile, and contents of the
alimentary canal, which reflected the routes of elimination for the
[.sup.14C]brincidofovir-drug-derived radioactivity after IV
administration.
[0182] Tissue concentrations in most tissues decreased steadily,
but at 168 h post-dose, elimination of drug related material was
not complete in most tissues; however all tissues containing
radioactivity had concentrations that were <1.0 .mu.g equiv/g.
At 840 h post-dose (last sampled time point), radioactivity was
still present in bone marrow, lymph node, spleen, and adrenal
gland, but they were approaching the LLOQ in these tissues, except
spleen. The t.sub.1/2 for all tissues (with sufficient data points,
and acceptable r.sup.2) ranged from 14.2 h for blood (cardiac) to
378 h for spleen. The tissues with the longest t.sub.1/2 were
spleen (378 h), lymph node (345 h), skin non-pigmented (310 h), and
bone marrow (276 h). The t.sub.1/2 for tissues with quantifiable
concentrations at 840 h ranged from 254 h (adrenal gland) to 378 h
(spleen).
[0183] A similar pattern of tissue distribution was observed in
rats given a 2-h IV infusion at the lower dose of 2 mg/kg. Most
tissue concentrations after the 2 mg/kg IV dose compared to 15
mg/kg dose, increased approximately proportional to dose (i.e.,
with increase in dose of 7.5, a change in concentration of 4 to 15
fold). However, in small intestine, a greater than proportional
increase in small intestine concentrations was seen with an
increase in dose from 2 to 15 mg/kg.
Distribution after Oral Administration
[0184] The C.sub.max of [.sup.14C]brincidofovir total radioactivity
in plasma after PO administration to male LE rats at 15 mg/kg was
1.5 .mu.g equiv/mL at a T.sub.max of 8 h, and the concentration
decreased to 0.180 .mu.g equiv/mL at 24 h post-dose. The
AUC.sub.last of [.sup.14C]brincidofovir total radioactivity was
21.0 .mu.g equivh/mL, and the t.sub.1/2 was not able to be
determined due to insufficient data points.
[0185] Blood to plasma ratios showed that
[.sup.14C]brincidofovir-derived radioactivity was similarly
distributed from 1 h to 24 h (ranged between 0.76-1.13) after
administration of a PO dose of [.sup.14C]brincidofovir to male LE
rats.
[0186] [.sup.14C]brincidofovir-derived radioactivity was well
distributed into most tissues of pigmented and non-pigmented male
rats after a PO administration at 15 mg/kg, and most tissues had
concentrations that were slightly higher plasma, especially for
excretory and alimentary canal tissues, which were much higher
(tissue AUC.sub.all:plasma ratio >4.1). The C.sub.max of
[.sup.14C]brincidofovir-derived radioactivity in most tissues were
found at 8 h post-dose (22 of 38 tissues) when most of the tissues
had concentrations that were between 0.7 and 17.8 .mu.g
equiv/g.
[0187] The tissues of pigmented male rats with the highest
concentrations (>3.3 .mu.g equiv/g) at the respective T.sub.max
after a PO dose at 15 mg/kg were observed in: small intestine,
liver kidney cortex, stomach (gastric mucosa), kidney medulla,
cecum, esophagus, large intestine. The tissues with the lowest
concentrations (<1.0 .mu.g equiv/g) at their respective
T.sub.max were: spinal cord, brain, bone, white adipose, and eye
lens.
[0188] The highest overall concentrations (>36.0 .mu.g equiv/g)
after a PO dose at 15 mg/kg determined by QWBA were observed in the
urinary bladder contents, bile, and contents of the alimentary
canal, which reflected the routes of elimination for the
[.sup.14C]brincidofovir-drug-derived radioactivity after PO
administration.
[0189] Tissue concentrations in most tissues of pigmented male rats
decreased steadily, but at 168 h post-dose, elimination of drug
related material was not complete in most tissues; however all
tissues had concentrations that were <0.4 .mu.g equiv/g, with
the exception of small intestine (5.9 .mu.g equiv/g). At 840 h
post-dose (last sampled time point) radioactivity was still present
in kidney cortex, liver, and small intestine, but was approaching
the LLOQ. The t.sub.1/2 for plasma was approximately 5 h. The
t.sub.1/2 for all tissues (with sufficient data points, and
acceptable r.sup.2) ranged from 37.2 h for esophagus to 234 h for
adipose (brown). The tissues with the longest t.sub.1/2 were
adipose brown (234 h), spleen (115 h), pituitary gland (106 h), and
lung (93 h). The only tissue with a reliable t.sub.1/2 and
quantifiable concentrations at 840 h was small intestine at 98.1
h.
Comparison of Distribution to Tissues after a PO Dose and a 2-h IV
Infusion
[0190] Following a 2-hour intravenous infusion or oral gavage of
[.sup.14C]brincidofovir to Long-Evans and Sprague-Dawley rats, the
AUC.sub.all blood to plasma ratio ranged between 1.1 and 1.4. For
individual points, the blood to plasma ratio ranged from 0.64 to
1.5 with a median value of 1.00. Without wishing to be bound by
theory, the similar distribution of radioactivity between blood and
plasma after both routes of administration indicated minimal
restriction of [.sup.14C] brincidofovir distribution from plasma
into blood cells.
[0191] Most tissues of rats given a single oral dose of
[.sup.14C]brincidofovir at 15 mg/kg attained maximal observed
concentrations at either 4 or 8 h, and peak concentrations were
generally about 10-25% of peak tissue concentrations observed in
rats given a 2-h IV infusion. In small intestine, however, a 6-fold
higher C.sub.max was observed after oral administration compared to
IV. The oral bioavailability of radioactivity (AUC.sub.all
PO/AUC.sub.all IV) was 48% (blood) to 61% (plasma). Lower tissue
AUC ratios (AUC.sub.PO/AUC.sub.IV<25%) were observed in
approximately 1/3 of the tissues after oral administration compared
to IV, notably in central nervous system, skin, skeletal muscle and
some endocrine and reproductive tissues. By both routes, tissues
with highest concentrations of radioactivity were associated with
excretory organs (i.e., liver, kidney, and intestine). The tissue
to plasma ratios were high (>30) for each of these tissues, and
in kidney cortex and liver the tissue/plasma ratios were similar
between the IV and oral routes of administration. Small intestinal
tissue to plasma ratio was highest after oral administration
(T/P=428) as expected based on the route of administration,
however, notable radioactivity was associated with small intestine
(tissue to plasma ratio 32) after IV infusion. Elimination from
tissues was incomplete at 840 h post oral-dose with radioactivity
present in kidney cortex, liver, and small intestines after PO
administration, which were different from those observed after a
2-h IV infusion at the same time point. In small intestine, the
residual concentration at 168 h was considerably higher for orally
dosed rats (5.9 equiv/g) than that for rats administered
[.sup.14C]brincidofovir by IV infusion (tissue concentrations BQL).
At 840 h, residual concentrations were much higher in
vascular/lymphatic tissue systems after a 2-h IV infusion compared
to the concentrations observed after a PO administration (BQL).
Distribution in Pigmented Tissues
[0192] A comparison of tissue distribution of radioactivity between
non-pigmented and pigmented tissues showed that concentrations in
the pigmented tissues (i.e., eye uveal tract and pigmented skin)
were similar to those concentrations observed in the non-pigmented
tissues, which suggested that there was no specific association of
[.sup.14C]brincidofovir-drug-derived radioactivity with
melanin.
[0193] Without wishing to be bound by theory, in most tissues,
higher concentrations of radioactivity were observed after IV
administration, which can be reflective of higher blood
concentrations. One exception was the gastro-intestinal tissue.
Tissues with the highest concentrations, ranked from highest to
lowest, were liver, kidney cortex, small intestine, and kidney
medulla for IV administration, and small intestine, liver, kidney
cortex, kidney medulla, and cecum for oral administration. The
tissues with residual drug after 840 hours were the adrenal glands,
spleen, lymph nodes and bone marrow for IV administration, and
small intestine, liver, and kidney cortex for oral
administration.
[0194] Thus, in some embodiments, lower dose of BCV administered
intravenously can provide similar plasma concentrations similar to
those observed using oral administration at higher doses.
Accordingly, the present disclosure provides treatment of a viral
infection in a subject in need thereof using a lower dose of BCV
than necessary with oral administration.
Pharmacokinetics of Brincidofovir IV Administration
[0195] As set forth in Example 11 below, brincidofovir was
administered to healthy subjects both orally and intravenously. It
was found that IV brincidofovir administration at 10 mg provided
similar exposure as orally administered brincidofovir at 100 mg.
Accordingly, the present disclosure teaches administration of
brincidofovir intravenously. In some embodiments, no drug-related
adverse events (e.g., no gastrointestinal events) are observed upon
IV administration of brincidofovir. In some embodiments, no graded
lab abnormalities (e.g., no hemolytic toxicity and no kidney
toxicity) are observed.
EXAMPLES
[0196] The disclosure is further illustrated by the following
examples which are not to be construed as limiting this disclosure
in scope or spirit to the specific procedures herein described. It
is to be understood that the examples are provided to illustrate
certain embodiments and that no limitation to the scope of the
disclosure is intended thereby. It is to be further understood that
resort may be had to various other embodiments, modifications, and
equivalents thereof which may suggest themselves to those skilled
in the art without departing from the spirit of the present
disclosure and/or scope of the appended claims.
[0197] This section provides abbreviations and definitions of terms
and concepts that may be commonly used throughout. [0198] AUC Area
Under the Time-Concentration Curve [0199] AUC.sub.all Area Under
the Time-Concentration Curve Including all Time Points [0200]
AUC.sub.inf_obs Area Under the Time-Concentration Curve
Extrapolated to Infinity Observed [0201] AUC.sub.last Area Under
the Time-Concentration Curve Including the last time point with
data above the lower limit of quantitation [0202] BQL Below
Quantification Limit [0203] .degree. C. Degrees Centigrade [0204]
c(BCV) Concentration of brincidofovir in a liquid or aqueous
formulation [0205] c(bulking agent) Concentration of bulking agent
in liquid or aqueous formulation [0206] ca Circa (approximately)
[0207] C.sub.max Maximum observed concentration [0208] cpm Counts
Per Minute [0209] % CV Percent Coefficient of Variance [0210] DMA
N,N-dimethylacetamide [0211] dpm Disintegrations Per Minute [0212]
EDTA edetate disodium [0213] FVC Femoral Vein Cannulated [0214] g
Gram [0215] h Hour [0216] HPLC High Performance Liquid
Chromatography [0217] HPbCD hydroxypropyl beta-cyclodextrin [0218]
IV Intravenous [0219] kg Kilogram [0220] L Liter [0221] LE
Long-Evans [0222] LLOQ Lower Limit of Quantification [0223] LSC
Liquid Scintillation Counting [0224] MB Mass Balance [0225] MBq
Megabecquerel [0226] mCi/mmol Millicurie Per Millimole [0227] MCD
MicroComputer Image Device [0228] MDC Molecular Dynamics Counts
[0229] mg Milligram [0230] mSv milli Sievert(s) [0231] .mu.g
equiv/g Microgram Equivalents per Gram of Tissue [0232] min Minutes
[0233] mL Milliliter [0234] mm Millimeter [0235] n Number of
Observations [0236] NA Not Applicable [0237] NI Not Identified
[0238] NC Not Calculated [0239] NS Not Sampled [0240] NMP
N-methyl-2-pyrrolidone [0241] PEG polyethyleneglycol [0242] PG
propylene glycol [0243] PK Pharmacokinetic [0244] PO per os (oral
dose) [0245] ROA Route of Administration [0246] SD Sprague Dawley
or Standard Deviation [0247] t.sub.1/2 Terminal Elimination
Half-Life [0248] T.sub.max Time of C.sub.max [0249] ULOQ Upper
Limit of Quantification [0250] QWBA Quantitative Whole-Body
Autoradiography
Example 1--Liquid Formulation Development
[0251] Various plain liquid formulations (i.e., pre-lyophilization
formulations, or bulk formulations) were prepared and tested for
the purpose of developing intravenous formulations of
brincidofovir.
[0252] pH Stability of Brincidofovir:
[0253] Brincidofovir sample stock solutions (50 .mu.g/mL) were
prepared in 50 mM phosphate buffer at pH 6.0-8.0 containing 20%
methanol. The samples were aliquoted and stored at two temperatures
(T=2-8.degree. C. and T=40.degree. C.), and monitored for pH and
recovery of brincidofovir at multiple time points over the course
of two weeks. Under both conditions, the samples maintained the pH
level throughout the time course of the experiment.
[0254] Storage at 2-8.degree. C.:
[0255] For samples stored at 2-8.degree. C., precipitation was
observed for pH 6.0 and 6.5 at t=2 days. For pH 7.0, precipitation
was observed at t=4 days. Samples prepared at a higher target pH
(pH=7.5 and pH=8.0) revealed no observable change in appearance
throughout the time course of the experiment, whereas the samples
prepared at pH 6.0, 6.5, and 7.0 showed a significant decrease in
brincidofovir concentration with respect to the initial time point.
The decrease may be related to the observed precipitation.
Variation for pH 7.0 is observed for t>8 days. One impurity
(RT=4.7 min) was detected that exceeded the set total peak area
threshold that was applied for peak integration. The percent of the
total peak area was below 0.4% for samples at pH 6 and 6.5, and
below 0.1% for samples at higher pH.
[0256] Storage at 40.degree. C.:
[0257] For samples stored at 40.degree. C., no physical change in
appearance was observed for samples at any pH. Minimal variation in
brincidofovir concentration was observed for all of the studied pH
conditions at 40.degree. C. While concentration of brincidofovir
showed some fluctuation, no significant trends can be observed. A
synoptic display of the HPLC chromatograms revealed differences in
impurity formation across the varying pH-conditions over the course
of the experiment. For the samples at lower pH, a peak associated
with the deamination product of brincidofovir, was observed,
encompassing an area % of 1.91 at pH 6.0, and 0.58 at pH 6.5. The
degradants peak is not observed at neutral pH and above.
##STR00005##
Preparation and Characterization of Brincidofovir Formulations
[0258] Preparation of Vehicles and Formulations:
[0259] Vehicles for brincidofovir formulations were prepared from
organic excipients, 5.times. sodium phosphate buffer stocks (pH
6.0, 6.5, 7.0, 7.5, and 8.0), and the stock solutions of
tonicifiers and EDTA. Solid brincidofovir was dissolved in the
vehicle yielding a concentration of c(BCV)=6.4 mg/mL. The
appearance (including precipitation upon storage at 2-8.degree. C.)
was evaluated, and the pH was measured. The results of the study
are summarized in Table 1.
TABLE-US-00001 TABLE 1 Precipitation status and pH of vehicles and
formulations. All formulations except formulations # 30-32 were
prepared with a Na phosphate buffer (100 nM). Precipitation
status.sup.1 Formulation composition Target Vehicle Form. Day Day
Day Day # Tonicifier Excipient #1 Excipient #2 pH pH pH init. 1 2 7
12 1 NaCl, 66.5 mM Cremophor .RTM., 40% DMA, 10% 6.00 6.05 6.03 N N
N N Y 2 NaCl, 66.5 mM Cremophor .RTM., 40% DMA, 10% 6.50 6.18 6.68
N N N N N 3 NaCl, 66.5 mM -- -- 7.00 6.87 6.83 Y Y Y Y Y 4 NaCl,
66.5 mM -- -- 7.50 7.33 7.23 N N N Y Y 5 NaCl, 66.5 mM -- -- 8.00
7.80 7.39 N N N Y Y 6 Dextrose, 68.8 nM Cremophor .RTM., 40% DMA,
10% 6.00 6.38 6.07 N N N N Y 7 Dextrose, 68.8 mM Cremophor .RTM.,
40% DMA, 10% 6.50 7.22 6.78 N N N N N 8 Dextrose, 68.8 mM -- --
7.00 6.88 6.83 N N N Y Y 9 Dextrose, 68.8 mM -- -- 7.50 7.35 7.21 N
N N Y Y 10 Dextrose, 68.8 mM -- -- 8.00 7.89 7.45 N N N N N 11
MgCl2, 49.8 mM -- -- 7.00 6.22 6.42 Y Y Y Y Y 12 Ca D-gluconate,
35.7 mM -- -- 7.00 6.49 6.63 Y Y Y Y Y 13 NaCl, 66.5 mM EDTA, 50
.mu.M -- 7.00 6.82 6.95 Y Y Y Y Y 14 NaCl, 66.5 mM HPbCD, 1.8% --
7.00 6.86 6.93 N N N Y Y 15 NaCl, 66.5 mM HPbCD, 6.4% -- 7.00 6.92
6.85 N N N Y Y 16 NaCl, 66.5 mM PEG 300, 10% -- 7.00 7.29 6.99 Y Y
Y Y Y 17 NaCl, 66.5 mM polysorbate 80, 2% -- 7.00 6.82 6.87 N N N N
Y 18 NaCl, 66.5 mM PEG 300, 10% polysorbate 80, 2% 6.00 6.28 6.20 Y
Y Y Y Y 19 NaCl, 66.5 mM PEG 300, 10% polysorbate 80, 2% 7.00 7.31
6.98 N N N N N 20 NaCl, 66.5 mM Ethanol, 10% -- 7.00 7.35 7.02 N N
N Y Y 21 NaCl, 66.5 mM Cremophor .RTM., 10% -- 7.00 6.93 6.87 N N N
N N 22 NaCl, 66.5 mM Cremophor .RTM., 10% Ethanol, 10% 6.00 6.19
6.03 N N N Y Y 23 NaCl, 66.5 mM Cremophor .RTM., 4% Ethanol, 10%
7.00 7.30 7.00 N N N N N 24 NaCl, 66.5 mM Cremophor .RTM., 10%
Ethanol, 10% 7.00 7.51 7.13 N N N N N 25 NaCl, 66.5 mM Glycerin,
10% -- 7.00 7.02 7.06 N N N Y Y 26 NaCl, 66.5 mM PG, 10% -- 7.00
7.36 7.08 Y Y Y Y Y 27 NaCl, 66.5 mM DMA, 10% -- 7.00 7.63 7.20 Y Y
Y Y Y 28 NaCl, 66.5 mM NMP, 1% -- 7.00 7.13 7.07 N N N Y Y 29 NaCl,
66.5 mM Albumin, 5% -- 7.00 7.24 6.95 N N N Y Y 30 -- Cremophor
.RTM., 50% Ethanol, 50% N/A N/A N/A Y Y Y Y Y 31 -- Cremophor
.RTM., 50% Ethanol, 44%; DMA N/A N/A N/A Y Y Y Y Y 32 -- Cremophor
.RTM., 50% Ethanol, 30%; N/A N/A N/A Y Y Y Y Y polysorbate 80, 8%;
Benzyl alcohol, 2% .sup.1Formulations were prepared at ambient
temperature and kept at ambient conditions until day 1 time point.
On day 1, the hemolysis assay was carried out after which the
samples were stored at 2-8.degree. C. .sup.2N: No precipitation
observed. .sup.3Y: Precipitation observed. .sup.4N/A: Not
applicable or not measured.
[0260] The vehicles were clear, colorless solutions. Precipitation
was observed for vehicles #11 and #12, and vehicle #29 was a clear
light yellow solution. Formulations #11 and #12 inherited
precipitation from their vehicles. Formulations #18, #30, #31, and
#32 were observed with precipitation on day 1 after preparation.
Formulations were stored at 2-8.degree. C. from day 2 onwards, and
the appearance was monitored over 12 days.
[0261] Brincidofovir Recovery:
[0262] The brincidofovir formulations of Table 1 were analyzed with
respect to c(BCV) on day 2 and day 7 after preparation.
Formulations that showed initial precipitation were not tested on
day 2. Formulations that showed precipitation up to day 7 were
subjected to centrifugation and HPLC recovery analysis. The results
are summarized in Table 2.
TABLE-US-00002 TABLE 2 HPLC recovery of brincidofovir formulations
Formulation Supernatant analysis (day 2) analysis (day 7) Target
Observed Observed c(BCV) c(BCV) Recovery c(BCV) Recovery Form. #
(mg/mL) (mg/mL) (%) (mg/mL) (%) 1 6.4 8.01 125 -- -- 2 6.4 8.06 126
-- -- 3 6.4 -- -- 1.15 18 4 6.4 7.20 112. 1.17 18 5 6.4 7.41 116
7.18 112 6 6.4 11.25 176 -- -- 7 6.4 8.70 136 -- -- 8 6.4 8.15 127
0.94 15 9 6.4 8.43 132 6.89 108 10 6.4 7.96 124 -- -- 11 6.4 -- --
0.52 8 12 6.4 -- -- 8.30 130 13 6.4 -- -- 0.88 14 14 6.4 9.78 153
1.41 22 15 6.4 10.12 158 4.58 72 16 6.4 -- -- 0.52 8 17 6.4 8.53
133 -- -- 18 6.4 -- -- 2.22 35 19 6.4 9.11 142 -- -- 20 6.4 7.66
120 1.85 29 21 6.4 7.79 122 -- -- 22 6.4 8.11 127 3.74 58 23 6.4
8.33 130 -- -- 24 6.4 8.15 127 -- -- 25 6.4 8.67 136 0.57 9 26 6.4
-- -- 1.14 18 27 6.4 -- -- 0.60 9 28 6.4 7.93 124 0.73 11 29 6.4
9.56 149 3.44 54 30 6.4 -- -- 0.70 11 31 6.4 -- -- 0.72 11 32 6.4
-- -- 1.12 18
[0263] Vehicle #11 and #12 were found to form precipitate upon
preparation of the formulation (i.e., addition of brincidofovir).
Formulations with a low pH formed precipitate in the presence of
solubilizing excipients such as Cremophor.RTM., PEG 300,
polysorbate 80, and ethanol (formulation #1, #6, #18, and #22),
while formulations with the same composition, but higher pH
(formulation #2, #7, #19, and #24) maintained the ability to
solubilize brincidofovir (Table 1). Purely organic formulations
(formulation #30, #31, and #32) showed limited solubilizing ability
toward brincidofovir.
[0264] The HPLC recovery data demonstrate that under storage
condition (2-8.degree. C.) and at c(BCV)=6.4 mg/mL, some
formulations are not suited to continuously solubilize
brincidofovir. Moreover, the data demonstrate that formulations
with pH greater than or equal to about 8.0 can solubilize
brincidofovir.
[0265] Further formulations (formulations #33-40 and #49-56) were
prepared and tested for brincidofovir equilibrium solubility and
filterability.
[0266] Formulation vehicles #33-40 and #49-56 were saturated with
brincidofovir and rotated at ambient conditions. The recovery of
brincidofovir in the sample supernatant was determined at different
time points to establish when the solubility equilibrium was
reached.
[0267] The saturated supernatants of the equilibrium solubility
study were subjected to filterability assessment. For this,
approximately 900 .mu.L of the saturated solutions were filtered
through a syringe filter (25 mm, 0.2 .mu.m PES membrane). The ease
of the filtration was recorded, the pH in the filtrate was
measured, and the brincidofovir recovery was determined by HPLC
analysis. In order to assess if sterile filtering introduces
impurities, the HPLC chromatograms of the saturated formulation
vehicles were qualitatively compared at the pre- and
post-filtration stage and are shown. The composition of each of
formulations #33-40 and #49-56, as well as their pH, c(BCV) at
solubility equilibrium and ease of filtration are summarized in
Table 3.
TABLE-US-00003 TABLE 3 Summary of brincidofovir solubility and
filterability assessment. The buffer in all formulations was 100 mM
Na phosphate. Observed c(BCV) Measured pH (mg/mL) BCV- Volume At
Form. Composition Target saturated filtered Ease of solubility
Post- # Tonicifier Excipient pH Vehicle filtrate (.mu.L) filtration
equilibrium filtration 33 Dextrose, 68.8 mM -- 8.0 8.1 6.9 900 Easy
27 27 34 NaCl, 66.5 mM Cremophor .RTM., 10% 7.5 7.6 6.5 900 Easy 30
31 35 NaCl, 66.5 mM Cremophor .RTM., 5% 7.5 7.6 6.6 900 Easy 27 29
36 Dextrose, 68.8 mM Cremophor .RTM., 5% 7.5 7.6 6.7 900 Easy 26 26
37 NaCl, 66.5 mM Cremophor .RTM., 2% 7.5 7.6 6.7 900 Easy 25 26 38
NaCl, 66.5 mM HPbCD, 4% 7.5 7.6 6.8 600 Easy 19 16 39 NaCl, 66.5 mM
SBEbCD, 7.5% 7.5 7.6 6.3 200 Easy 4 4 40 NaCl, 66.5 mM -- 7.5 7.6
6.8 900 Easy 22 22 49 Dextrose, 68.8 mM -- 7.5 7.6 6.9 1000 Easy 25
24 50 Dextrose, 68.8 mM -- 7.5 7.6 6.9 1000 Easy 24 25 51 Dextrose,
68.8 mM -- 7.5 7.6 6.9 1000 Easy 27 25 52 NaCl, 66.5 mM -- 7.5 7.6
6.8 1000 Easy 29 27 53 NaCl, 66.5 mM -- 7.5 7.6 6.8 1000 Easy 27 27
54 NaCl, 66.5 mM -- 7.5 7.6 6.8 1000 Easy 24 29 55 Dextrose, 68.8
mM -- 8.0 8.0 6.9 1000 Easy 28 31 56 Dextrose, 68.8 mM -- 8.0 8.0
6.9 1000 Easy 29 30
[0268] The equilibrium solubility of brincidofovir in the tested
formulations ranged from 4 to 30 mg/mL with an average solubility
of c(BCV)=25 mg/mL within the range of apparent pH=6.3-6.9 (ambient
temperature). In some embodiments, the relatively high
brincidofovir concentrations observed in equilibrium solubility
testing led to a significant shift of the vehicle pH. Filtration of
the formulations saturated with brincidofovir through the selected
syringe filters did not reveal any issues regarding the ease of
filtration, loss of brincidofovir, or introduction of
impurities.
Example 2--Brincidofovir Formulation Testing
[0269] A formulation vehicle comprising 100 mM phosphate and 69 mM
dextrose (vehicle/Formulation #57), was prepared and tested further
with respect to the a) equilibrium solubility, and b)
filterability.
a) Brincidofovir Solubility Assessment
[0270] Two samples of vehicle #57, were saturated with
brincidofovir. One sample vehicle was saturated with brincidofovir
without further pH adjustment (leading to a formulation pH of 6.9)
and the pH of the second sample was re-adjusted to pH=8.0 using a
sodium hydroxide solution (1 N). The pH drifted towards lower pH
values upon dissolution of brincidofovir which in return changed
the brincidofovir solubility until an equilibrium was reached.
Consequently, the pH had to be brought up close to the target pH
before more brincidofovir was added. The concentration of
brincidofovir at equilibrium solubility conditions was not reached
for the pH adjusted sample. After supplementing the sample with
approximately 300 mg/mL of additional brincidofovir solid, the
addition of solid brincidofovir was stopped.
[0271] The recovery of brincidofovir (HPLC assay) in both sample
solutions/supernatants was determined at equilibrium and after 24
h. At ambient temperature, the equilibrium solubility of
brincidofovir in formulation 57 was c(BCV)=25 mg/mL at an apparent
pH of pH=6.9 and 26 mg/mL after 24 h. For the sample at pH=8 the
observed brincidofovir concentration was 348 mg/mL after 24 h, and
the equilibrium solubility was not reached with c(BCV)>270
mg/mL.
b) Filterability Assessment
[0272] The filterability of formulation #57 was tested at
c(BCV)=20, 10, and 0.5 mg/mL. The testing solution at c(BCV)=20
mg/mL was adjusted to pH=8.0 using a NaOH-solution (1 N) and was
applied as a stock solution to prepare the testing solutions at
lower concentration by dilution with D5W. The testing solutions
were filtered (5 mL filter pass) through a syringe filter (25 mm,
0.2 .mu.m PES membrane). The ease of the filtration was observed
and the pH as well as brincidofovir recovery (HPLC assay) were
recorded at the pre- and post-filtration stage. No significant
change in pH, loss of brincidofovir, or introduction of impurities
was observed (Table 4).
TABLE-US-00004 TABLE 4 Filterability assessment of brincidofovir in
the formulation 57 vehicle Observed Measured pH c(BCV) (mg/mL)
c(BCV) Pre Post Pre Post prepared to filtration filtration
filtration filtration 20 mg/mL 8.0 8.0 23 26 10 mg/mL 8.1 8.1 13 12
0.5 mg/mL 8.0 8.1 0.8 0.9
Example 3--Formulation Studies and Assessments
[0273] A compounding process for preparation of brincidofovir
formulations was developed and has been specified for the
tonicifier (dextrose)-free formulation. For stability studies and
testing, the following brincidofovir formulations were prepared on
a scale of up to 0.5 L: [0274] 10 mg/mL brincidofovir, 200 mM
sodium phosphate, pH=8.0, 69 mM dextrose [0275] 15 mg/mL
brincidofovir, 200 mM sodium phosphate, pH=8.0, 69 mM dextrose
[0276] 10 mg/mL brincidofovir, 200 mM sodium phosphate, pH=8.0
(tonicifier-free) Tonicifier-Containing Formulation
(w/Dextrose)
[0277] A brincidofovir formulation containing dextrose as
tonicifier was applied in stability study I as well as in the
infusion vehicle compatibility assessment. The outline of the
corresponding formulation preparation is as follows: [0278] 1. 500
mM (2.5.times.) monobasic sodium phosphate solution and 500 mM
(2.5.times.) dibasic sodium phosphate solution were prepared by
dissolving the corresponding sodium phosphate species in DI water
using a volumetric flask. [0279] 2. The 500 mM (2.5.times.)
monobasic and dibasic sodium phosphate solutions were mixed under
pH-control to prepare a 500 mM (2.5.times.) sodium phosphate buffer
solution at pH=8.0. [0280] 3. A vehicle stock solution of 400 mM
(2.times.) sodium phosphate, 138 mM (2.times.) dextrose at pH=8.0
was prepared by dissolving solid dextrose in the 500 mM
(2.5.times.) sodium phosphate solution at pH=8.0 and Q.S. with
water. [0281] 4. Brincidofovir was dissolved in the vehicle stock
solution (400 mM (2.times.) sodium phosphate, 138 mM (2.times.)
dextrose solution, pH=8.0) at 2.times. of the desired final
concentration using a volumetric flask. Under pH control a sodium
hydroxide solution was added to adjust the solution to pH=8.0. DI
water was used to Q.S. yielding a formulation of the desired
brincidofovir concentration containing 200 mM (1.times.) sodium
phosphate, 69 mM (1.times.) dextrose, pH=8.0.
Tonicifier Free Formulation (w/o Dextrose)
[0282] A brincidofovir formulation lacking dextrose was applied in
stability study II, as well as in the adsorption assessment, and
the majority of experiments associated with material compatibility
assessments. The tonicifier-free formulation was prepared as
follows: [0283] 1. A 400 mM (2.times.) monobasic sodium phosphate
solution and a 400 mM (2.times.) dibasic sodium phosphate solution
were prepared by dissolving the corresponding sodium phosphate
species in water (volumetric flaks). [0284] 2. The 400 mM
(2.times.) monobasic and dibasic sodium phosphate solutions were
mixed under pH-control to prepare a 400 mM (2.times.) sodium
phosphate buffer solution at pH=8.0 (vehicle solution). [0285] 3.
Brincidofovir was dissolved in the vehicle solution (400 mM
(2.times.) sodium phosphate solution at pH 8.0) at 2.times. of the
final concentration using a 500 mL volumetric flask. A 1 N sodium
hydroxide solution was added at a volume of V.sub.NaOH
[mL]=1.65.times.M.sub.BCV [g]. Q.S. with DI water yielded the
formulation at the desired brincidofovir concentration in 200 mM
sodium phosphate buffer at pH=8.0. [0286] 4. For preparation of
lower brincidofovir concentrations the prepared formulation was
diluted with the corresponding vehicle to the desired strength.
Stability Study I: Brincidofovir Formulation Containing Tonicifier
(Dextrose)
[0287] Design and Setup:
[0288] In this study, the chemical and physical stability of
formulation 57 was evaluated. Testing of the chemical degradation
was performed in an accelerated stability study setup applying heat
as an accelerator/stressor. An Arrhenius analysis was carried out
providing an API half-life and a degradation rate at lower
temperatures. Physical stability was evaluated at the desired
storage temperature (2-8.degree. C.); a benchmark/reference
condition was likewise included in the study. The setup of the
study is specified below:
Testing Solutions:
[0289] 10 mg/mL brincidofovir, 200 mM sodium phosphate, 69 mM
dextrose, pH=8.0, [0290] 15 mg/mL brincidofovir, 200 mM sodium
phosphate, 69 mM dextrose, pH=8.0, [0291] Vehicle control 1: 200 mM
sodium phosphate, 69 mM dextrose, pH=8.0 (only at 75.degree. C.),
[0292] Vehicle control 2: 200 mM sodium phosphate, pH=8.0 (only at
75.degree. C.).
Stability Station Temperatures:
[0292] [0293] Accelerated stability study: 50.degree. C.,
60.degree. C., 65.degree. C., 70.degree. C., 75.degree. C., [0294]
Physical degradation and benchmark stability study: 2-8.degree. C.,
25.degree. C.
Time Points:
[0294] [0295] Accelerated stability study: initial, 2, 6, 9, 14
days, [0296] Physical degradation and benchmark stability study:
initial, 14 and 28 days, [0297] Vehicle control: initial, 1, 5, 8,
and 14 days.
Tests:
[0297] [0298] Brincidofovir formulation: appearance, pH, c(BCV)
recovery (HPLC), purity (HPLC), and liquid particle counting
(physical degradation and benchmark stability study), [0299]
Vehicle control: appearance and pH.
[0300] The stability testing results (accelerated conditions),
i.e., the changes of pH and trends of brincidofovir recovery over
the course of the stability study are summarized in Table 8 and
Table 9 for the formulations at 10 mg/mL and 15 mg/mL respectively.
The accelerated stability study with strong biphasic rate trends
did not allow for an Arrhenius analysis and calculation of a
brincidofovir half-life or extrapolation to degradation rates at
typical storage conditions. The test results on the vehicle
controls in the accelerated stability study (75.degree. C. station
only) are summarized in Table 5.
TABLE-US-00005 TABLE 5 Accelerated stability study results of the
10 mg/mL brincidofovir formulation (Study I) Sample conditions
Station Mea- Observed BCV BCV temp. Time Appear- sured c(BCV).sup.2
recovery.sup.2,3 purity (.degree. C.) point ance.sup.1 pH (mg/mL)
(%) (%) 50 0 Clear, 8.1 10.1 100 99 Yellow (0) 2 Clear, 7.8 9.0 89
91 Yellow (0) 6 Clear, 7.5 9.0 89 96 Yellow (0) 9 Clear, 7.4 8.9 88
96 Yellow (1) 14 Clear, 7.4 8.8 87 95 Yellow (1) 60 0 Clear, 8.1
10.1 100 99 Yellow (0) 2 Clear, 7.5 9.0 89 98 Yellow (0) 6 Clear,
7.3 9.2 91 96 Yellow (2) 9 Clear, 7.4 9.2 91 96 Yellow (2) 14
Clear, 7.3 9.1 90 96 Yellow (2) 65 0 Clear, 8.1 10.1 100 99 Yellow
(0) 2 Clear, 7.5 9.5 94 99 Yellow (1) 6 Clear, 7.3 9.4 93 97 Yellow
(3) 9 Clear, 7.3 9.3 92 96 Yellow (3) 14 Clear, 7.2 9.0 89 95
Yellow (4) 70 0 Clear, 8.1 10.1 100 99 Yellow (0) 2 Clear, 7.4 9.3
92 97 Yellow (4) 6 Clear, 7.2 9.3 92 96 Yellow (5) 9 Clear, 7.1 9.2
91 95 Yellow (6) 14 Clear, 7.0 8.7 86 93 Yellow (7) 75 0 Clear, 8.1
10.1 100 99 Yellow (0) 2 Clear, 7.3 9.4 93 98 Yellow (6) 6 Clear,
6.9 9.1 90 93 Yellow (8) 9 Clear, 6.9 8.7 86 90 Yellow (8) 14
Clear, 6.8 7.9 78 84 Yellow (9) .sup.1The yellow color of the
solution was expressed on a scale of 0 to 10: 0, colorless; 5,
mustard; 10, amber. .sup.2Average value of three (3) replicates.
.sup.3BCV recovery = c(BCV).sub.Day n/c(BCV).sub.Day 0 .times.
100%.
TABLE-US-00006 TABLE 6 Accelerated stability study results of the
15 mg/mL brincidofovir formulation (Study I) Sample conditions
Station Time Mea- Observed BCV BCV temp. point Appear- sured
c(BCV).sup.2 recovery.sup.2,3 purity (.degree. C.) (day) ance.sup.1
pH (mg/mL) (%) (%) 50 0 Clear, 8.1 15.0 100 99 Yellow (0) 2 Clear,
7.8 13.6 90 98 Yellow (0) 6 Clear, 7.5 13.6 90 97 Yellow (0) 9
Clear, 7.5 13.3 88 98 Yellow (2) 14 Clear, 7.4 13.7 91 97 Yellow
(2) 60 0 Clear, 8.1 15.0 100 99 Yellow (0) 2 Clear, 7.6 13.8 92 98
Yellow (0) 6 Clear, 7.3 13.8 92 97 Yellow (2) 9 Clear, 7.4 13.7 91
97 Yellow (2) 14 Clear, 7.3 13.8 92 96 Yellow (3) 65 0 Clear, 8.1
15.0 100 99 Yellow (0) 2 Clear, 7.5 14.0 93 99 Yellow (1) 6 Clear,
7.3 14.0 93 97 Yellow (3) 9 Clear, 7.3 13.8 92 96 Yellow (4) 14
Clear, 7.2 13.1 87 95 Yellow (5) 70 0 Clear, 8.1 15.0 100 99 Yellow
(0) 2 Clear, 7.4 13.9 93 98 Yellow (3) 6 Clear, 7.2 13.9 92 96
Yellow (5) 9 Clear, 7.2 13.8 91 95 Yellow (5) 14 Clear, 7.1 13.1 87
93 Yellow (7) 75 0 Clear, 8.1 15.0 100 99 Yellow (0) 2 Clear, 7.3
14.1 94 98 Yellow (5) 6 Clear, 6.9 13.7 90 92 Yellow (8) 9 Clear,
6.9 12.9 86 89 Yellow (9) 14 Clear, 6.7 11.7 78 83 Yellow (10)
.sup.1The yellow color of the solution was expressed on a scale of
0 to 10: 0, colorless; 5, mustard; 10, amber. .sup.2Average value
of three (3) replicates. .sup.3BCV recovery = c(BCV)Day n/c(BCV)Day
0 .times. 100%. It was defined as 100% at the initial time
point.
TABLE-US-00007 TABLE 7 Accelerated stability study: Results of
vehicle controls (Study I) Sample conditions Station Time Measured
Sample temp. (.degree. C.) point (day) Appearance.sup.1 pH Vehicle
75 0 Clear, Yellow (0) 8.1 control 1 1 Clear, Yellow (5) 7.3
(dextrose- 5 Clear, Yellow (6) 7.1 added) 8 Clear, Yellow (7) 6.9
14 Clear, Yellow (9) 6.9 Vehicle 75 0 Clear, Yellow (0) 8.1 control
2 1 Clear, Yellow (0) 8.0 (dextrose- 5 Clear, Yellow (0) 8.0 free)
8 Clear, Yellow (0) 8.0 14 Clear, Yellow (0) 8.0 .sup.1The yellow
color of the solution was expressed on a scale of 0 to 10: 0,
colorless; 5, mustard; 10, amber.
[0301] The study showed degradation of brincidofovir of 16%
(50.degree. C.) to 22% (75.degree. C.) over the course of two
weeks. While some loss in brincidofovir recovery (assay loss) was
observed, the purity of brincidofovir remained at 95% and 93%
respectively for temperatures up to 70.degree. C. At the 75.degree.
C. station, up to 16% impurities were observed at both starting
concentrations. Degradation at higher temperatures was accompanied
by a strong pH shift towards lower pH values and coloration of the
solutions. The pH shifts and the degradation rates were higher for
the initial time point, and after 2 days the degradation and pH
shifting slowed down significantly (bi-phasic trends not following
a first-order decomposition).
Physical Degradation and Benchmark Stability Study
[0302] The results of the physical/benchmark stability for both
tested formulations are summarized in Table 11 and Table 12,
respectively. The purities of the formulations at different
temperatures were determined by comparing chromatograms obtained at
different temperatures and time points during the study.
Degradation between 6% (2-8.degree. C.) and 8% (25.degree. C.) was
observed over one month while the purity of brincidofovir stagnated
at 99% indicating a brincidofovir mass deficit.
TABLE-US-00008 TABLE 8 Physical degradation and benchmark stability
study results of the 10 mg/mL brincidofovir formulation (Study I)
Sample conditions Station Time Mea- Observed BCV BCV temp. point
Appear- sured c(BCV).sup.1 recovery.sup.1,3 purity (.degree. C.)
(day) ance pH (mg/mL) (%) (%) 2-8 0 Clear, 8.1 10.1 100 99
Colorless 14 Clear, 8.0 9.6 95 100 Colorless 28 Clear, 8.1 9.5 94
99 Colorless 25 0 Clear, 8.1 10.1 100 99 Colorless 14 Clear, 7.9
9.3 92 99 Colorless 28 Clear, 8.0 9.3 92 99 Colorless .sup.1Average
value of three (3) replicates. .sup.2Integration region: 2.00-13.00
min. .sup.3BCV recovery = c(BCV)Day n/c(BCV)Day 0 .times. 100%. It
was defined as 100% at the initial time point.
TABLE-US-00009 TABLE 9 Physical degradation and benchmark stability
study results of the 15 mg/mL brincidofovir formulation (Study I)
Sample conditions Station Time Mea- Observed BCV BCV temp. point
Appear- sured c(BCV).sup.1 recovery.sup.1,3 purity (.degree. C.)
(day) ance pH (mg/mL) (%) (%) 2-8 0 Clear, 8.1 15.0 100 99
Colorless 14 Clear, 8.0 14.3 95 100 Colorless 28 Clear, 8.1 14.6 97
100 Colorless 25 0 Clear, 8.1 15.0 100 99 Colorless 14 Clear, 7.9
13.8 92 99 Colorless 28 Clear, 8.0 13.9 93 99 Colorless
.sup.1Average value of three (3) replicates. .sup.2Integration
region: 2.00-13.00 min. .sup.3BCV.sub.recovery = c(BCV).sub.Day
n/c(BCV).sub.Day 0 .times. 100%. It was defined as 100% at the
initial time point.
[0303] Light Obstruction Measurement:
[0304] To prepare samples for liquid particle counting, under best
clean conditions the formulation samples from six serum vials (1.0
mL filling) were carefully combined (avoiding foaming) into a
particle-free centrifuge tube. The tube was then capped and
sonicated for 1 min before the sample was subjected to liquid
particle counting. Four sample strokes of 1 mL were analyzed. The
drawing rate of the liquid particle counter was 10 mL/min. No
significant increase in particulate matter was observed over the
course of the study. The test results of the test are summarized in
Table 10.
TABLE-US-00010 TABLE 10 Liquid particle counting results of the 10
and 15 mg/mL brincidofovir formulations (Study I) Stability station
temperature (.degree. C.) 2-8 25 10 .mu.m 25 .mu.m 10 .mu.m 25
.mu.m Time point particle particle particle particle Formulation
(day) count count count count 10 mg/mL 0 1 0 1 0 brincidofovir 14
11 1 7 0 formulation 28 4 0 5 0 15 mg/mL 0 0 0 0 0 brincidofovir 14
7 1 4 0 formulation 28 3 1 3 0
Stability Study II: Brincidofovir Formulation without Tonicifier
(Dextrose)
[0305] Design and Setup:
[0306] The stability study I, designed to evaluate the chemical and
physical degradation properties of the liquid brincidofovir
formulation was repeated without the presence of dextrose. A color
change of the solution (potential Maillard browning) and a pH-shift
could be correlated with the presence of dextrose that consequently
might have influenced the degradation routes and impaired the
observed rates for brincidofovir degradation. The study setup for
this repeated analysis was modified with regard to the tested
formulation as well as stability station parameters. The exact
parameters are specified below:
Testing Solutions:
[0307] 15 mg/mL brincidofovir, 200 mM sodium phosphate, pH=8.0,
[0308] Vehicle control 1: 200 mM sodium phosphate, pH=8.0.
Stability Station Temperatures:
[0308] [0309] Accelerated stability study: 50.degree. C.,
60.degree. C., 70.degree. C., and 75.degree. C. [0310] Physical
degradation and benchmark stability study: 2-8.degree. C. and
25.degree. C.
Time Points:
[0310] [0311] Accelerated stability study: initial, 3, 6, 10, 14
days, [0312] Physical degradation and benchmark stability study:
initial, 14 and 28 days.
Tests:
[0313] Brincidofovir formulation: appearance, pH, c(BCV) recovery
(HPLC), purity (HPLC), and liquid particle counting (physical
degradation and benchmark stability study samples only),
[0314] Vehicle control: appearance, pH, and liquid particle
counting (physical degradation and benchmark stability study
samples only).
[0315] Results:
[0316] The stability testing results on brincidofovir recovery and
purity of the tested formulation and the vehicle control are
summarized in Table 11 and Table 12, respectively.
TABLE-US-00011 TABLE 11 Accelerated stability study results of the
15 mg/mL brincidofovir formulation (Study II) Sample conditions
Station Time Mea- Observed BCV BCV temp. point Appear- sured
c(BCV).sup.a recovery.sup.a,b purity (.degree. C.) (day) ance pH
(mg/mL) (%) (%) 50 0 Clear, 8.0 15.4 100 99 Colorless 3 Clear, 8.0
15.0 98 98 Colorless 6 Clear, 8.0 14.6 95 98 Colorless 10 Clear,
8.0 14.6 95 97 Colorless 14 Clear, 7.9 14.2 92 97 Colorless 60 0
Clear, 8.0 15.4 100 99 Colorless 3 Clear, 7.9 14.8 96 98 Colorless
6 Clear, 8.0 14.2 92 96 Colorless 10 Clear, 8.0 14.3 93 95
Colorless 14.sup.c -- -- -- -- -- 70 0 Clear, 8.0 15.4 100 99
Colorless 3 Clear, 7.9 14.6 95 96 Colorless 6 Clear, 7.9 13.8 90 94
Colorless 10 Clear, 7.9 14.0 91 92 Colorless 14 Clear, 7.9 13.2 86
90 Colorless 75 0 Clear, 8.0 15.4 100 99 Colorless 3 Clear, 7.9
14.3 93 95 Colorless 6 Clear, 7.9 13.4 87 93 Colorless 10 Clear,
7.9 13.3 86 90 Colorless 14 Clear, 7.9 12.9 84 88 Colorless
.sup.aAverage value of three (3) replicates. .sup.bIntegration
region: 0.00-13.00 min. .sup.cData of this time point were
compromised due to oven malfunction, thus not included in the
following graphic representations or calculations
TABLE-US-00012 TABLE 12 Accelerated stability study results of the
vehicle control (Study II) Sample conditions Station temp.
(.degree. C.) Time point (day) Appearance Measured pH n.a..sup.a 0
Clear, colorless 8.0 50 3 Clear, colorless 8.0 6 Clear, colorless
8.1 10 Clear, colorless 8.0 14 Clear, colorless 8.0 60 3 Clear,
colorless 8.0 6 Clear, colorless 8.0 10 Clear, colorless 8.0
14.sup.b -- -- 70 3 Clear, colorless 8.0 6 Clear, colorless 8.1 10
Clear, colorless 8.0 14 Clear, colorless 8.1 75 3 Clear, colorless
8.0 6 Clear, colorless 8.0 10 Clear, colorless 8.0 14 Clear,
colorless 8.0 .sup.aNot applicable. .sup.bData of this time point
were compromised due to oven malfunction, thus not included in the
following graphic representations.
[0317] It is noted that the values for brincidofovir recovery and
purity do not align. This is at least partially attributed to the
fact that the injection peak area of the chromatographic trace
(0.00-2.1 min) was not integrated, which would could overestimate
the purity value, since the non-integrated retention time area
likely accommodates known degradants. Additionally, a baseline
separation between brincidofovir and its deamination product could
not be achieved with the existing method, which might affect
recovery values (degradation) and purity values.
[0318] The obtained recovery data were applied to determine the
degradation rate at the different temperatures via an Arrhenius
analysis, i.e., by logarithmically plotting the brincidofovir
recovery as a function of time, and determining the rate constant
through a linear fit. The half-life for the dextrose-free 15 mg/mL
brincidofovir formulation was so determined to 729 days and 295
days at stability station temperatures of 5.degree. C. and
25.degree. C. respectively. The Arrhenius analysis is illustrated
in FIG. 2.
Physical Degradation and Benchmark Stability Study
[0319] The results of the physical/benchmark stability for the
studied formulation and the vehicle control are summarized in Table
13 and Table 14, respectively.
TABLE-US-00013 TABLE 13 Physical degradation and benchmark
stability study results of the 15 mg/mL brincidofovir formulation
(Study II) Sample Station Time Mea- Observed BCV BCV temp. point
Appear- sured c(BCV).sup.1 recovery.sup.1,3 purity (.degree. C.)
(day) ance pH (mg/mL) (%) (%) 2-8 0 Clear, 8.0 15.4 100 99
Colorless 14 Clear, 8.0 14.9 97 100 Colorless 28 Clear, 8.1 15.2 99
99 Colorless 25 0 Clear, 8.0 15.4 100 99 Colorless 14 Clear, 8.0
14.9 97 99 Colorless 28 Clear, 8.0 15.2 99 99 Colorless
.sup.1Average value of three (3) replicates. .sup.3BCV recovery =
c(BCV).sub.Day n/c(BCV).sub.Day 0 .times. 100%. It was defined as
100% at the initial time point.
TABLE-US-00014 TABLE 14 Physical degradation and benchmark
stability study results of the vehicle control (Study II) Sample
conditions Station temp. (.degree. C.) time point (day) Appearance
Measured pH n.a..sup.1 0 Clear, colorless 8.0 2-8 14 Clear,
colorless 8.1 28 Clear, colorless 8.1 25 14 Clear, colorless 8.1 28
Clear, colorless 8.0 .sup.1Not applicable.
[0320] For the analyzes of particulate matter, the formulation
sample from one serum vial (5.0 mL filling) was sonicated for 1 min
before decrimping of the vial. The tonicifier-free sample was
subjected to liquid particle counting as described above for the
tonicifier containing formulations. The Results of the analysis are
summarized in Table 15.
TABLE-US-00015 TABLE 15 Liquid particle counting results of the
tonicifier-free 15 mg/mL brincidofovir formulation and vehicle
control (Study II) Stability station temperature (.degree. C.) 2-8
25 Time 10 .mu.m 25 .mu.m 10 .mu.m 25 .mu.m point particle particle
particle particle Solution (day) count count count count 15 mg/mL 0
0 0 0 0 brincidofovir 14 80 2 45 0 formulation 28 87 0 4 0 Vehicle
0 0 0 0 0 control 14 0 0 0 0 28 0 0 0 0
[0321] The degradation of brincidofovir in the tonicifier-free
formulation at storage conditions (2-8.degree. C. and 25.degree.
C.) is negligible over the course of one month. The nominal
degradation of 1% is within the error of brincidofovir
quantification; a mass deficit or particulate matter were not ob
served.
Assessment of Brincidofovir Adsorption to Vials and Stoppers
[0322] Design and Setup:
[0323] An adsorption assessment on brincidofovir was performed to
test for adsorption of brincidofovir to vials and/or stoppers. For
this, different storage conditions that might have an impact (e.g.
vial orientation) were assessed. Solutions of brincidofovir were
studied at the formulation strength (10 mg/mL) and at a low
concentration (0.2 mg/mL).
[0324] To test for adsorption, a treatment with an organic solvent
(methanol) that is suited to re-solubilize adsorbed material was
implemented at the different time points and compared to treatment
with water or untreated samples. An observation of higher
brincidofovir concentration in the samples containing methanol
(after normalization for dilution) would have been indicative of an
adsorption effect. Differences of an imposed volume contraction
between the applied solvents are not considered. The study was
conducted using solutions of 10 mg/mL and 0.2 mg/mL of
brincidofovir in 200 mM sodium phosphate (pH=8.0), at temperatures
of 2-8.degree. C. and 25.degree. C. Appearance, pH, brincidofovir
recovery (HPLC), and purity (HPLC) of the solutions were tested
initially and after 1, 2, and 7 days. Some samples were further
treated by addition of methanol (1.0 mL) or water (1.0 mL). The
results of the study are summarized in Table 16 and Table 17.
TABLE-US-00016 TABLE 16 Test results of the 10 mg/mL brincidofovir
formulation in adsorption assessment Sample conditions Station Time
Observed BCV BCV temp. Vial point Measured c(BCV).sup.a
Recovery.sup.a,b purity (.degree. C.) orientation Treatment (day)
Appearance pH (mg/mL) (%) (%) 2-8 Upright No 0 Clear, Colorless 8.0
10.5 100.00 99.80 treatment 1 Clear, Colorless 8.0 10.5 100.36
99.55 2 Clear, Colorless 8.0 10.5 99.57 99.67 7 Clear, Colorless
8.0 10.6 99.93 99.61 Add 0 Clear, Colorless 8.3 10.4 100.00 99.90
methanol 1 Clear, Colorless 8.4 10.2 98.10 99.52 2 Clear, Colorless
8.4 10.3 98.90 99.73 7 Clear, Colorless 8.4 10.5 100.41 99.43 Add
water 0 Clear, Colorless 8.1 10.5 100.00 99.32 1 Clear, Colorless
8.1 10.3 98.56 99.72 2 Clear, Colorless 8.0 10.6 101.14 99.47 7
Clear, Colorless 8.1 10.6 100.68 99.61 Inverted No 0.sup.c Clear,
Colorless 8.0 10.5 100.00 99.80 treatment 1 Clear, Colorless 8.1
10.5 100.11 99.74 2 Clear, Colorless 8.0 10.4 99.18 99.78 7 Clear,
Colorless 8.0 10.5 99.41 99.77 Add 0.sup.c Clear, Colorless 8.3
10.4 100.00 99.90 methanol 1 Clear, Colorless 8.4 10.6 102.34 99.46
2 Clear, Colorless 8.4 10.7 103.30 99.64 7 Clear, Colorless 8.4
10.7 102.56 99.44 Add water 0.sup.c Clear, Colorless 8.1 10.5
100.00 99.32 1 Clear, Colorless 8.1 10.4 99.50 99.46 2 Clear,
Colorless 8.0 10.4 99.53 99.47 7 Clear, Colorless 8.0 10.6 100.21
99.54 25 Upright No 0 Clear, Colorless 8.0 10.5 100.00 99.80
treatment 1 Clear, Colorless 8.1 10.5 99.78 99.47 2 Clear,
Colorless 8.0 10.4 99.02 99.66 7 Clear, Colorless 8.0 10.4 98.72
99.73 Add 0 Clear, Colorless 8.3 10.4 100.00 99.90 methanol 1
Clear, Colorless 8.4 10.6 101.66 99.57 2 Clear, Colorless 8.3 10.3
98.83 99.61 7 Clear, Colorless 8.4 10.5 100.52 99.74 Add water 0
Clear, Colorless 8.1 10.5 100.00 99.32 1 Clear, Colorless 8.1 10.6
101.21 99.61 2 Clear, Colorless 8.1 10.6 101.00 99.76 7 Clear,
Colorless 8.1 10.6 100.13 99.70 Inverted No 0.sup.c Clear,
Colorless 8.0 10.5 100.00 99.80 treatment 1 Clear, Colorless 8.1
10.6 101.40 99.57 2 Clear, Colorless 8.0 10.6 101.08 99.82 7 Clear,
Colorless 8.0 10.5 99.22 99.60 Add 0.sup.c Clear, Colorless 8.3
10.4 100.00 99.90 methanol 1 Clear, Colorless 8.4 10.7 102.92 99.60
2 Clear, Colorless 8.4 10.6 101.97 99.61 7 Clear, Colorless 8.4
10.5 100.47 99.81 Add water 0.sup.c Clear, Colorless 8.1 10.5
100.00 99.32 1 Clear, Colorless 8.0 10.5 100.55 99.47 2 Clear,
Colorless 8.0 10.5 100.56 99.63 7 Clear, Colorless 8.0 10.5 99.92
99.68 .sup.1Average value of the three (3) replicates of samples
that received treatment. .sup.2BCV recovery = c(BCV).sub.Day
n/c(BCV).sub.Day 0 .times. 100%. It was defined as 100% at the
initial time point. BCV recovery was defined as 100.00% on the
initial time point for samples with and without treatment.
.sup.3Not applicable for samples without treatment. .sup.4The
sample for the initial time point was not subjected to any
conditions.
TABLE-US-00017 TABLE 17 Test results of the 0.2 mg/mL brincidofovir
formulation in adsorption assessment Sample conditions Station Time
Observed BCV BCV temp. Vial point Measured c(BCV).sup.a
Recovery.sup.a,b purity (.degree. C.) orientation Treatment (day)
Appearance pH (mg/mL) (%) (%) 2-8 Upright No 0 Clear, Colorless 8.0
0.19 100.00 99.37 treatment 1 Clear, Colorless 8.1 0.19 100.23
99.37 2 Clear, Colorless 8.1 0.19 100.01 99.45 7 Clear, Colorless
8.1 0.20 100.26 99.52 Add 0 Clear, Colorless 8.3 0.20 100.00 99.28
methanol 1 Clear, Colorless 8.4 0.20 100.91 99.45 2 Clear,
Colorless 8.4 0.20 100.79 99.55 7 Clear, Colorless 8.4 0.20 101.74
99.57 Add water 0 Clear, Colorless 8.0 0.19 100.00 99.48 1 Clear,
Colorless 8.1 0.20 100.38 99.27 2 Clear, Colorless 8.1 0.20 100.78
99.35 7 Clear, Colorless 8.1 0.20 100.30 99.37 Inverted No 0.sup.c
Clear, Colorless 8.0 0.19 100.00 99.37 treatment 1 Clear, Colorless
8.1 0.19 99.21 99.38 2 Clear, Colorless 8.1 0.19 99.68 99.50 7
Clear, Colorless 8.0 0.19 99.61 99.66 0.sup.c Clear, Colorless 8.3
0.20 100.00 99.28 Add 1 Clear, Colorless 8.5 0.20 100.09 99.51
methanol 2 Clear, Colorless 8.4 0.20 100.17 99.54 7 Clear,
Colorless 8.4 0.20 100.04 99.66 Add water 0.sup.c Clear, Colorless
8.0 0.19 100.00 99.48 1 Clear, Colorless 8.1 0.19 99.81 99.30 2
Clear, Colorless 8.0 0.19 99.67 99.42 7 Clear, Colorless 8.1 0.19
99.27 99.62 25 Upright No 0 Clear, Colorless 8.0 0.19 100.00 99.37
treatment 1 Clear, Colorless 8.1 0.19 100.18 99.46 2 Clear,
Colorless 8.0 0.19 100.12 99.27 7 Clear, Colorless 8.1 0.19 99.92
99.42 Add 0 Clear, Colorless 8.3 0.20 100.00 99.28 methanol 1
Clear, Colorless 8.4 0.20 101.00 99.54 2 Clear, Colorless 8.4 0.20
101.25 99.45 7 Clear, Colorless 8.4 0.20 101.87 99.53 Add water 0
Clear, Colorless 8.0 0.19 100.00 99.48 1 Clear, Colorless 8.1 0.20
100.60 99.54 2 Clear, Colorless 8.1 0.20 100.22 99.31 7 Clear,
Colorless 8.1 0.20 100.45 99.44 Inverted No 0.sup.c Clear,
Colorless 8.0 0.19 100.00 99.37 treatment 1 Clear, Colorless 8.1
0.19 99.38 99.45 2 Clear, Colorless 8.1 0.19 98.91 99.55 7 Clear,
Colorless 8.1 0.19 98.96 99.62 Add 0.sup.c Clear, Colorless 8.3
0.20 100.00 99.28 methanol 1 Clear, Colorless 8.5 0.20 100.25 99.62
2 Clear, Colorless 8.4 0.20 99.70 99.39 7 Clear, Colorless 8.4 0.20
99.76 99.52 Add water 0.sup.c Clear, Colorless 8.0 0.19 100.00
99.48 1 Clear, Colorless 8.1 0.19 99.28 99.37 2 Clear, Colorless
8.1 0.19 99.31 99.43 7 Clear, Colorless 8.1 0.19 98.99 99.53
.sup.aAverage value of the three (3) replicates of samples that
received treatment. .sup.bBCV recovery = c(BCV).sub.Day
n/c(BCV).sub.Day 0 .times. 100%. It was defined as 100% at the
initial time point. BCV recovery was defined as 100.00% on the
initial time point for samples with and without treatment.
.sup.cThe sample for the initial time point was not subjected to
any conditions.
[0325] A comparison of differently treated samples did not reveal
any evidence for an adsorption phenomenon; no significant
differences in the studied parameters, i.e., no significant change
in appearance or purity and no significant loss of brincidofovir,
were observed. The observed mass deficits were within the range of
the sample preparation errors as expressed by the standard
deviation of replicate preparations.
Formulation Compatibility Assessments
[0326] An array of materials was tested for compatibility with the
formulation. This includes testing materials from the manufacturing
stage, pre-clinical toxicology as well as clinical tests. The
applied compatibility tests evaluate 1) a potential loss of
brincidofovir by adsorption or precipitation (appearance,
brincidofovir recovery by HPLC) and 2) a shift in pH that could
compromise stability. Materials that were tested include the
infusion vehicle, filters for sterilization, product vials and
stoppers, infusion systems for testing animals (rats) and infusion
bags and IV systems for clinical applications.
[0327] A brincidofovir formulation comprising 10 mg/mL
brincidofovir, 200 mM sodium phosphate, and 69 mM dextrose (pH=8.0)
was diluted with the testing infusion vehicle (5% dextrose
solution) to yield c(BCV)=0.5 and 1.0 mg/mL solutions. The diluted
solutions were stored at ambient conditions, and samples were
collected at initially at the beginning of the experiment, and
after 2 h, 8 h, and 24 h and evaluated for appearance, pH, and
recovery of c(BCV) (via HPLC). No significant changes in
appearance, pH, or c(BCV) were observed for the tested solutions
over 24 hours at ambient conditions. Detailed results of the study
are summarized below (Table 18).
TABLE-US-00018 TABLE 18 Results of infusion vehicle compatibility
assessment c(BCV) in infusion Time Observed BCV vehicle point
Measured c(BCV).sup.a Recovery.sup.a,b (mg/mL) (h) Appearance pH
(mg/mL) (%) 0.5 0 Clear, colorless 8.2 0.51 100 2 Clear, colorless
8.3 0.51 100 8 Clear, colorless 8.2 0.51 100 24 Clear, colorless
8.2 0.49 99 1.0 0 Clear, colorless 8.2 1.0 100 2 Clear, colorless
8.2 1.0 99 8 Clear, colorless 8.2 1.0 99 24 Clear, colorless 8.2
1.0 100 .sup.aAverage value of the three (3) replicates. .sup.bBCV
recovery = c(BCV)Hour n/c(BCV)Hour 0 .times. 100%. It was defined
as 100% for samples at initial time point.
Sterilization Filter Compatibility
[0328] Formulations of the disclosure were evaluated for
sterilization filter compatibility. Aliquots of a testing
brincidofovir formulation were filtered through the corresponding
syringe filters as follows:
A: Filter: 0.2 .mu.m, 25 mm, syringe filter, Supor.RTM. membrane
(PES), sterile; testing solution: 10 mg/mL brincidofovir, 200 mM
sodium phosphate, pH=8.0; testing volume: 10 mL. B: Filter: 0.2
.mu.m, 25 mm, syringe filter, Posidyne.RTM. membrane, sterile;
testing solution: 10 mg/mL brincidofovir, 200 mM sodium phosphate,
pH=8.0; testing volume: 10 mL. C: Filter: 0.22 .mu.m, 33 mm,
syringe filter, Durapore.RTM. PVDF membrane, .gamma.-irradiated;
testing solution: 2 mg/mL brincidofovir, 4 mM sodium phosphate,
pH-8, approx. 5% dextrose (50.times. dilution of 10 mg/mL
brincidofovir, 200 mM sodium phosphate, pH=8.0 with 5% dextrose
solution; testing volume: 50 mL.
[0329] The first and last 10% of the filtrate were collected.
Samples of the collected filtrates, along with samples of the pre
filtration formulation, were assessed for their appearance, pH, and
c(BCV).
TABLE-US-00019 TABLE 19 Results of sterilization filter
compatibility assessment Observed BCV Filter Measured c(BCV).sup.a
Recovery.sup.a,b membrane Conditions Appearance pH (mg/mL) (%) PES
Pre filtration Clear, 8.1 9.9 100 colorless First 10% filtrate
Clear, 8.0 9.8 99 volume colorless Last 10% filtrate Clear, 8.0 9.8
99 volume colorless Posidyne Pre filtration Clear, 8.1 9.9 100
colorless First 10% filtrate Clear, 8.0 9.7 98 volume colorless
Last 10% filtrate Clear, 8.0 9.8 99 volume colorless PVDF Pre
filtration Clear, 8.0 0.20 100 colorless First 10% filtrate Clear,
8.1 0.20 99 volume colorless Last 10% filtrate Clear, 8.1 0.20 100
volume colorless .sup.aAverage value of the three (3) replicates.
.sup.bBCV recovery = c(BCV).sub.Post filtration/c(BCV).sub.Pre
filtration .times. 100%; defined as 100% for pre filtration
samples.
[0330] No significant changes in appearance, pH, or c(BCV) were
observed when comparing the filtrate solutions with the
corresponding pre-filtration samples and no impurity was introduced
during filtration.
Drug Product Vial and Stopper Compatibility
[0331] Formulations of the disclosure were evaluated for
sterilization filter compatibility. The testing formulation (5 mL,
10 mg/mL brincidofovir, 200 mM sodium phosphate, pH=8.0) was
dispensed into a set of Afton sterilized, Ready-To-Fill.RTM. vials.
The vials were stoppered with West NovaPure.RTM. stoppers and
crimped with Afton Ready-To-Fill.RTM. sterilized seals, and stored
for 6 hours at ambient conditions in upright and inverted
orientations. Samples of the filling solution were collected before
filling and after 6 hours of storage under the respective
condition. The collected samples were assessed for their
appearance, pH, and c(BCV). No significant changes in appearance,
pH, or c(BCV) were observed for the tested formulation after 6
hours of contact with the tested vials and stoppers. No additional
impurity was introduced following contact.
TABLE-US-00020 TABLE 20 Results of drug product vial and stopper
compatibility assessment Observed BCV Storage Measured c(BCV).sup.a
Recovery .sup.a,b condition Appearance pH (mg/mL) (%) Pre filling
Clear, colorless 8.0 9.5 100 Upright, 6 h Clear, colorless 8.0 9.5
100 Inverted, 6 h Clear, colorless 8.0 9.4 99 .sup.aAverage value
of the three (3) replicates. .sup.bBCV recovery = c(BCV)Hour
n/c(BCV)Pre filling .times. 100%. It was defined as 100% for pre
filling samples.
Preclinical Material Compatibility Assessment (Rats)
Infusion System Assembly (Rats)
[0332] The compatibility of the following brincidofovir
formulations with an infusion system assembly for dosing of rats
(consisting of a syringe, tether, catheter and syringe pump) was
tested
a.) 0.2 mg/mL brincidofovir, 4 mM sodium phosphate, pH .about.8.0,
approx. 4.90% dextrose; b.) 1.5 mg/mL brincidofovir, 30 mM sodium
phosphate, pH=8.0, approx. 4.25% dextrose.
[0333] Both solutions were prepared via dilution of the developed
brincidofovir formulation (10 mg/mL brincidofovir, 200 mM sodium
phosphate, pH=8.0) with a 5% dextrose solution. The testing
procedure can be summarized as follows: [0334] 1. The appearance,
pH, and c(BCV) of the bulk testing solutions (0.2 mg/mL BCV, 4 mM
sodium phosphate, pH-8.0, approx. 4.90% dextrose, and 1.5 mg/mL
BCV, 30 mM sodium phosphate, pH .about.8.0, approx. 4.25% dextrose
were assessed pre-contact. [0335] 2. Approximately 5 mL of the
testing solution was drawn into a syringe which was then connected
to the assembled infusion system (Vascular Access Harness (VAH)
tether kit, the VAH port for rat jacket, and catheter). [0336] 3.
The infusion system was manually filled with the testing solution.
Approximately 200 .mu.L of the testing solution were pushed out of
the infusion system and collected as an end-of-line sample
(t.sub.0) for the assessments of appearance and c(BCV). [0337] 4.
The syringe of the testing infusion system was mounted onto the
syringe pump, and a flow rate was set to a flow of 1.2 mL/h (20
.mu.L/min) for a total dispensing volume of 2.4 mL (120 min).
[0338] 5. The entire flow-through was collected over the course of
the experiment. At the respective time points (t.sub.10min,
t.sub.105 min), end-of-line samples (approx. 200 .mu.L) were
collected for analysis. [0339] 6. The pH of the bulk flow-through
was measured at the end of the experiment (tin min), since the
volume of samples acquired from previous time points (200 .mu.L)
was too small for a standard pH measurement. The results of the
study are summarized in Table 21.
TABLE-US-00021 [0339] TABLE 21 Results of preclinical infusion
system compatibility assessment Testing solution Observed BCV
c(BCV) Measured c(BCV).sup.a Recovery .sup.a,b (mg/mL) Conditions
Appearance pH (mg/mL) (%) 0.2 Pre contact (bulk Clear, colorless
8.0 0.21 100 (Test #1) solution) t.sub.0 Clear, colorless n.a. 0.19
90 t.sub.10 min Clear, colorless n.a. 0.16 93 t.sub.105 min Clear,
colorless n.a. 0.20 94 t.sub.120 min (bulk solution) Clear,
colorless 8.0 n.a. n.a. 1.5 Pre contact (bulk Clear, colorless 8.2
1.5 100 (Test #2) solution) t.sub.0 Clear, colorless n.a. 1.5 100
t.sub.10 min Clear, colorless n.a. 1.5 100 t.sub.105 min Clear,
colorless n.a. 1.5 100 t.sub.120 min (bulk solution) Clear,
colorless 8.2 n.a. n.a. .sup.aAverage value of the three (3)
replicates. .sup.bBCV recovery = c(BCV)Minute n/c(BCV)Pre contact
.times. 100%, defined as 100% for pre contact samples. n.a.: Not
available or not applicable.
[0340] For test #1 (0.2 mg/mL brincidofovir), an initial 10% loss
in c(BCV) was observed, which can be assigned to absorption to the
testing infusion system. At low concentrations an initial
adsorption effect (surface saturation) is frequently observed, in
the present case a full recovery of the observed loss is not
observed over the time of testing; at t=105 min a brincidofovir
loss of 6% was observed. No significant changes in appearance or pH
were observed. For test #2, no significant loss in c(BCV) or
changes in appearance or pH were observed over the course of the
experiment.
Clinical Material Assessment
Infusion Bag Compatibility
[0341] A standard mini-infusion bag (100 mL) was subjected to
material compatibility testing with the following formulation:
[0342] 15 mg/mL brincidofovir, 200 mM sodium phosphate, pH=8.0. The
tested formulation strengths after dilution into the infusion bag
were approximately: [0343] 0.1 mg/mL brincidofovir, 5.3 mM sodium
phosphate, pH.apprxeq.8.0 (in 5% dextrose), [0344] 1.0 mg/mL
brincidofovir, 13.3 mM sodium phosphate, pH.apprxeq.8.0 (in 5%
dextrose).
[0345] The testing clinical infusion bag is specified as follows:
[0346] 5% dextrose for injection, USP, 100 mL VIAFLEX infusion
bag
[0347] The developed brincidofovir formulation (15 mg/mL
brincidofovir, 200 mM sodium phosphate, pH=8.0) was diluted to
c(BCV)=0.1 and c(BCV)=1.0 mg/mL by injection into the infusion
bags. The infusion bags were then stored at ambient conditions, and
samples were collected from the infusion bags at the time points
(t.sub.0, t.sub.1h, t.sub.8h, and t.sub.24h) for assessments of
appearance, pH, and c(BCV). As a control, the 5% dextrose solution
was removed from a separate infusion bag, and external dilutions
(standard laboratory glassware) of the testing formulation with the
5% dextrose solution were performed applying identical dilution
factors. Samples of the external diluted solution were likewise
analyzed. The results are summarized in Table 22.
TABLE-US-00022 TABLE 22 Results of clinical infusion bag
compatibility assessment c(BCV) in testing infusion Observed BCV
bag Appear- Measured c(BCV).sup.a Recovery .sup.a,b (mg/mL)
Conditions ance pH (mg/mL) (%) 0.1 External Clear, 7.9 0.098 106
dilution colorless t.sub.0 Clear, 7.9 0.092 100 colorless t.sub.1 h
Clear, 7.9 0.093 101 colorless t.sub.8 h Clear, 7.9 0.093 101
colorless t.sub.24 h Clear, 7.9 0.093 101 colorless 1.0 External
Clear, 8.2 0.94 108 dilution colorless t.sub.0 Clear, 8.2 0.87 100
colorless t.sub.1 h Clear, 8.2 0.87 100 colorless t.sub.8 h Clear,
8.2 0.87 100 colorless t.sub.24 h Clear, 8.1 0.86 99 colorless
.sup.aAverage value of the three (3) replicates. .sup.bBCV recovery
= c(BCV).sub.Hour n/c(BCV).sub.hour 0 .times. 100%. It was defined
as 100% for t.sub.0 samples.
[0348] No significant change in appearance, pH, or c(BCV) was
observed for the diluted solutions in the testing infusion bag at
either c(BCV). The higher brincidofovir recoveries obtained from
the external dilutions most likely resulted from the fact that
infusion bags are generally overfilled (105-115 mL with an average
of 110 mL, for 100 mL bags--information communicated by Baxter). By
applying an identical dilution factor and using the to time point
(dilution into the infusion bag) for normalization (defined as
100%) the concentrations determined for the external dilution are
overestimated. An internal study on a single infusion bag assessed
the volume of the infusion solution in a 100 mL infusion bag with
.apprxeq.109 mL.
Clinical Infusion System Compatibility
[0349] Two infusion systems (Baxter Non-DEHP CONTINUO-FLO solution
set with 2 INTERLINK injection sites, male Luer-lok adaptor; and
Brown Rate Flow.RTM. regulator IV set with 15 micron filter, 1 non
needle free injection site) were tested for compatibility with the
following formulation: [0350] 15 mg/mL brincidofovir, 200 mM sodium
phosphate, pH=8.0. The tested formulation strengths after dilution
into the infusion bag were approximately: [0351] 0.1 mg/mL
brincidofovir, 5.3 mM sodium phosphate, pH.apprxeq.8.0 (in 5%
dextrose), [0352] 1.0 mg/mL brincidofovir, 13.3 mM sodium
phosphate, pH.apprxeq.8.0 (in 5% dextrose). The testing procedure
can be summarized as follows: [0353] 1. The developed brincidofovir
formulation (15 mg/mL brincidofovir, 200 mM sodium phosphate, pH
8.0), was injected into infusion bags (reservoir function)
mentioned above to yield the testing solutions at c(BCV)=0.1 or 1.0
mg/mL. A sample was collected from the infusion bag for assessment
of c(BCV) (t.sub.24 h, bag). [0354] 2. The infusion bag was
incubated ("aged") for 24 h at ambient conditions before another
sample (t.sub.0, bag) was collected from the infusion bag for
assessment (appearance, pH, c(BCV)). [0355] 3. The tested infusion
system was attached to the infusion bag and manually filled with
solution from the infusion bag. Approximately 1 mL (triplicates) of
the testing solution was collected as an end-of-line sample for
analysis (t.sub.0, eol). [0356] 4. The testing infusion system was
shut off, and stored at ambient conditions until the next time
point. Approximately 17 mL of the testing solution (volume of the
infusion system) was drained before another end-of-line sample (1
mL, triplicates) was collected for analysis (t.sub.10 min, eol,
t.sub.3 h, eol, and t.sub.6 h, eol).
TABLE-US-00023 [0356] TABLE 23 Results of clinical infusion system
compatibility assessment c(BCV) Testing in infusion Observed BCV
infusion bag Measured c(BCV).sup.a Recovery.sup.a,b system (mg/mL)
Conditions Appearance pH (mg/mL) (%) Baxter 0.1 t.sub.-24 h, bag
n.a. n.a. 0.090 100 t.sub.0, bag Clear, colorless 7.7 0.091 100
t.sub.0, eol Clear, colorless 7.7 0.088 96 t.sub.10 min, eol Clear,
colorless 7.7 0.092 100 t.sub.3 h, eol Clear, colorless 7.7 0.091
100 t.sub.6 h, eol Clear, colorless 7.7 0.092 100 1.0 t.sub.-24 h,
bag n.a. n.a. 0.85 100 t.sub.0, bag Clear, colorless 8.1 0.86 100
t.sub.0, eol Clear, colorless 8.1 0.85 100 t.sub.10 min, eol Clear,
colorless 8.1 0.86 100 t.sub.3 h, eol Clear, colorless 8.1 0.86 100
t.sub.6 h, eol Clear, colorless 8.1 0.86 100 B. Braun 0.1 t.sub.-24
h, bag n.a. n.a. 0.090 100 t.sub.0, bag Clear, colorless 7.8 0.091
100 t.sub.0, eol Clear, colorless 7.7 0.086 94 t.sub.10 min, eol
Clear, colorless 7.7 0.091 100 t.sub.3 h, eol Clear, colorless 7.7
0.090 99 t.sub.6 h, eol Clear, colorless 7.7 0.091 100 1.0
t.sub.-24 h, bag n.a. n.a. 0.86 100 t.sub.0, bag Clear, colorless
8.1 0.86 100 t.sub.0, eol Clear, colorless 8.1 0.86 100 t.sub.10
min, eol Clear, colorless 8.1 0.86 100 t.sub.3 h, eol Clear,
colorless 8.1 0.86 100 t.sub.6 h, eol Clear, colorless 8.1 0.86 100
.sup.aAverage value of the three (3) replicates. .sup.bBCV recovery
= c(BCV).sub.Hour n/c(BCV).sub.Hour 0 .times. 100%. It was defined
as 100% for t.sub.0, bag samples.
[0357] An initial loss in c(BCV) (up to 6%) was observed for the
testing solution with low c(BCV)=0.1 mg/mL in both testing clinical
infusion systems. This observation can most likely be explained
with an initial saturating adsorption of the tubing material. The
brincidofovir concentration recovered to approx. 100% over the
course of the experiment (6 h). No significant overall changes in
appearance, pH, or c(BCV) were observed for both of the tested
infusion systems.
Example 4--Stability and Characteristics of Lyophilized and
Reconstituted Formulations
[0358] Pre-lyophilization formulations were prepared as set forth
in Table 24, below and subjected to the lyophilization process.
TABLE-US-00024 TABLE 24 Pre-lyophilization Formulations. c(bulking
Form. c(buffer) c(BCV) Target Bulking agent) (w/v) # Buffer [mM]
[mg/mL] pH agent [%] 1 Na-Phosphate 200 10.0 8.0 Mannitol 5 2
Na-Phosphate 200 10.0 8.0 Sucrose 9 3 Arginine 200 10.0 8.0
Mannitol 5 3a Arginine 100 10.0 8.0 Mannitol 5 3b Arginine 50 10.0
8.0 Mannitol 5 3c Arginine 25 10.0 8.0 Mannitol 5 4 Arginine 200
10.0 8.0 Sucrose 9 5 Tromethamine (Tris) 200 10.0 8.0 Mannitol 5 6
Tromethamine (Tris) 200 10.0 8.0 Sucrose 9 7 water (pH-adjusted) --
10.0 8.0 Mannitol 5 8 water (pH-adjusted) -- 10.0 8.0 Sucrose 9
[0359] The glass transition temperature T.sub.g' (amorphous), the
eutectic melting point T.sub.eu (crystalline), the onset
temperature of melting T.sub.melt, onset, and the freezing
temperature T.sub.freeze of the formulations 1-8 of Table 27 were
determined by differential scanning calorimetry (DSC) analysis. For
this, 5 .mu.L of each formulation was dispensed into an aluminum
sample pan and hermetically sealed. DSC scans were performed by
down and up scanning in the temperature interval of +25.degree. C.
to -65.degree. C. to +25.degree. C. with a ramp rate of 5.degree.
C./min. The tested formulations displayed a sharp freezing point
and a well-defined melting transition. The results of the DSC study
are summarized in Table 25.
TABLE-US-00025 TABLE 25 Results of the DSC analysis Form. Bulking
T.sub.g'/T.sub.eu T.sub.freeze T.sub.melt T.sub.melt, onset #
Buffer agent [.degree. C.] [.degree. C.] [.degree. C.] [.degree.
C.] 1 Na-Phosphate Mannitol -45.0 -20.0 -0.5 -3.0 2 Na-Phosphate
Sucrose -32.0 -18.0 -0.2 -3.4 3 Arginine Mannitol -45.0 -20.0 -0.5
-4.2 4 Arginine Sucrose -32.0 -18.0 -0.2 -4.2 5 Tromethamine
Mannitol -16.0 -21.0 -0.4 -5.2 6 Tromethamine Sucrose -43.0 -20.0
-0.4 -5.2 7 DI water (pH- Mannitol -30.5 -21.2 -0.01 -2.2 adjusted)
8 DI water (pH- Sucrose -34.8 -16.6 -0.8 -2.9 adjusted)
Lyophilization Cycle
[0360] A conservative lyophilization cycle was applied for
lyophilization of brincidofovir formulations including parameters
informed by the DSC analysis (see Table 28). The cycle consisted of
the following steps: freezing, annealing, primary drying and a
secondary drying. The set point temperature, ramp rate, step time
and load time for each step in an exemplary lyophilization cycle
are summarized in Table 26.
TABLE-US-00026 TABLE 26 Lyophilization cycle parameters (Cycle # 1)
T.sub.shelf Ramp Step Total (Set point) rate time time Phase Type
[.degree. C.] [.degree. C./h] [min] [h] Loading Hold 5 N/A 0 0
Freeze/Anneal Hold 5 0 60 1 Freeze/Anneal Rate -50 20 165 3.8
Freeze/Anneal Hold -50 N/A 180 6.8 Freeze/Anneal Rate -8 20 174 9.7
Freeze/Anneal Hold -8 N/A 240 13.7 Freeze/Anneal Rate -35 20 81
15.0 Freeze/Anneal Hold -35 N/A 60 16.0 Primary Drying Hold -35 N/A
1200 36.0 Secondary drying Rate 20 2.5 1300 57.7 Secondary drying
Hold 20 N/A 360 63.7
[0361] Sample vials (5 mL vial size, 1 mL filling volume) were
processed under best clean conditions in a biosafety cabinet. For
each formulation, nine samples were prepared. For each formulation
condition, one sample vial was equipped with a product probe to
monitor the product temperature (T.sub.product) along with the
shelf temperature of the lyophilizer (T.sub.shelf) throughout the
lyophilization cycle. The end of the primary drying was determined
as the time when T.sub.product.apprxeq.T.sub.shelf is observed. At
the end of the lyophilization cycle, the sample vials were
back-filled nitrogen (N.sub.2), stoppered and removed from the
lyophilizer to be inspected and analyzed.
[0362] The step time for the primary drying phase was set to 20 h.
FIG. 1 displays the product temperature profiles of Formulations
1-8 during this step. In the tested formulations, after activation
of the vacuum pump and drop of the product temperature to
T.sub.(product)<T.sub.(shelf) at around 16 hours of total cycle
time, temperature jumps were observed in the different formulations
within 15 hours. The temperature jump is taken to be indicative of
successful sample drying.
[0363] The lyophilized products were evaluated based on structure
and uniformity of the lyophilization cakes. The observation of a
`melt-back` or `cake collapses` led to a lowered ranking. Four
vials from each formulation were randomly selected for the
appearance evaluation. The assessment and ranking are summarized in
Table 27.
TABLE-US-00027 TABLE 27 Assessment of lyophilized products and
reconstitution performance Reconstitution Foam. Formulation Dissip.
Ranking App. Lyophilized Product Reconst. time Meas. c(BCV) Appear.
Reconst. # Buffer Bulking agent pH Cake appearance time [s] Foam*
[min] pH [mg/mL] ranking ranking 1 Na-Phosphate Mannitol (5%) 7.9
White uniform solid. 50 + 7 7.8 10.4 2 4 Minor melt-back 2
Na-Phosphate Sucrose (9%) 7.9 White uniform solid. 80 ++ 45 7.8
10.5 5 7 Minor melt-back. Minor detachment from vial. 3 Arginine
Mannitol (5%) 8.0 White uniform solid. 60 - -- 7.9 10.3 3 1 Minor
melt-back. Minor detachment from bottom (cake collapse). 4 Arginine
Sucrose (9%) 8.0 Major melt-back. 45 ++ 35 7.9 9.8 7 5 Significant
cake collapse. 5 Tris Mannitol (5%) 8.0 Minor-melt back and 90 - --
8.1 10.6 4 2 detachment from bottom. Minor cake collapse. 6 Tris
Sucrose (9%) 8.0 Extensive melt-back 15 ++ 45 8.0 10.4 8 6 and cake
collapse. 7 DI water(pH- Mannitol (5%) 8.3 White uniform solid. 45
+ 3 8.4 10.6 1 3 adjusted) Minimal melt-back. 8 DI water(pH-
Sucrose (9%) 8.3 Strong melt back and 60 ++ 60 8.3 10.3 6 4
adjusted) cake collapse. Cake detached from vial.
[0364] Of the tested formulations, the mannitol based formulations
(Formulations 1, 3, 5 and 7) yielded a more compact and uniform
lyophilization cake, and the sucrose based formulations
(Formulations 2, 4, 6 and 8) revealed partial cake collapses as
well as an increased melt-back.
Reconstitution Assessment of Lyophilized Formulations
[0365] Lyophilization product vials were weighed before and after
lyophilization in order to determine the reconstitution solution
volume. The lyophilized products were reconstituted with DI water.
After addition of the solvent the vials were gently swirled and the
reconstitution appearance as well as the reconstitution time were
recorded. The reconstituted product was further analyzed for its pH
and brincidofovir recovery (determined via HPLC). For a
reconstitution ranking the described parameters were complemented
by recording the intensity of foaming as well as the foam
dissipation time. The results of the reconstitution assessment for
Formulations 1-8 are summarized in Table 30.
[0366] Formulation 3 was found to lack foaming upon reconstitution,
and Formulation 7 was found to be able to be reconstituted with
different buffers or infusion vehicles.
Example 5--Lyophilization Process Development
[0367] For the development of a lyophilized drug product of
brincidofovir, five different activities have been executed: i)
lyophilization feasibility assessment, ii) lyophilization process
development, iii) short-term stability studies, iv) preparation of
drug product batches for stability testing, and v) material
compatibility assessment.
[0368] A project overview capturing the purpose, results and the
outcome of each activity is summarized in Table 28. The details of
the lyophilization cycle parameters for certain lyophilization runs
during process development can be found in Table 29.
TABLE-US-00028 TABLE 28 Project overview and decision rationale.
Activity Cycle # Purpose Formula Results Feasibility 1
Lyophilization of 1-8 Tromethamine and sucrose containing
assessment starting matrix formulations formulations did not have
sufficient (feasibility); 1 mL fill stability. volume, 5 mL vials 2
Scale of product unit 1, 3, 7 .sub.i) Scale-up prevented completion
of drying size (5 mL fill, 20 mL during the prim,. drying phase.
vials) .sub.ii) Less foaming (reconstitution) observed for # 3. 3
.sub.i) Cycle parameter 1, 3, 7 Products obtained w/2% volatiles. A
adjustment. reconstitution analysis was not performed .sub.ii)
Obtain products w/ for this lyophilization cycle. acceptable
residual moisture. Process 4 --- Cycle fail --- Development 5
.sub.i) Investigate the extent 1, 3, 3a, .sub.i) Comparable foaming
for #3, #3a. of foaming 3b, 3c .sub.ii) No loss of brincidofovir
was observed .sub.ii) Evaluate the within 48 h after reconstitution
(all reconstitution process formulations) incl. 48 h reconst.
stability Short-term 6 Evaluate the stability of 1, 3, 3a (i)
Increased residual moisture for #1 and stability the lyophilized
products therefore stronger degradation. (2 weeks) (solid) (ii)
Superior stability of #3a vs. #3. (iii) Loss in brincidofovir
recovery observed for #1, #3 pre/post lyophilization (pot. handling
errors, high assay variability) DP 7 DP preparation for 3a
Preparation of 200 vials (5 mL filling, 20 preparation stability
testing mL vial). A loss in recovery (not degradation) observed
pre/post lyophilization, (pot. handling errors, high assay
variability) Material NA Evaluate the 3a The formulation (3a) was
compatible with compatibility compatibility of sterile all tested
materials. An initial assessment filters, vials, stoppers,
adsorption/saturation effect was observed infusion bags, IV for the
IV systems at low c(BCV). systems. Samples at c(BCV) = 0.1 mg/mL
showed a more significant pH shift.
TABLE-US-00029 TABLE 29 Lyophilization cycle parameters (Cycles #2,
#3, #5, #6) Cycle #2 Cycle #3 Cycle #5 Cycle #6 Ramp Total Ramp
Total Ramp Total Ramp Total rate T.sub.shelf Time Time rate
T.sub.shelf Time Time rate T.sub.shelf Time Time rate T.sub.shelf
Time Time Step Type [.degree. C./h] [.degree. C.] [min] [h]
[.degree. C./h] [.degree. C.] [min] [h] [.degree. C./h] [.degree.
C.] [min] [h] [.degree. C./h] [.degree. C.] [min] [h] Loading Hold
N/A 5 0 0 N/A 5 0 0 N/A 5 0 0 N/A 5 0 0 Freeze/ Hold 0 5 60 1 0 5
60 1 0 5 60 1 0 5 60 1 Anneal Freeze/ Rate 20 -50 165 3.8 20 -50
165 3.8 20 -50 165 3.8 20 -50 165 3.8 Anneal Freeze/ Hold N/A -50
180 6.8 N/A -50 180 6.8 N/A -50 180 6.8 N/A -50 180 6.8 Anneal
Freeze/ Rate 20 -4 174 9.7 20 -4 174 9.7 20 -4 174 9.7 20 -4 174
9.7 Anneal Freeze/ Hold N/A -4 240 13.7 N/A -4 240 13.7 N/A -4 240
13.7 N/A -4 240 13.7 Anneal Freeze/ Rate 20 -38 81 15 20 -35 81 15
20 -35 81 15 20 -35 81 15 Anneal Freeze/ Hold N/A -38 60 16 N/A -35
60 16 N/A -35 180 18 N/A -35 180 18 Anneal Primary Hold N/A -38
1200 36 N/A -35 2160 52 N/A -35 2160 54 N/A -35 2160 54 Drying
Secondary Rate 2.5 20 1300 57.7 30 30 130 54.2 30 30 130 56.2 30 30
390 60.5 drying Secondary Hold N/A 20 360 63.7 N/A 30 360 60.2 N/A
30 360 62.2 N/A 30 360 66.5 drying
i) Feasibility Assessment: Compounding and Reconstitution
[0369] To further evaluate the formulations upon reconstitution
following lyophilization according to the different lyophilization
cycles, liquid fill solutions were prepared and evaluated as
outlined below. For lyophilization cycles #1, #2, and #5, liquid
fill solutions were prepared comprising: [0370] a) Brincidofovir
stock solution at c(BCV)=10 mg/mL (pH adjustment to pH=8.0), [0371]
b) 10.times. buffer stock solutions or DI water (pH adjustment to
pH=8.0), and [0372] c) 12.5% (w/v) mannitol stock solution (pH
adjustment to pH=8.0) [0373] d) DI water.
[0374] For cycles #6 and #7, liquid fill solutions were prepared
comprising: [0375] a) brincidofovir (solid), [0376] b) mannitol
(solid), [0377] c) 10.times. arginine stock solution (pH adjustment
to pH=8.0), [0378] d) NaOH-solution (1M), and [0379] e) DI
water.
[0380] For cycles #1, #2, and #5 the applied reconstitution volumes
were determined by gravimetric analysis of the product vials before
and after lyophilization for each of the formulations. In case of
cycles #1 and #2 the specific measured volumes were applied. For
reconstitution of lyophilized products of cycle #5 an average
reconstitution volume was applied to all formulations. For the
lyophilized product of lyophilization cycle #7, the liquid fill
volume was scaled in accordance to the previously determined
difference between liquid fill and reconstitution volume.
[0381] After each lyophilization cycle the lyophilized products
were evaluated for their cake structure, uniformity and defects in
appearance. Following reconstitution with DI water, the appearance
of the solution during and after reconstitution (e.g. foaming
intensity, foam dissipation time), pH, and recovery of
brincidofovir were evaluated. The recovery of brincidofovir was
determined by HPLC analysis. The fraction of volatiles was
determined by thermogravimetric analysis (TGA). The results of the
reconstitution assessments are summarized in Table 30.
TABLE-US-00030 TABLE 30 Assessment of the drug product and
reconstitution process. Average Fraction Average Foam. Pre lyo Post
lyo Purity Cycle Form. Volatiles Reconst. dissip. time* c(BCV)
c(BCV) c(BCV) # # [%] Average Appear. Time [s] [min] pH [mg/mL]
[mg/mL] [%] 1 1 N/A Minor melt-back 50 7 7.8 N/A 10.43 N/A 2 N/A
Minor melt-back 80 45 7.8 N/A 10.53 N/A 3 N/A Minor melt-back 60
<1 7.9 N/A 10.33 N/A 4 N/A Cake collapse 45 35 7.9 N/A 9.83 N/A
5 N/A Cake collapse 90 <1 8.1 N/A 10.63 N/A 6 N/A Cake collapse
15 45 8.0 N/A 10.43 N/A 7 N/A Porous, uniform 45 3 8.4 N/A 10.63
N/A 8 N/A Cake collapse 60 60 8.3 N/A 10.33 N/A 2 1 0.3 Little
cracks 100 32 8.0 9.8 9.6 N/A 3 1.5 Porous, uniform 45 8 8.0 9.9
9.6 N/A 7 3.8 Minor melt-back 105 30 8.4 9.5 9.5 N/A 3 1 0.3 Little
cracks N/A N/A N/A N/A N/A N/A 3 2.0 Porous, uniform N/A N/A N/A
N/A N/A N/A 7 0.8 Minor melt-back N/A N/A N/A N/A N/A N/A 5 1 N/A
Minor cracks 45 >30 (minor 7.9 N/A 10.4 N/A foaming) 3 N/A
Porous, uniform 45 <20 8.0 N/A 10.2 N/A 3a N/A Porous, uniform
130 <20 7.9 N/A 10.1 N/A 3b N/A Porous, uniform 95 >30 (minor
7.9 N/A 10.0 N/A foaming) 3c N/A Porous, uniform 80 >30 (minor
7.9 N/A 10.0 N/A foaming) 7 N/A Minor melt-back 15 >30 8.2 N/A
10.2 N/A 6 1 4.2 Several cracks 40 >30 (minor 7.9 10.3 9.8 N/A
foaming) 3 1.0 Porous, uniform 30 <20 7.9 10.7 9.5 99.7 3a 0.3
Porous, uniform 91 >30 (minor 7.9 10.5 9.6 99.6 foaming) 7 3a
0.5 Porous, uniform 83 <20 8.0 10.7 9.9 N/A *Average value of
three vials. For cycle #1-2 the approximate dissipation time was
recorded (constant observation). For cycle #5-7 the foaming
intensity was evaluated at 10, 20 and 30 min after reconstitution.
<20 indicates that the foaming dissipation time was between the
10 and 20 min interval. <30 indicates that the foaming
dissipation time was between the 20 and 30 min interval. If foaming
still persisted over 30 min, the dissipation time was indicated by
>30 min.
ii) Process Development: Stability Studies of Lyophilized
Formulations--48 h Study
[0382] To evaluate the physical and chemical stability of the
reconstituted solutions the appearance and pH of each of
Formulations 1, 3, 3a, 3b, 3c, and 7, as well as recovery of c(BCV)
at the time of reconstitution (t.sub.0), after 1 day (t.sub.24h)
and after 2 days 4480 was assessed at ambient temperature. The
recovery of c(BCV) was determined via HPLC.
[0383] All tested formulations (Formulations #1, 3, 3a, 3b, 3c, and
7), were clear and colorless in appearance and yielded complete
recovery of brincidofovir within 48 h after reconstitution (i.e.,
no significant loss of brincidofovir, variations in recovery are
within the error range). Formulations 3 and 3a were found to
display faster dissipation of foam/bubbles than Formulations 3b,
and 3c (Table 33).
iii) Short-Term Stability Study: Stability Studies of Lyophilized
Formulations--2 Week Study
[0384] The stability of Formulations 1, 3, and 3a was assessed at
25.degree. C. and 60.degree. C. by evaluating appearance and pH of
each formulation, as well as recovery of c(BCV), at the time of
reconstitution (t.sub.0), after 2 days (t.sub.2 days), 7 days
(t.sub.7 days), 10 days (t.sub.10 days), and 14 days (t.sub.14
days). In addition, the foaming intensity was evaluated at 10, 20
and 30 minutes after reconstitution.
[0385] The recovery of brincidofovir was determined via HPLC.
Degradants observed in the HPLC chromatograms were identified as
the deamination product, likely Cidofovir, as well as additional
degradants No shift or changes in pH were associated with
degradation. Of the tested formulations, Formulation 3a showed the
least degree of degradation under the applied conditions with a
purity of 99.2% after 14 days at 60.degree. C. The results of the
experiments are summarized in Table 31, Table 32, and Table 36.
TABLE-US-00031 TABLE 31 Stability study results of the formulation
1 (200 mM Phosphate) Conditions Station Average Average Average
Observed BCV BCV temp. Cake Average Recon. Foam. Time c(BCV).sup.a
Reovery .sup.b Purity (.degree. C.) Day appearance Recon. Appear.
Time [s] [min]* pH (mg/mL) [%] [%] Pre lyo N/A N/A N/A N/A 7.9 10.3
0.4 N/A Pre lyo 25 0** Several cracks Clear, colorless 40 >30
(minor 7.9 9.8 100 N/A foaming) 2 Several cracks Clear, colorless
41 >30 (minor 8.0 9.9 101 N/A foaming) 7 Several cracks Clear,
colorless 33 >30 (minor 8.0 10.0 102 N/A foaming) 10 N/A N/A N/A
N/A N/A N/A N/A N/A 14 N/A N/A N/A N/A N/A N/A N/A N/A 60 0**
Several cracks Clear, colorless 40 >30 (minor 7.9 9.8 100 N/A
foaming) 2 Major collapse Clear, colorless 30 >30 (minor 8.0 9.8
100 N/A foaming) 7 Major collapse Clear, colorless 32 >30 (minor
8.0 9.6 98 N/A foaming) 10 N/A N/A N/A N/A N/A N/A N/A N/A 14 N/A
N/A N/A N/A N/A N/A N/A N/A *Average value of three vials. The
foaming intensity was evaluated at 10, 20 and 30 min after
reconstitution. <20 indicates that the foaming dissipation time
was between the 10 and 20 min interval. <30 indicates that the
foaming dissipation time was between the 20 and 30 min interval. If
foaming still persisted over 30 min, the dissipation time was
indicated by >30 min. .sup.aAverage value of the three
replicates of samples that received treatment. .sup.b BCV recovery
= c(BCV).sub.Day n/c(BCV).sub.Day 0 .times. 100%. **The sample for
t.sub.0 at T = 25.degree. C. and t.sub.0 at T = 60.degree. C. is
identical. Note: HPLC sample variability accounts for the assay
variability between samples. Vial to vial variability accounts for
the variability between drug product vials.
TABLE-US-00032 TABLE 32 Stability study results of the formulation
3 (200 mM Arginine) Conditions Station Average Average Average
Observed BCV BCV temp. Cake Average Recon. Foam. Time c(BCV).sup.a
Recovery.sup.b Purity (.degree. C.) Day appear. Recon. Appear. Time
[s] [min]* pH [mg/mL] [%] [%] Pre lyo N/A N/A N/A N/A 8.0 10.7 N/A
N/A 25 0** Uniform Clear, colorless 30 10 7.9 9.5 100 99.7 2
Uniform Clear, colorless 24 10 7.9 9.6 101 99.6 7 Uniform Clear,
colorless 24 <10 7.9 9.7 102 99.7 10 Uniform Clear, colorless 18
<10 8.0 9.9 104 99.8 14 Uniform Clear, colorless 16 <10 8.0
9.8 103 99.7 60 0** Uniform Clear, colorless 30 10 7.9 9.5 100 99.7
2 Uniform Clear, colorless 34 10 7.9 9.5 100 99.6 7 Uniform Clear,
colorless 20 10 7.9 9.6 101 99.0 10 Uniform Clear, colorless 18
<30 (minor 7.9 9.7 102 98.9 foaming) 14 Uniform Yellow tint 20**
<20 7.9 9.6 101 95.9 *Average value of three vials. The foaming
intensity was evaluated at 10, 20 and 30 min after reconstitution.
<20 indicates that the foaming dissipation time was between the
10 and 20 min interval. <30 indicates that the foaming
dissipation time was between the 20 and 30 min interval. If foaming
still persisted over 30 min, the dissipation time was indicated by
>30 min. .sup.aAverage value of the three replicates of samples
that received treatment. .sup.bBCV recovery = c(BCV).sub.Day
n/c(BCV).sub.Day 0 .times. 100%. **The sample for t.sub.0 at T =
25.degree. C. and t.sub.0 at T = 60.degree. C. is identical. Note:
HPLC sample variability accounts for the assay variability between
samples. Vial to vial variability accounts for the variability
between drug product vials.
TABLE-US-00033 TABLE 33 Stability study results of the formulation
#3a (100 mM Arginine) Conditions Station Average Average Average
Observed BCV BCV temp. Cake Average Recon. Foam. Time c(BCV).sup.a
Recovery.sup.b Purity (.degree. C.) Day appear Recon. Appear. Time
[s] [min]* pH (mg/mL) (%) (%) Pre lyo N/A N/A N/A N/A 7.9 10.5 N/A
N/A 25 0** Uniform Clear, colorless 91 >30 (minor 7.9 9.6 100
99.6 foaming) 2 Uniform Clear, colorless N/A N/A N/A N/A N/A N/A 7
Uniform Clear, colorless N/A N/A N/A N/A N/A N/A 10 Uniform Clear,
colorless 80 >30 (minor 8.0 10.0 104 99.7 foaming) 14 Uniform
Clear, colorless 65 >30 (minor 8.0 9.9 103 99.7 foaming) 60 0**
Uniform Clear, colorless 91 >30 (minor 7.9 9.6 100 99.6 foaming)
2 Uniform Clear, colorless N/A N/A N/A N/A N/A N/A 7 Uniform Clear,
colorless N/A N/A N/A N/A N/A N/A 10 Uniform Clear, colorless 49
>30 (minor 7.9 9.8 102 99.8 foaming) 14 Uniform Clear, colorless
26 >30 (minor 8.0 9.8 102 99.2 foaming) .sup.aAverage value of
the three replicates of samples that received treatment. .sup.bBCV
recovery = c(BCV).sub.Day n /c(BCV).sub.Day 0 .times. 100%. **The
sample for t.sub.0 at T = 25.degree. C. and t.sub.0 at T =
60.degree. C. is identical. Note: HPLC sample variability accounts
for the assay variability between samples. Vial to vial variability
accounts for the variability between drug product vials
iv) Preparation of Drug Product Batches for Stability Testing
[0386] Preparation of Pre-Lyophilization Formulations: Formulation
3a was compounded according to the following procedure: The pH of a
1M buffer solution of arginine in DI water was adjusted to 8 using
Hydrochloric acid (35-37%) under pH-control before Q.S. Then
Mannitol was added to the buffer solution followed by BCV. The pH
of the resulting solution was adjusted to 8 using sodium hydroxide.
DI water was used to Q.S. the solution in separate volumetric
flasks. The two solutions were then mixed and sterile filtered to
yield the desired Formulation 3a. The procedure yielded Formulation
3a with an observed c(BCV) of 9.9 mg/mL.
[0387] Preparation of Lyophilized Formulations:
[0388] Under best clean conditions approximately 200 vials of
liquid Formulation 3a, made according to the procedure outlined
above, were vials were stoppered in lyophilization position and
subjected to lyophilization according to lyophilization cycle #7.
The resulting lyophilized product was tested for appearance,
reconstitution appearance, pH, and recovery of brincidofovir. The
recovery of brincidofovir was determined via HPLC, and
thermogravimetric analysis. The results of the testing of
appearance, reconstitution appearance and recovery of brincidofovir
in the lyophilized formulation prepared according to the procedure
outlined herein are summarized in Table 34.
TABLE-US-00034 TABLE 34 Appearance, reconstitution and recovery of
formulations batch pre and post Lyophilization. Foam. dissip.
Average Fraction Reconst. time c(BCV).sup.b Condition Volatiles
Appear. time [s] [min]* pH [mg/mL] Pre lyo. N/A N/A N/A N/A 8.0
10.7 Release 0.5 Porous, 83 <20 8.0 9.9 *Average value of three
vials. The foaming intensity was evaluated at 10, 20 and 30 min
after reconstitution. <20 indicates that the foaming dissipation
time was between the 10 and 20 min interval. <30 indicates that
the foaming dissipation time was between the 20 and 30 min
interval. If foaming still persisted over 30 min, the dissipation
time was indicated by >30 min. .sup.aScan range = 25.degree. C.
to 150.degree. C., ramp rate = 10.degree. C./min. .sup.bAverage
value of the three (3) replicates of samples that received
treatment. indicates data missing or illegible when filed
v) Compatibility of Lyophilized Formulations
[0389] The liquid fill solution for the lyophilized Formulation 3a
was tested for compatibility with infusion vehicles as well as a
series of materials from the manufacturing and clinical stage. The
results of the infusion vehicle compatibility testing are
summarized in Table 38. No precipitation, pH shift or significant
loss in recovery of brincidofovir were observed at the end of the
study.
Infusion Vehicle Compatibility
[0390] The Formulation 3a was diluted with the infusion vehicle (5%
dextrose) solution to yield c(BCV)=0.5 mg/mL and 1.0 mg/mL
solutions. The appearance, pH, and brincidofovir recovery
(determined via HPLC) were evaluated at the time of preparing the
test solution (t.sub.0), and after 2 h (t.sub.2h), 8 h (t.sub.8h),
and 24 h (t.sub.24h)
TABLE-US-00035 TABLE 35 Results of the infusion vehicle
compatibility assessment. c(BCV) in infusion Time Observed BCV
vehicle point Measured c(BCV).sup.a Recovery.sup.a,b (mg/mL) (h)
Appearance pH (mg/mL) (%) 0.5 t.sub.0 Clear, 7.9 0.492 100
Colorless t.sub.2 h Clear, 7.9 0.493 100 Colorless t.sub.8 h Clear,
7.7 0.492 100 Colorless t.sub.24 h Clear, 7.7 0.492 100 Colorless
1.0 t.sub.0 Clear, 7.9 0.967 100 Colorless t.sub.2 h Clear, 7.8
0.966 100 Colorless t.sub.8 h Clear, 7.8 0.969 100 Colorless
t.sub.24 h Clear, 7.7 0.967 100 Colorless .sup.aAverage value of
the three replicates. .sup.bBCV recovery = c(BCV).sub.Hour
n/c(BCV).sub.Hour 0 .times. 100%. The value for BCV recovery at
t.sub.0 was defined as 100%.
Sterilization Filter Compatibility
[0391] The liquid Formulation 3a was tested for compatibility with
two syringe filters for aseptic processing. 10 mL of the liquid
Formulation 3a, were filtered through one of the filters a (0.2
.mu.m, 25 mm, syringe filter, Posidyne.RTM. membrane) orb (0.2
.mu.m, 25 mm, syringe filter, Supor.RTM. membrane (PES)), and the
first and last 10% filtrate were evaluated for appearance, pH and
recovery of brincidofovir.
[0392] The results of the evaluation are summarized in Table 36. No
precipitation, pH shift or significant loss in brincidofovir
recovery were observed. Samples appeared clear and colorless.
Degradant peaks were not observed.
TABLE-US-00036 TABLE 36 Results of the sterilization filter
compatibility assessment of formulation 3a. Observed Filter
Measured c(BCV).sup.a BCV membrane Conditions Appearance pH (mg/mL)
Recovery.sup.a,b Posidyne .RTM. Pre filtration Clear, 7.9 10.25 100
Colorless First 10% filtrate Clear, 7.9 10.16 99 volume Colorless
Last 10% filtrate Clear, 7.9 10.12 99 volume Colorless PES .RTM.
Pre filtration Clear, 7.9 10.25 100 Colorless First 10% filtrate
Clear, 7.9 10.21 100 volume Colorless Last 10% filtrate Clear, 7.9
10.15 99 volume Colorless .sup.aAverage value of the three
replicates. .sup.bBCV recovery = c(BCV).sub.Hour n/c(BCV).sub.Pre
filling .times. 100%. The value for BCV recovery at t.sub.0 was
defined as 100%.
Drug Product Vial and Stopper Compatibility
[0393] The liquid Formulation 3a was tested for compatibility with
vials and stoppers. The vials were filled with 5 mL of the liquid
Formulation 3a, stoppered, crimped, and stored for 6 hours at
ambient conditions in upright and inverted orientations. The vials
used for testing were clear, 20 mL, Class A borosilicate glass
serum vials, and the stoppers were 20 mm Novapure.RTM. stoppers,
with FluroTec.RTM. coating.
[0394] The results of the study are summarized in Table 37. No
precipitation, pH shift or significant loss in brincidofovir
recovery was observed throughout the study. No degradant peaks were
observed in HPLC.
TABLE-US-00037 TABLE 37 Results of the drug product vial and
stopper compatibility assessment. Observed BCV Measured
c(BCV).sup.a Recovery.sup.a,b Conditions Appearance pH (mg/mL) (%)
Pre filling Clear, 7.9 10.25 100 Colorless Upright, 6 h Clear, 7.9
10.15 99 Colorless Inverted, 6 h Clear, 7.9 10.20 100 colorless
.sup.aAverage value of the three replicates. .sup.bBCV recovery =
c(BCV).sub.Hour n/c(BCV).sub.Pre filling .times. 100%. The value
for BCV recovery at t.sub.0 was defined as 100%.
Infusion Bag Compatibility
[0395] The liquid Formulation 3a was tested for compatibility with
a 100 mL infusion bag. Solutions of Formulation 3a, in a 5%
dextrose injection vehicle, were prepared at two different
formulation strengths (1.0 mg/mL and 0.1 mg/mL brincidofovir), and
stored in the infusion bag at ambient conditions. Samples form the
infusion bags and from an externally diluted standard were
evaluated for appearance, pH and brincidofovir recovery (determined
via HPLC) at the beginning of the study (t.sub.0), after 8 h
(t.sub.8h), and after 24 h (t.sub.24h). The results of the infusion
bag compatibility assessment are summarized in Table 38.
TABLE-US-00038 TABLE 38 Results of the infusion bag compatibility
assessment c(BCV) Bag BCV in testing repli- Mea- Observed Recov-
infusion cate Condi- Appear- sured c(BCV).sup.a ery.sup.a,b bag #
tions ance pH (mg/mL) (%) 0.1 1 External Clear, 7.2 0.0909 101
dilution Colorless t.sub.0 Clear, 7.3 0.0896 100 Colorless t.sub.1
h Clear, 7.1 0.0893 100 Colorless t.sub.8 h Clear, 7.3 0.0909 101
Colorless t.sub.24 h Clear, 7 0.0906 101 Colorless 2 t.sub.0 N/A
N/A 0.0886 100 t.sub.24 h Clear, 7 0.0883 100 Colorless 3 t.sub.0
N/A. N/A 0.111 100 t.sub.24 h Clear, 7 0.0891 81 Colorless 1 1
External Clear, 7.9 0.927 101 dilution Colorless t.sub.0 Clear, 7.9
0.915 100 Colorless t.sub.1 h Clear, 7.7 0.908 99 Colorless t.sub.8
h Clear, 7.8 0.914 100 Colorless t.sub.24 h Clear, 7.7 0.915 100
Colorless 2 t.sub.0 N/A N/A 1.108 100 t.sub.24 h Clear, 7.7 1.106
100 Colorless 3 t.sub.0 N/A. N/A 0.903 100 t.sub.24 h Clear, 7.7
0.903 100 Colorless .sup.aAverage value of the three replicates.
.sup.bBCV recovery = c(BCV).sub.Hour n/c(BCV).sub.Hour 0 .times.
100%. The value for BCV recovery at t.sub.0 was defined as
100%.
[0396] The samples appeared clear and colorless. For both sample
concentrations, no significant loss in brincidofovir recovery was
observed over 24 h. For one bag replicate at c(BCV)=0.1 mg/mL an
apparent loss of brincidofovir (19%) is observed; It was further
observed that a dilution of the brincidofovir formulation into the
infusion vehicle, yielding a concentration of c(BCV)=0.1 mg/mL
caused a significant pH shift to pH=7. No degradation was observed
in the experiment.
Infusion System Compatibility
[0397] The brincidofovir formulation (10 mg/mL brincidofovir, 100
mM arginine, 5% mannitol, pH=8.0) was tested for compatibility with
two infusion systems (Baxter, Non-DEHP CONTINU-FLO; B Braun, Rate
Flow.RTM. regulator IV set). The liquid formulation was diluted
into infusion bags as previously described (tested final
concentrations: 1.0 mg/mL and 0.1 mg/mL) and an aliquot was
collected from the infusion bag for analysis of brincidofovir
recovery after 24 h (t0, bag). The infusion systems were then
attached to the infusion bag and manually filled with solution from
the infusion bag. Aliquots were collected as an end-of-line sample
for analysis (t0, eol). The IV lines were closed between time
points with the infusate resting within the infusion system and
stored at ambient temperature. At testing time points,
approximately 17 mL (approx. volume of the IV system) of infusate
solution were drained, which led to a replacement of the solution
in the IV system from the bag reservoir. Following this
replacement, another end-of-line sample was collected for the
assessments (t10 min, eol, t3 h, eol, and t6 h, eol). The samples
were evaluated for appearance, pH, and brincidofovir recovery. The
results of infusion system compatibility testing are summarized in
Table 42.
[0398] An initial adsorption/saturation effect was observed for
both IV systems at low c(BCV), indicated by a loss in brincidofovir
recovery of 7-11%. This loss partially recovered over the following
6 h. The appearance of degradants was not observed over the course
of this study. As previously noted, samples at c(BCV)=0.1 mg/mL
showed a pH-shift by approximately one pH-unit to pH=7 upon
dilution with 5% dextrose solution. The samples appeared clear and
colorless over the course of the study.
TABLE-US-00039 TABLE 39 Results of the infusion system
compatibility assessment. c(BCV) in BCV Testing infusion Observed
Recov- infusion bag Appear- c(BCV).sup.a ery.sup.a,b system (mg/mL)
Conditions ance pH (mg/mL) (%) Baxter 0.1 t.sub.0, bag Clear, 7
0.0883 100 Colorless t.sub.0, eol Clear, 6.9 0.0826 93 Colorless
t.sub.10 min, eol Clear, 6.9 0.0868 98 Colorless t.sub.3 h, eol
Clear, 7 0.0876 99 Colorless t.sub.6 h, eol Clear, 7 0.0863 98
Colorless 1 t.sub.0, bag Clear, 7.7 1.106 100 Colorless t.sub.0,
eol Clear, 7.7 1.095* 99 Colorless t.sub.10 min, eol Clear, 7.7
1.121 101 Colorless t.sub.3 h, eol Clear, 7.7 1.136 103 Colorless
t.sub.6 h, eol Clear, 7.7 1.107 100 Colorless B Braun 0.1 t.sub.0,
bag Clear, 7 0.0891 100 Colorless t.sub.0, eol Clear, 6.9 0.0796 89
Colorless t.sub.10 min, eol Clear, 6.9 0.0854 96 Colorless t.sub.3
h, eol Clear, 7 0.0849 95 Colorless t.sub.6 h, eol Clear, 6.9
0.0844 95 Colorless 1 t.sub.0, bag Clear, 7.7 0.903 100 Colorless
t.sub.0, eol Clear, 7.7 0.9 100 Colorless t.sub.10 min, eol Clear,
7.6 0.908 101 Colorless t.sub.3 h, eol Clear, 7.7 0.909 101
Colorless t.sub.6 h, eol Clear, 7.7 0.909 101 Colorless
.sup.aAverage value of the three replicates. .sup.bBCV recovery =
c(BCV).sub.Hour n/c(BCV).sub.Hour 0 .times. 100%. The value for BCV
recovery at t.sub.0 was defined as 100%.
Example 6--Stability Testing
[0399] The bulk liquid formulations and lyophilized formulations of
the disclosure were further tested for stability. A liquid
formulation comprising arginine, mannitol, and brincidofovir at a
concentration of 10 mg/mL was kept in a 10 mL glass vial, stoppered
(rubber stopper or 20 mm serum stopper) and sealed (aluminum
seals), and kept at about 5.degree. C. or about 25.degree. C. for
up to 12 months. The appearance, pH and brincidofovir content of
the stored formulation were monitored along with impurities and
particulate matter, at various time intervals (i.e., after 2 weeks,
after 1 month, after 2 months, after 3 months, after 6 months,
after 9 months and after 12 months). Impurities were evaluated via
HPLC and are identified herein by their relative retention time.
The same experiment was repeated for a solution of the lyophilized
powder, comprising 10 mg/mL of brincidofovir. For the lyophilized
formulations, a 20 mL glass vial with a 20 mm rubber stopper was
used. Furthermore, the appearance of the lyophilized powder (i.e.,
the lyophilization cake) was recorded.
[0400] The results of the stability study of the bulk liquid
formulations at 5.degree. C. and 25.degree. C. are summarized in
Tables 40A and 40B, respectively. The results of the stability
study of the lyophilized formulations at 5.degree. C. and
25.degree. C. are summarized in Tables 41A, 41B, and 42,
respectively.
TABLE-US-00040 TABLE 40A Stability of liquid formulation at
5.degree. C. (.+-.3.degree. C.) Specifications Acceptance Stability
Intervals Test Criteria Initial 2 week 1 month 2 month 3 month 6
month 9 month 12 month Appearance Clear, colorless; Conforms
Conforms Conforms Conforms Conforms Conforms Does not Conforms free
of visible conform particulates Brincidofovir 90.0 to 110.0% 100.3
100.7 100.4 100.0 101.2 101.3 99.9 99.8 Content - Average Drug
Related Report Results <0.05 <0.05 <0.05 0.05 0.06 0.07
0.12 0.15 Impurities - RRT 0.80 Drug Related Report Results
<0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05
<0.05 Impurities - RRT 1.13 Drug Related Report Results <0.05
<0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05
Impurities - RRT 1.19 Drug Related Report Results <0.05 <0.05
<0.05 <0.05 <0.05 <0.05 <0.05 <0.05 Impurities -
RRT 1.34 Drug Related Report Results <0.05 <0.05 <0.05
<0.05 <0.05 <0.05 <0.05 <0.05 Impurities - RRT 1.42
Drug Related Report Results <0.05 <0.05 <0.05 0.05 0.06
0.07 0.12 0.15 Impurities - Total Impurities pH - Average 7.5 to
8.5 8.0 8.0 7.9 7.8 8.0 8.0 7.8 7.9 Particulate .ltoreq.6000 148
N/T 274 N/T 181 135 N/T N/T Matter - 10 .mu.m Particulate
.ltoreq.600 1 N/T 1 N/T 2 2 N/T N/T Matter - 25 .mu.m
TABLE-US-00041 TABLE 40B Stability of liquid formulation at
25.degree. C. (.+-.2.degree. C.) and 60% relative humidity (.+-.5%
relative humidity) Specifications Acceptance Stability Intervals
Test Criteria Initial 2 week 1 month 2 month 3 month 6 month 12
month Appearance Clear, colorless; Conforms Conforms Conforms
Conforms Conforms Conforms Conforms free of visible particulates
Brincidofovir 90.0 to 110.0% 100.3 100.9 100.1 99.4 98.9 99.9 97.5
Content - Average Drug Related Report Results <0.05 0.10 0.21
0.37 0.54 1.0 2.0 Impurities - RRT 0.80 Drug Related Report Results
<0.05 <0.05 0.06 0.07 0.12 0.29 0.60 Impurities - RRT 1.13
Drug Related Report Results <0.05 <0.05 0.05 <0.05
<0.05 <0.05 <0.05 Impurities - RRT 1.19 Drug Related
Report Results <0.05 <0.05 <0.05 <0.05 0.05 <0.05
<0.05 Impurities - RRT 1.34 Drug Related Report Results <0.05
<0.05 <0.05 <0.05 <0.05 <0.05 0.05 Impurities - RRT
1.42 Drug Related Report Results <0.05 0.10 0.32 0.45 0.71 1.3
2.7 Impurities - Total Impurities pH - Average 7.5 to 8.5 8.0 8.0
7.9 7.9 8.0 8.0 7.9 Particulate .ltoreq.6000 148 N/T N/T N/T N/T
325 N/T Matter- 10 .mu.m Particulate .ltoreq.600 1 N/T N/T N/T N/T
7 N/T Matter - 25 .mu.m
TABLE-US-00042 TABLE 41A Stability of lyophilized formulation at
5.degree. C. (.+-.3.degree. C.) Specifications Acceptance Stability
Intervals Test Criteria Initial 2 week 1 month 2 month 3 month 6
month 9 month 12 month Appearance of Report White White White White
White White White White Lyophilized Results porous porous uniform
uniform porous cake in porous porous Cake-Appearance uniform
uniform cake with cake with uniform the uniform uniform structure
structure some deposited structure bottom of structure structure
with some with some deposited material with some the bottle with
some with some solid solid material above on solid solid solid
deposits on deposits above the wall. deposits deposits deposits
vial above above the One cake above the above the above the the
cake cake on at -20.degree. C., cake in cake in cake on the vials.
both the vial. the vial. the vial. The vials cakes at 5.degree. had
some C., and cake one cake particulates at 40.degree. C., are on
the removable stoppers from the of the bottom of vials. the vial.
Completeness Meets the N/T N/T N/T Conforms Not tested Conforms
Conforms Conforms and Clarity of requirements Solution of USP
<1> Brincidofovir 90.0 to 110.0% 100.3 100.4 98.6 98.7 98.2
100.1 98.4 98.2 Content - Average Drug Related Report None None
None None None None None None Impurities - Results Detected
Detected Detected Detected Detected Detected Detected Detected
Synergi imp Drug Related Report None None None None None None None
None Impurities - Results Detected Detected Detected Detected
Detected Detected Detected Detected XBridge imp Drug Related Report
None None None None None None None None Impurities - Total Results
Detected Detected Detected Detected Detected Detected Detected
Detected Impurities = XBridge + Synergi pH 7.5 to 8.5 7.9 7.9 7.9
7.9 7.9 7.8 7.9 7.9 Reconstitution Report 90 N/T N/T 136 129 105
152 160 Time - Prep 1 Results Reconstitution Report 95 N/T N/T 160
90 125 148 130 Time - Prep 2 Results Particulate .ltoreq.6000 188
N/T 52 N/T N/T 84 N/T N/T Matter - 10 .mu.m Particulate .ltoreq.600
4 N/T 0 N/T N/T 3 N/T N/T Matter - 25 .mu.m Moisture Report N/T N/T
N/T 0.3 0.8 0.5 N/T 0.5 Average Results
TABLE-US-00043 TABLE 41B Stability of lyophilized formulation at
25.degree. C. (.+-.2.degree. C.) and 60% relative humidity (.+-.5%
relative humidity) Specifications Acceptance Stability Intervals
Test Criteria Initial 2 week 1 month 3 month 6 month 9 month 12
month Appearance of Report White White White White White cake White
White Lyophilized Results porous porous uniform porous in the
porous porous Cake-Appearance uniform uniform cake with uniform
bottom of uniform uniform structure structure some structure the
bottle structure structure with some with some deposited with some
with some with some solid solid material solid solid solid deposits
on deposits above deposits deposits deposits vial above above the
above the above the above the the cake cake on the cake on cake on
cake on vials. The the vial. the vial. the vial. vials had some
cake particulates on the stoppers of the vials. Completeness Meets
the N/T N/T N/T Not tested Conforms Conforms Conforms and Clarity
of requirements Solution of USP <1> Brincidofovir 90.0 to
110.0% 100.3 98.0 98.5 98.4 101.2 98.2 98.5 Content - Average Drug
Related Report None None None None None None None Impurities -
Results Detected Detected Detected Detected Detected Detected
Detected Synergi imp Drug Related Report None None None None None
None None Impurities - Results Detected Detected Detected Detected
Detected Detected Detected XBridge imp Drug Related Report None
None None None None None None Impurities - Total Results Detected
Detected Detected Detected Detected Detected Detected Impurities =
XBridge + Synergi pH 7.5 to 8.5 7.9 7.9 7.9 7.9 7.8 7.9 7.9
Reconstitution Report 90 N/T N/T 92 111 147 177 Time - Prep 1
Results Reconstitution Report 95 N/T N/T 91 103 135 189 Time - Prep
2 Results Particulate .ltoreq.6000 188 N/T N/T N/T 83 N/T N/T
Matter-10 .mu.m Particulate .ltoreq.600 4 N/T N/T N/T 2 N/T N/T
Matter-25 .mu.m Moisture Report N/T N/T N/T 0.6 0.5 0.6 0.6 Average
Results
TABLE-US-00044 TABLE 42 Stability of lyophilized formulation at
40.degree. C. (.+-.2.degree. C.) and 60% relative humidity (.+-.5%
relative humidity) Specifications Acceptance Stability Intervals
Test Criteria Initial 2 week 1 month 2 month 3 month 6 month
Appearance of Report White White White White White White cake in
Lyophilized Results porous porous uniform uniform porous the bottom
of Cake-Appearance uniform uniform cake with cake with uniform the
bottle structure structure some deposited structure with some with
some deposited material with some solid solid material above on
solid deposits deposits above the wall. deposits on vial above the
One cake above the above the cake on at -20.degree. C., cake in
cake the vials. both the vials. The vials cakes at 5.degree. had
some C., and cake one cake particulates at 40.degree. C. are on the
removable stoppers of from the the vials. bottom of the vial.
Completeness Meets the Not tested Not tested Not tested Conforms
Not tested Conforms and Clarity of requirements per protocol per
protocol Solution of USP <1> Brincidofovir 90.0 to 110.0%
100.3 97.5 99.1 98.8 98.5 99.6 Content - Average Drug Related
Report None None None None None None Impurities - Results detected
detected detected detected detected detected Synergi imp Drug
Related Report None None None None None None Impurities - Results
detected detected detected detected detected detected XBridge imp
Drug Related Report None None None None None None Impurities -
Total Results detected detected detected detected detected detected
Impurities = XBridge + Synergi pH 7.5 to 8.5 7.9 7.9 7.9 7.9 7.9
7.8 Reconstitution Report 90 Not tested Not tested 135 82 99 Time -
Prep 1 Results per protocol per protocol Reconstitution Report 95
Not tested Not tested 126 77 106 Time - Prep 2 Results per protocol
per protocol Particulate .ltoreq.6000 188 Not tested Not tested Not
tested Not tested Not tested Matter - 10 .mu.m per protocol per
protocol per protocol per protocol per protocol Particulate
.ltoreq.600 4 Not tested Not tested Not tested Not tested Not
tested Matter - 25 .mu.m per protocol per protocol per protocol per
protocol per protocol Moisture Report Not tested Not tested Not
tested 0.6 0.6 Average Results per protocol per protocol per
protocol
Example 7--Determination of Hemocompatibility in Rat and Human
Whole Blood Introduction
[0401] Each concentration of test item for the rat was mixed
separately in a 1:4 ratio with rat whole blood while each
concentration of test item for the human was mixed separately in a
1:4 ratio with human whole blood. These samples plus control
samples [vehicle control (vehicle+whole blood, 1 for rat and 1 for
humans), positive control (1% Saponin+whole blood), untreated whole
blood control and negative control (saline+whole blood) were
analyzed for hemolysis (supernatant hemoglobin concentration).
[0402] The rat and human whole blood samples were incubated in test
or control items at 37.+-.1.degree. C. for 20 minutes and then
immediately (within 2 minutes) centrifuged at 3500.+-.100 rpm (2740
g) at room temperature for 5-10 minutes. The supernatant was
extracted and subjected to a hemoglobin analysis using the
cyanmethemoglobin method employed on the Siemens Advia 120
hematology analyzer. If the test item has a potential for causing
hemolysis, then hemoglobin released from lysed red blood cells into
the plasma supernatant can be quantified via the reaction of heme
iron in solution being oxidized from a ferrous to a ferric state in
the presence of potassium cyanide. The reaction results in the
formation of methemoglobin, which then combines with cyanide to
form a stable cyanmethemoglobin. When measured
spectrophotometrically at 546 nm the absorbance of
cyanmethemoglobin follows Beer's Law and is directly proportional
to the concentration of hemoglobin released into solution. In
addition, the formulated test item at each concentration assessed
was mixed in a 1:4 ratio with saline and then directly analyzed to
ensure that the test item formulations did not interfere with the
Siemens Advia measurement of hemoglobin.
Materials and Methods
[0403] The testing was conducted in 2 phases as shown below:
TABLE-US-00045 TABLE 43 Phase 1, Brincidofovir in Rat Blood Rat
Test Item Positive Tube Blood* (BCV).sup.a Vehicle.sup.b
Control.sup.c Saline.sup.d 1.sup.e + +0.2, 0.5 and - - - 1.6 mg/mL
2.sup.f - +0.2, 0.5 and - - + 1.6 mg/mL 3.sup.g + - + - - 4.sup.h +
- - + - 5.sup.i + - - - - 6.sup.j + - - - + *1.0 mL added to Tubes
1 (separate tube for each concentration) and Tubes 3-6 .sup.a250
.mu.L added to Tubes 1-2 (separate tube for each concentration)
.sup.b250 .mu.L added to Tube 3 .sup.c250 .mu.L added to Tube 4
.sup.d1.0 mL added to Tube 2; 250 .mu.L added to Tube 6 .sup.eTest
item assay for hemolysis: Tube 1A contained 0.2 mg/mL Tube 1B
contained 0.5 mg/mL Tube 1C contained 1.6 mg/mL .sup.fTest item
interference control: Tube 2A contained 0.2 mg/mL Tube 2B contained
0.5 mg/mL Tube 2C contained 1.6 mg/mL .sup.gVehicle control for
hemolysis .sup.hPositive control for hemolysis; 1% Saponin was used
as a positive control .sup.iUntreated whole blood control for
hemolysis .sup.jNegative control for hemolysis + Added to the tube
- Not added to the tube Note: Tubes 3-6 for each phase were run
once per phase.
TABLE-US-00046 TABLE 44 Phase 2, Brincidofovir in Human Blood Human
Test Item Positive Tube Blood* (BCV).sup.a Vehicle.sup.b
Control.sup.c Saline.sup.d 1.sup.e + +0.2, 0.5 and - - - 1.6 mg/mL
2.sup.f - +0.2, 0.5 and - - + 1.6 mg/mL 3.sup.g + - + - - 4.sup.h +
- - + - 5.sup.i + - - - - 6.sup.j + - - - + *1.0 mL added to Tubes
1 (separate tube for each concentration) and Tubes 3-6 .sup.a250
.mu.L added to Tubes 1-2 (separate tube for each concentration)
.sup.b250 .mu.L added to Tube 3 .sup.c250 .mu.L added to Tube 4
.sup.d1.0 mL added to Tube 2; 250 .mu.L added to Tube 6 .sup.eTest
item assay for hemolysis: Tube 1A contained 0.2 mg/mL Tube 1B
contained 0.5 mg/mL Tube 1C contained 1.6 mg/mL .sup.fTest item
interference control: Tube 2A contained 0.2 mg/mL Tube 2B contained
0.5 mg/mL Tube 2C contained 1.6 mg/mL .sup.gVehicle control for
hemolysis .sup.hPositive control for hemolysis; 1% Saponin was used
as a positive control .sup.iUntreated whole blood control for
hemolysis .sup.jNegative control for hemolysis + Added to the tube
- Not added to the tube Note: Tubes 3-6 for each phase were run
once per phase.
Control Item (Vehicle)
[0404] The control items for dose formulations was 2 sodium
phosphate buffer solution (400 mM, pH=8.0); 1.0N sodium hydroxide
solution; sterilized water for injection; and 5% dextrose
solution.
Test Preparation and Analysis
[0405] Brincidofovir injectable liquid stock formulation was
prepared to 15 mg/mL brincidofovir, compounded from stock solutions
inclusive of three (3) steps: (1) 2.times. sodium phosphate buffer
solution (400 mM, pH=8.0) was prepared; (2) 1.0 N sodium hydroxide
solution was purchased; brincidofovir injectable liquid stock
formulation (15 mg/mL brincidofovir, 200 mM sodium phosphate,
pH=8.0) from solid brincidofovir, 1.0 N sodium hydroxide solution,
2.times. sodium phosphate buffer solution and WFI (water for
injection) was prepared.
[0406] The brincidofovir injectable liquid stock formulation is
stable for 28 days under refrigerated conditions (2 to 8.degree.
C.). Brincidofovir injectable liquid stock formulation was diluted
with 5% dextrose to yield the desired dose concentration.
[0407] Fresh dose formulations were prepared once on the day of
testing. Dose formulations were refrigerated (2 to 8.degree. C.)
when not in use.
[0408] Saponin was diluted with saline to the appropriate
concentration on the day of testing.
Test System
[0409] Whole blood (.about.20 mL) from rats (samples from four
animals were pooled for testing) was collected in presence of
K.sub.2EDTA anticoagulant and inverted to mix.
[0410] Human whole blood (.about.20 mL) was collected for use in
this study from one human volunteer in the presence of K2EDTA
anticoagulant.
[0411] The blood samples were held for no more than 4 hours at room
temperature following collection, for use in hemolytic potential
testing in this study.
Test Methodology
[0412] Each concentration of test item for the rat was mixed
separately in a 1:4 ratio with rat whole blood while each
concentration of test item for the human was mixed separately in a
1:4 ratio with human whole blood. These samples plus control
samples [vehicle control (vehicle+whole blood, 1 for rat and 1 for
humans), positive control (1% Saponin+whole blood), untreated whole
blood control and negative control (saline+whole blood) were
analyzed for hemolysis (supernatant hemoglobin concentration).
[0413] The rat and human whole blood samples were incubated in test
or control items at 37.+-.1.degree. C. for 20 minutes and then
immediately (within 2 minutes) centrifuged at 3500.+-.100 rpm (2740
g) at room temperature for 5-10 minutes. The supernatant was
extracted and subjected to a hemoglobin analysis using the
cyanmethemoglobin method employed on the Siemens Advia 120
hematology analyzer. Without wishing to be bound by theory, if the
test item has a potential for causing hemolysis, then hemoglobin
released from lysed red blood cells into the plasma supernatant can
be quantified via the reaction of heme iron in solution being
oxidized from a ferrous to a ferric state in the presence of
potassium cyanide. The reaction results in the formation of
methemoglobin, which then combines with cyanide to form a stable
cyanmethemoglobin. When measured spectrophotometrically at 546 nm
the absorbance of cyanmethemoglobin follows Beer's Law and is
directly proportional to the concentration of hemoglobin released
into solution.
[0414] In addition, the formulated test item at each concentration
assessed was mixed in a 1:4 ratio with saline and then directly
analyzed to ensure that the test item formulations did not
interfere with the Siemens Advia measurement of hemoglobin.
[0415] The testing was conducted in 2 phases.
[0416] Two independent analytical runs composing 1 batch were
performed using whole blood from 4 normal rats and 1 human donor.
The R&D Systems whole blood quality control sets were run
before and after the batch to verify operation performance of the
instrument using established criteria for this control set per the
stated manufacturer's ranges for the control material. An
independent (Pointe Scientific) set of Low, Mid, and High controls
containing hemoglobin in solution was run once to verify the
control rank order of the measurement of hemoglobin in solution
phase. The negative and positive control, and test samples were run
in singlet.
Hemolytic Potential Criteria
Run Criteria:
[0417] The 1% Saponin positive control must demonstrate hemoglobin
value>(Negative Control+0.5 g/dL) for the assay run to be
considered acceptable.
[0418] Untreated whole blood must demonstrate hemoglobin
values<(Negative Control+0.5 g/dL).
[0419] Saline+untreated whole blood (Negative Control) must
demonstrate hemoglobin values <0.5 g/dL.
[0420] R&D Systems whole blood control set should be within the
defined manufacturer's target range.
[0421] The Pointe Scientific liquid hemoglobin controls have no
established target range for the ADVIA instrument but should
demonstrate a rank order in response (based on concentration) of
High>Mid>Low.
Sample Analysis Criteria
[0422] The test item+saline sample must demonstrate hemoglobin
value<(Negative Control+0.5 g/dL) for the assay run to be
considered valid.
[0423] The vehicle control sample must demonstrate hemoglobin
value<(Negative Control+0.5 g/dL) for the assay to be considered
valid
[0424] The (Pointe Scientific) liquid QC control set containing
hemolysates prepared from human erythrocytes ranging from Low, Mid,
and High concentrations were used to verify the ability of the
Advia to measure liquid hemolysates using the Advia
cyanmethemoglobin procedure. The Low, Mid, and High liquid controls
from Phase 1 returned values of: 7.9, 11.7, and 15.6 g/dL,
respectively, as measured on the Siemens Advia. The Low, Mid, and
High liquid controls from Phase 2 returned values of: 8.1, 11.9,
and 15.6 g/dL, respectively, as measured on the Siemens Advia. The
rank order of the control sets meets the protocol criteria.
[0425] The (R&D System) whole blood quality controls were used
to verify the operational performance of the Siemens Advia 120
hematology instrument. The QC values recorded in the raw data were
within the defined manufacturer's acceptable ranges for the Advia
instrument.
[0426] The amphipathic glycoside Saponin, which complexes with
cholesterol to form pores in cell membrane bilayers and cause red
blood cell lysis, was utilized for the positive hemolytic control.
The 1% Saponin-treated positive controls for rat and human whole
blood hemolysis generated a mean hemoglobin concentration of 3.9
and 11.0 g/dL in the supernatant, respectively (Tables 47, 48, 49
and 50). These values met the protocol defined assay acceptability
criterion of hemoglobin concentration>(Negative Control+0.5
g/dL).
[0427] The vehicle control (5% Dextrose) in rat and human whole
blood in Phases 1 and 2 generated a mean hemoglobin concentration
of 0.0 g/dL (Tables 47, 48, 49 and 50). The vehicle control from
both phases met the protocol defined sample analysis criterion of
<(Negative Control+0.5 g/dL).
[0428] The plasma and negative controls from both phases exhibited
a mean hemoglobin concentration of 0.0 g/dL (Tables 47, 48, 49 and
50). These values met the protocol defined assay acceptability
criteria of untreated whole blood hemoglobin concentration
<(Negative Control+0.5 g/dL) and saline with untreated whole
blood hemoglobin concentration <0.5 g/dL.
[0429] Vehicle controls were negative and method controls provided
the expected targeted values. All control checks indicate
acceptable runs in Phases 1 and 2.
Brincidofovir Hemolytic Potential
[0430] Brincidofovir in rat whole blood at 0.2, 0.5 and 1.6 mg/mL
exhibited mean hemoglobin concentrations of 0.0, 0.25 and 1.35
g/dL, respectively (Tables 47, 48, 49 and 50) and as
summarized:
TABLE-US-00047 TABLE 45 Brincidofovir Hemolysis in Rat Whole Blood
Mean HgB Concentration Estimated % Tube Contents (g/dL) Hemolysis*
1A Test Item (0.2 mg/mL) + 0.0 -- whole blood 1B Test Item (0.5
mg/mL) + 0.25 2.4% whole blood 1C Test Item (1.6 mg/mL) + 1.35
13.0% whole hood *Calculated as: (HgB plasma)/(normal HgB whole
blood .times. dilution factor) * 100 Based on normal whole blood
rat hemoglobin value of 13 g/dL (as per our HCD)
[0431] Point scientific controls are system tests to verify the
measurement of hemoglobin in solution phase. As set forth herein,
brincidofovir demonstrated a hemolytic potential effect in rat
whole blood that appears to be concentration dependent. Presumptive
evidence of this effect was observed at the 0.5 mg/mL concentration
and more distinct evidence of hemolysis was observed at the 1.6
mg/mL concentration.
[0432] The estimated % hemolysis for detectable levels of
hemoglobin in this study can be calculated using the mean normal
hemoglobin concentration for rat whole blood and the appropriate
dilution factor. For instance, a rat whole blood normal hemoglobin
value of 13 g/dL corrected for dilution by test article, results in
a value of 10.4 g/dL (13.times.0.8). The calculated % hemolysis for
hemoglobin values of 0.25 and 1.35 g/dL was 2.4% and 13.0%
respectively. Formulations with a hemolysis value of <10% are
considered to be non-hemolytic while values >25% are considered
to be at risk for hemolysis (Amin and Dannenfelser, 2006). Without
wishing to be bound by theory, the 0.2 mg/mL concentration showed
no detectable hemoglobin and is thus considered non-hemolytic.
Concentrations of 0.5 and 1.6 mg/mL resulted in detectable
hemoglobin levels in rat whole blood, but the levels noted were not
considered a hemolytic risk.
[0433] Results of two (n=2) independent analytical runs from Phase
2 indicated that there was no effect from brincidofovir on
hemocompatibility with human whole blood at the concentrations of
0.2 and 0.5 and 1.6 mg/mL and as summarized:
TABLE-US-00048 TABLE 46 Brincidofovir Hemolysis in Human Whole
Blood Mean HgB Concentration Estimated % Tube Contents (g/dL)
Hemolysis* 1A Test Item (0.2 mg/mL) + 0.0 -- whole blood 1B Test
Item (0.5 mg/mL) + 0.0 -- whole blood 1C Test Item (1.6 mg/mL) +
0.0 -- whole blood *Calculated as: (HgB plasma)/(normal HgB whole
blood .times. dilution factor) * 100 Based on normal whole blood
human female hemoglobin value of 14 g/dL (as per Mayo Clinic
on-line data)
[0434] In addition, brincidofovir was not found to interfere with
hemoglobin concentration determinations in both Phases 1 and 2
(Tables 47, 48, 49 and 50). Test item controls met the protocol
defined sample analysis criterion which indicates acceptable runs
in Phases 1 and 2.
[0435] Results of two (n=2) independent analytical runs from Phase
1 indicated without wishing to be bound by theory that
brincidofovir demonstrated a hemolytic potential effect in rat
whole blood that appears to be concentration dependent. Without
wishing to be bound by theory, presumptive evidence of this effect
was observed at the 0.5 mg/mL concentration and clear evidence of
hemolysis was observed at the 1.6 mg/mL concentration. Formulations
with a hemolysis value of <10% are considered to be
non-hemolytic while values >25% are considered to be at risk for
hemolysis. The 0.2 mg/mL concentration showed no detectable
hemoglobin and is considered non-hemolytic. Concentrations of 0.5
and 1.6 mg/mL resulted in detectable hemoglobin levels in rat whole
blood, but the levels noted were not considered a hemolytic
risk.
[0436] Results of two (n=2) independent analytical runs from Phase
2 indicated that there was no effect from brincidofovir on
hemocompatibility with human whole blood at the concentrations of
0.2, 0.5 or 1.6 mg/mL.
[0437] Without wishing to be bound by theory, not all species react
the same to the positive hemolytic reagents which may represent RBC
fragility differences between species.
TABLE-US-00049 TABLE 47 Summary of Hemolytic Potential Results;
Brincidofovir in Rat Blood Phase 1 Mean HgB Concentration Estimated
% Tube Contents (g/dL) Hemolysis* 1A Test Item (0.2 mg/mL) + 0.0 --
whole blood 1B Test Item (0.5 mg/mL) + 0.25 2.4% whole blood 1C
Test Item (1.6 mg/mL) + 1.35 13.0% whole blood 2A Test Item (0.2
mg/mL) + 0.0 -- saline 2B Test Item (0.5 mg/mL) + 0.0 -- saline 2C
Test Item (1.6 mg/mL) + 0.0 -- saline 3 Test Item Vehicle + whole
0.0 -- blood 4 Positive Control (whole 3.9 37.5% blood + 1.0%
saponin) 5 Plasma Control (whole blood) 0.0 -- 6 Negative (saline +
0.0 -- whole blood) *Calculated as: (HgB plasma)/(normal HgB whole
blood .times. dilution factor) * 100 Based on normal whole blood
rat hemoglobin value of 13 g/dL (as per our HCD)
TABLE-US-00050 TABLE 48 Summary of Hemolytic Potential Results;
Brincidofovir in Human Blood Phase 2 Mean HgB Concentration
Estimated % Tube Contents (g/dL) Hemolysis* 1A Test Item (0.2
mg/mL) + 0.0 -- whole blood 1B Test Item (0.5 mg/mL) + 0.0 -- whole
blood 1C Test Item (1.6 mg/mL) + 0.0 -- whole blood 2A Test Item
(0.2 mg/mL) + 0.0 -- saline 2B Test Item (0.5 mg/mL) + 0.0 --
saline 2C Test Item (1.6 mg/mL) + 0.0 -- saline 3 Test Item Vehicle
+ 0.0 -- whole blood 4 Positive Control (whole 11.0 98.2% blood +
1.0% saponin) 5 Plasma Control (whole blood) 0.0 -- 6 Negative
(saline + 0.0 -- whole blood) *Calculated as: (HgB plasma)/(normal
HgB whole blood .times. dilution factor) * 100 Based on normal
whole blood human female hemoglobin value of 14 g/dL (as per Mayo
Clinic on-line data)
TABLE-US-00051 TABLE 49 Hemolytic Potential Results (Hemoglobin
Concentration in g/dL): Brincidofovir in Rat Blood Phase 1 Tube
Number Test Samples 1A 1B 1C 2A 2B 2C 3 Run 1 0.0 0.2 1 0.0 0.0 0.0
0.0 Run 2 0.0 0.3 1.7 0.0 0.0 0.0 Mean 0.0 0.25 1.35 0.0 0.0 0.0
0.0 SD 0.0 0.1 0.5 0.0 0.0 0.0 Tube Number Controls 4 5 6 Run 1 3.9
0.0 0.0
TABLE-US-00052 TABLE 50 Hemolytic Potential Results (Hemoglobin
Concentration in g/dL): Brincidofovir in Human Blood Phase 2 Tube
Number Test Samples 1A 1B 1C 2A 2B 2C 3 Run 1 0.0 0.0 0.0 0.0 0.0
0.0 0.0 Run 2 0.0 0.0 0.0 0.0 0.0 0.0 Mean 0.0 0.0 0.0 0.0 0.0 0.0
0.0 SD 0.0 0.0 0.0 0.0 0.0 0.0 Tube Number Controls 4 5 6 Run 1
11.0 0.0 0.0 1A = Test Item assay for hemolysis at 0.2 mg/mL 1B =
Test Item assay for hemolysis at 0.5 mg/mL 1C = Test Item assay for
hemolysis at 1.6 mg/mL 2A = Test Item interference control at 0.2
mg/mL 2B = Test Item interference control at 0.5 mg/mL 2C = Test
Item interference control at 1.6 mg/mL 3 = Vehicle control for
hemolysis 4 = Hemolytic positive control; 1% Saponin used as a
positive control 5 = Untreated whole blood control for hemolysis 6
= Negative control for hemolysis SD = standard deviation
Example 8--Maximum Tolerated Dose and 7-Day Dose Range-Finding
(DRF) Study in Rats
[0438] The purpose of this 2-phase study was to determine the
maximum tolerated dose (MTD) of brincidofovir, when administered
via a single 2-hour intravenous infusion to rats and to assess the
toxicity, as well as the single and repeat and toxicokinetic
profile of brincidofovir when administered via 2-hour intravenous
infusion to rats on Days 1, 4 and 7.
[0439] The results of this study can allow for a decision regarding
the dose levels for subsequent toxicity studies of brincidofovir
when administered via intravenous infusion.
[0440] The study design incorporated elements of general regulatory
guidelines for toxicity studies.
Summary
[0441] This study was comprised of two phases; Phase 1 was a
maximum tolerated dose (MTD) study and Phase 2 was a repeat dose
range-finding (DRF) study.
[0442] Parameters evaluated during Phase 1 were: viability and
clinical observations.
[0443] For Phase 1, Sprague-Dawley CD.RTM. rats (2/sex/dosing
interval) were dosed with brincidofovir via 2-hour IV infusion once
at 2, 4, 10 or 15 mg/kg. The maximum feasible dose (MFD) was
limited to 15 mg/kg because of increased hemolytic potential when
the concentration of brincidofovir exceeds 1 mg/mL in the dosing
solution. The dose volume was 10 mL/kg/hr for all dosing intervals.
Each dose administration was followed by a 2-3 day observation
period. At the end of the Phase 1 treatment period, all animals
were euthanized and discarded without macroscopic examination.
[0444] Parameters evaluated during Phase 2 were: viability,
clinical observations, body weights, food consumption, hematology
(termination of dosing), clinical chemistry (termination of dosing)
and macroscopic observations.
[0445] For Phase 2, Sprague-Dawley CD.RTM. rats (5/sex/group) were
dosed with brincidofovir via 2-hour IV infusion with 0 [2.times.
sodium phosphate buffer solution (400 mM, pH 8.0.+-.0.04)], 1 or 15
mg/kg on Days 1, 4 and 7. The dose volume was 10 mL/kg/hr for all
groups. At the end of the Phase 2 treatment period, all animals
were euthanized and necropsied. Phase 2 satellite animals
(3/sex/Group 1 and 6/sex/Group 2-3) were similarly dosed and blood
samples collected on Days 1 and 7 for toxicokinetic analysis of
brincidofovir and one of its metabolites, cidofovir.
TABLE-US-00053 List of Abbreviations and Definitions of Terms
Abbreviation or Specialist Term Explanation AUC.sub.inf Area under
the plasma concentration-time curve from time 0 extrapolated to
infinity AUC.sub.inf % Extrap Percentage of the AUC.sub.inf that
occurred post T.sub.last AUC.sub.last Area under the plasma
concentration-time curve from time 0 to time of last measurable
plasma concentration AUC.sub.last/D Area under the plasma
concentration-time curve from time 0 to time of last measurable
plasma concentration normalized to dose BLQ Below the limit of
quantitation CL Apparent total body clearance C.sub.max Maximum
concentration observed during a dosing interval Brincidofovir
Hexadecyloxypropyl-cidofovir CMX021 Brincidofovir metabolite:
Cidofovir % CV Coefficient of variation (percent) DRF Dose range
finding K.sub.2EDTA Anticoagulant LLOQ Lower limit of
quantification M/P ratio: The ratio of exposure parameters
(C.sub.max and/or AUC.sub.last) of metabolite versus parent. The
M/P ratio is calculated by first converting the relevant parameters
to molar units and then dividing the molar exposure value of the
metabolite by the molar exposure value of the parent compound. MTD
Maximum tolerated dose IV Intravenous dosing TK Toxicokinetic(s)
t.sub.1/2 Apparent terminal half-life (aka t.sub.1/2elim)
T.sub.last Time of last measurable concentration T.sub.max Time to
maximum plasma concentration V.sub.z Apparent volume of
distribution, terminal phase
Phase 1
[0446] All animals administered a single dose of brincidofovir at
2, 4, 10 or 15 mg/kg via 2-hour IV infusion survived until their
scheduled euthanasia.
[0447] Administration of brincidofovir at 2, 4, or 15 mg/kg
resulted in intermittent clinical signs in some male and female
animals. Clinical signs were not observed in animals dosed at 10
mg/kg brincidofovir. The intermittent clinical signs consisted of
rapid breath and hunched posture with and without piloerection,
partially closed eyes, and decreased activity, or irregular
breathing, or decreased activity with or without irregular
breathing, piloerection, and bilateral partially closed eyes. The
clinical signs were reversible and resolved at the end of infusion
or within 45 minutes of after the end of the infusion in the Group
1 and 2 animals that displayed signs. The clinical signs were
reversible and resolved within 3 hours and 22 minutes of after the
end of the infusion in the Group 4 that displayed signs.
[0448] Under the conditions of the study, all doses were well
tolerated. Therefore, a maximally tolerated dose level was not
determined, and 15 mg/kg was considered the maximum feasible dose
(MFD).
Phase 2
[0449] All animals administered three total doses of brincidofovir
at 1 and 15 mg/kg/day over a 7 day period survived until their
scheduled euthanasia.
[0450] Administration of brincidofovir at 1 mg/kg/day or 15
mg/kg/day did not result in any brincidofovir-related clinical
signs in either male or female animals.
[0451] Plasma samples (108) were collected 2 hours following the
initiation of dosing on Day 1 and Day 7 (Group 1), or at 1, 2, 8,
and 24 hours following the initiation of dosing on Day 1, and prior
to administration and at 2, 8, and 24 hours following the
initiation of dosing on Day 7 (Groups 2 and 3), and analyzed to
determine the concentrations of brincidofovir and the metabolite,
cidofovir), by LC/MS/MS.
[0452] Peak concentrations for brincidofovir were generally
observed at the end of the 2 hour IV infusion, and decreased
rapidly. In general, the mean brincidofovir C.sub.max and AUC
increased approximately proportional to the increase in dose from 1
to 15 mg/kg, however the AUC following 1 mg/kg was based on limited
concentration-time data. The brincidofovir C.sub.max and
AUC.sub.last values on Day 7 showed a trend toward lower values on
Day 7 when compared to Day 1 [Day 7/Day 1 accumulation ratios (AR)
of 0.44 to 0.54]. No sex differences in brincidofovir TK parameters
were observed. Overall, the metabolite cidofovir increased less
than proportional to the increase in brincidofovir dose. There were
no apparent sex differences in cidofovir TK parameters and no
change in TK parameters after repeat administration.
[0453] Brincidofovir-related hematologic findings were limited to
slight decreases in reticulocytes in males and females administered
15 mg/kg brincidofovir (-33% and -30% controls, respectively;
statistically significant in males only), associated with increases
in mean cell hemoglobin concentration (MCHC) in males (+2.1%
controls, statistically significant). Decreases red cell mass
(hemoglobin, hematocrit, red blood cells) were negligible (to -4.2%
controls) at the end of the 7-day dosing phase
[0454] There were minimal brincidofovir-related increases in total
calcium in males administered 15 mg/kg brincidofovir (+3.8%
controls; not statistically significant) and females administered
.gtoreq.1 mg/kg brincidofovir (+4.9% controls; statistically
significant) and minimal increases in urea (BUN) in females
administered 15 mg/kg (+29% controls; statistically
significant).
[0455] Administration of 1 and 15 mg/kg/day brincidofovir via
2-hour IV infusion on Days 1, 4 and 7 are well tolerated.
Route, Duration, and Frequency of Administration
[0456] In Phase 1, each animal was administered a single dose of
brincidofovir via 2-hour intravenous infusion. At each dose
interval, an escalated dose was administered to naive animals to
determine the maximum tolerated dose (MTD). MTD data were used to
select doses for Phase 2.
[0457] In Phase 2, the selected doses were administered via 2-hour
intravenous infusion on Days 1, 4 and 7 to evaluate the toxicity of
repeated doses of the brincidofovir and to aid in the selection of
doses for subsequent toxicity studies. A seven-day study can be
appropriate for selecting doses for subsequent repeat dose GLP
toxicity studies.
Number of Animals
[0458] The number of animals in this study was considered to be the
minimum necessary for statistical, regulatory and scientific
reasons.
[0459] The number of animals (2/sex/dose interval) in Phase 1 (MTD)
was the minimum number that would be required to determine the
maximum tolerated dose in male and female rats. Four dose levels of
the brincidofovir were expected to be sufficient to determine the
maximum tolerated dose. Although there was a 2-3 day observation
period, new animals were used at each dose level to eliminate
possible additive effects of repeat dosing that could confound
determination of a maximum tolerated single dose level.
[0460] The number of main study animals (5/sex/group) in Phase 2
(DRF) was the minimum number that would control for the expected
variability among animals. The negative control group and the two
brincidofovir-treated groups receiving a low and high multiple of
the proposed human dose were considered the minimum number of
groups necessary to establish a baseline and provide a range of
effects and allow for extrapolation of results for additional
repeat dose studies.
[0461] The number of satellite toxicokinetic study animals
(6/sex/treated group) in Phase 2 (DRF) toxicokinetic evaluations
was considered the minimum number necessary to provide meaningful
data, given the inherent variability in distribution, metabolism
and excretion processes. A control group with 3 animals per sex
confirmed absence of brincidofovir exposure.
Dose Selection
[0462] The starting dose for Phase 1 of this study was 2 mg/kg.
[0463] The low and high doses for Phase 2 of this study (1 and 15
mg/kg) were selected based on the study results of Phase 1 of the
present study. In some embodiments, the low and high doses were
selected by the lack of clinical signs during the
post-administration observation period in animals that were
administered 15 mg/kg during Phase 1 of the present study.
Experimental Outline
[0464] For Phase 1 (MTD), up to four dose levels of brincidofovir
were administered as escalating single doses in naive male and
female rats via intravenous infusion (2-hour). Each dose
administration was followed by a 2-3 day observation period. Each
subsequent dose level was increased and administered to naive
animals based on the response to the preceding doses until the
maximum tolerated dose (MTD) was identified, or until the maximum
feasible dose (MFD) based on prior evidence of hemolysis of 15
mg/kg was achieved.
TABLE-US-00054 TABLE 51 Phase 1 (MTD) Study Consisted of 4 Dosing
Intervals Dose Number of Concen- Volume Interval Treat- Dose
animals tration Dose (Group) ment (mg/kg).sup.a Male Female (mg/mL)
(mL/kg/hr) 1 BCV 2 2 2 0.1 10 2 BCV 4 2 2 0.2 10 3 BCV 10 2 2 0.5
10 4 BCV 15 2 2 0.75 10 .sup.aDoses represent active ingredient
[0465] For Phase 2 (DRF), the test and control articles were
administered, via intravenous 2-hour IV infusion to rats on Days 1,
4, and 7.
TABLE-US-00055 TABLE 52 Phase 2 Study: 1 Control and 2 Treated
Groups Number of animals Volume Dose Main study Satellite
study.sup.b Concentration Dose Group Treatment (mg/kg).sup.a Male
Female Male Female (mg/mL) (mL/kg/hr) 1 Control 0 5 5 3 3 0 10 2
BCV 1 5 5 6 6 0.05 10 3 BCV 15 5 5 6 6 0.75 10 .sup.aDoses
represent active ingredient .sup.bSatellite animals used for
toxicokinetic blood sampling only.
[0466] A vehicle solution of 2.times. sodium phosphate buffer
solution (400 mM, pH 8.0.+-.0.04) was prepared by mixing the
appropriate amounts of monobasic sodium phosphate solution (400 mM)
and dibasic sodium phosphate solution (400 mM). The pH of the
solution was adjusted with monobasic sodium phosphate solution (400
mM), when necessary. The solution was filtered through a 0.22 .mu.m
Millex.RTM.-GP filter under a laminar flow hood into a sterile
vessel.
[0467] The vehicle (control article) was stored refrigerated
2-8.degree. C. Fresh vehicle solution was prepared once prior to
each phase, and used within one month of preparation.
[0468] A brincidofovir stock of 15 mg/mL was perpared by mixing the
appropriate amount of brincidofovir with 2.times. sodium phosphate
buffer solution, sterile water for injection, USP, and 1N sodium
hydroxide (NaOH). The solution was filtered through a 0.22 .mu.m
Millex.RTM.-GP filter under a laminar flow hood into a sterile
vessel.
[0469] Dose formulations for Phase 1 and 2 were prepared by
diluting the appropriate amounts of the brincidofovir stock (15
mg/mL) or of the stock solution vehicle (2.times. sodium phosphate
buffer solution) with the appropriate amounts of 5% dextrose for
injection, USP, into a sterile vial under a laminar flow hood and
inverting 10 times to mix. During Phase 1 and 2, fresh dose
formulations were prepared on each dosing day and were stored
refrigerated 2-8.degree. C. and protected from light when not in
use.
Method of Administration
Route
[0470] Intravenous infusion over 2-hours. Treated at constant doses
in 20 mL/kg/2 hours.
Infusion Catheter Implantation Procedure
[0471] Catheters for infusion were implanted approximately 1-2
weeks prior to dose administration. All animals, including the
spares, were surgically implanted with a catheter.
[0472] The surgical site was prepared as per Testing Facility's SOP
for aseptic, recovery surgical procedures. Animals received
analgesics pre-emptively (flunixin meglumine (USP) 2 mg/kg,
subcutaneously).
[0473] All animals were anesthetized (isoflurane) and shaved in the
inguinal and dorsal regions. A small incision was made and the
femoral vein isolated. A small incision in the vessel was performed
and a sterile catheter was placed into the vessel. The catheter was
inserted via the femoral vein and passed retrograde into the
inferior vena cava, just caudal to the kidneys. The catheter was
secured with sutures and passed subcutaneously so as to exit at the
nape of the neck. A small pocket was made at the exteriorization
site in which a loop of catheter remained. The inguinal region was
closed by sutures.
[0474] The animals were placed in jackets and the implanted
catheters were attached to pins with capped septum connectors. The
catheters were locked with taurolidine citrate locking solution.
Each animal received enrofloxacin 5.0 mg/kg intramuscularly on the
day of surgery.
Infusion Catheter Maintenance Procedure
[0475] Implanted femoral vein catheters were assessed for patency
as per Testing Facility's SOP prior to placing animals on
study.
Volume Dose (Rate)
[0476] 20 mL/kg/2 hours (10 mL/kg/hour).
Individual Dose Concentration
[0477] The individual animal concentrations were calculated from
the most recently recorded scheduled bodyweight.
Frequency and Duration
[0478] During Phase 1, each animal received a single dose
administered via intravenous infusion (2-hours), followed by a 2-3
day observation period after each dose interval. Following the
single intravenous (2-hour) infusion, the animals had their jackets
and dosing sets removed and were not returned to saline
maintenance.
[0479] During Phase 2, each animal received brincidofovir
administrations via intravenous infusion (2-hours) on Days 1, 4 and
7. Animals were maintained on sterile saline at a rate of 0.5
mL/hour between doses. Catheters were tied off after the completion
of dose administration on Day 7 (a knot was placed in the catheter
and the catheter was receded under the skin) and jackets were
removed.
Dose Site
[0480] Surgically implanted cannula in the femoral vein/vena
cava.
Dosing Procedure
[0481] One to two days prior to dose initiation, the lock solution
was withdrawn from each animal's implanted catheter (if possible)
and the catheter was flushed with saline before connecting to a
tether and infusion dosing set. After connecting the animals'
catheters, the animals were infused with sterile saline (0.9% NaCl,
USP) at a rate of 0.5 mL/hr by a calibrated Medfusion syringe pump
until dose administration on Day 1.
[0482] A saline flush was infused to deliver the brincidofovir in
the infusion lines and to ensure the delivery of a complete dose
(an additional .about.0.5 to 1 mL volume, at the same rate as the
brincidofovir, was administered to flush the catheter line).
Dosing Accuracy
[0483] The accountability (confirmation of actual dose
administered) for each day of infusion was calculated and reviewed
for adjustments daily. Pumps were checked for accuracy prior to
dose initiation and following end of dose administration for each
phase.
Collection Times and Number of Animals
[0484] Blood samples were obtained for the determination of plasma
concentrations of brincidofovir during Phase 2.
TABLE-US-00056 TABLE 53 Collection Times and Number of Animals
Interval Timepoints Number of Animals Day 1 2 hours after the
initiation of 3 animals/sex/Group 1 dose administration 1, 2, 8 and
24 hours after the 3 animals/sex/Groups 2-3 initiation of dose
administration Day 7 2 hours after the initiation of 3
animals/sex/Group 1 dose administration Predose, and 2, 8 and 24
hours 3 animals/sex/Groups 2-3 after the initiation of dose
administration
[0485] On Days 1 and 7, blood samples were obtained for
toxicokinetic determinations at the above timepoints.
Collection Procedures
[0486] Approximately 0.4 mL of whole blood was obtained via the
tail vein from each animal at each time point. Animals were not
fasted prior to blood collection. Blood was collected into tubes
containing K.sub.2EDTA anticoagulant and placed on wet ice in an
upright position, per facility SOP prior to processing. Plasma was
separated by centrifugation (10 minutes at approximately 2000 g, at
approximately 2-8.degree. C.). Approximately 0.10 mL of plasma was
transferred into a single cryotube appropriately labeled with study
number, animal number, time point, date of sampling and sample
type. Remaining plasma was transferred into a second cryotube and
retained as a backup sample. Plasma frozen at approximately
-80.+-.10.degree. C. within approximately 2 hours after collection
of each blood sample until analysis. Animals were euthanized
(CO.sub.2 inhalation) after the final blood collection.
[0487] All plasma sample tubes were stored frozen
(-80.+-.10.degree. C.) and shipped (frozen, on dry ice) to Pyxant
Labs, Colorado Springs, Colorado for analysis. Samples were shipped
within 1 month of collection.
[0488] In general, TK parameters were estimated using mean plasma
concentrations of brincidofovir and cidofovir derived from the
composite blood sampling design using Phoenix WinNonLin (V
6.3).
[0489] Animals were observed in their cages twice daily for
mortality and signs of severe toxic or pharmacologic effects.
[0490] For dose observations, Phase 1: Signs of poor health or
toxic or pharmacologic effects (e.g., abnormalities in general
condition, appearance, activity, behavior, respiration, etc.)
observed during infusion period were recorded.
[0491] Animals were removed from their cages and examined once
pretest. Examinations included observations of general condition,
skin and fur, eyes, nose, oral cavity, abdomen and external
genitalia as well as evaluations of respiration.
Body Weight
[0492] Phase 1: Animals were removed from their cages and weighed
twice pretest and prior to each dose.
[0493] Phase 2: Animals were removed from their cages and weighed
twice pretest, prior to each dose and following the last dose
administered during the afternoon of Day 7. Terminal, fasted body
weights were obtained just prior to necropsy.
[0494] Food consumption was measured (weighed) weekly, beginning
one week prior to dosing for Phase 2.
[0495] Blood obtained via the orbital sinus (retrobulbar venous
plexus) as a terminal procedure under light isoflurane anesthesia
was used to analyze hematology and clinical chemistry parameters
for all animals at termination of dosing. Animals were fasted
overnight prior to blood collection. Animals were not allowed to
recover from anesthesia after blood collection.
Hematology
[0496] Blood samples (approximately 0.25 mL) were collected into
tubes containing K.sub.2EDTA anticoagulant and analyzed for the
following using a Siemens ADVIA 120 Hematology Analyzer: Hemoglobin
concentration (HGB); Hematocrit (HCT); Erythrocyte count (RBC);
Platelet count (PLT); Mean corpuscular volume (MCV); Mean
corpuscular hemoglobin (MCH); Mean corpuscular hemoglobin
concentration (MCHC); Red cell distribution width (RDW); Total
leukocyte count (WBC); Reticulocyte count (RETIC); Differential
leukocyte count (Manual differential leukocyte counts were
performed for verification and absolute values were calculated if
necessary); Neutrophils (ANEU); Lymphocytes (ALYM); Eosinophils
(AEOS); Basophils (ABASO); Monocytes (AMONO); Large unstained cells
(ALUC);
[0497] A peripheral blood smear was prepared for each animal at
each blood collection interval and was available for confirmation
of automated results and/or other evaluations deemed necessary by
the Clinical Pathologist.
Clinical Chemistry
[0498] Blood samples (approximately 1 mL) were collected into tubes
with no anticoagulant, allowed to clot, centrifuged to obtain serum
and analyzed for the following using a Siemens ADVIA 1800 Chemistry
Analyzer: Aspartate aminotransferase (AST) (Kinetic--Modified
Bergmeyer); Alanine aminotransferase (ALT) (Kinetic--Modified
Bergmeyer); Alkaline phosphatase (ALKP) (Kinetic--Tietz AMP
Buffer); Blood urea nitrogen (BUN) (Enzymatic Roch-Ramek with
Urease); Creatinine (CREAT) (Jaffe Picric Acid in Alkaline Medium);
Glucose (GLU) (Glucose Hexokinase II Method); Cholesterol (CHOL)
(Enzymatic esterase/oxidase Trinder Endpoint); Triglycerides (TRIG)
(Fossati Three Step Enzymatic--Trinder Endpoint); Total protein
(TP) (Biuret Technique); Albumin (ALB) (Bromocresol Green Method);
Total bilirubin (TBILI) (Oxidation with Vandate); Sodium (NA.sup.+)
(Ion Selective Electrode); Potassium (K.sup.+) (Ion Selective
Electrode); Chloride (Cl.sup.-) (Ion Selective Electrode); Calcium
(Ca.sup.++) (Michaylova & Ilkova, Arsenazo III); Inorganic
phosphorus (PHOS) (Phosphomolybdate--UV Method).
Postmortem
[0499] Phase 1: All animals were euthanized by exsanguination
following carbon dioxide inhalation and discarded without
macroscopic examination after the completion of the observation
period.
[0500] Phase 2: Main DRF study animals were euthanized by
exsanguination following isoflurane inhalation and TK satellite
animals via carbon dioxide inhalation.
[0501] Necropsy was performed on the Phase 2 main study animals on
Day 8. Animals were fasted overnight prior to necropsy.
[0502] Complete macroscopic examinations were performed on all
Phase 2 main study animals. The macroscopic examination included
examination of the external surface and all orifices; the external
surfaces of the brain and spinal cord; the organs and tissues of
the cranial, thoracic, abdominal and pelvic cavities and neck; and
the remainder of the carcass for the presence of macroscopic
morphologic abnormalities. Animals were discarded after
examination; no tissues were collected or preserved.
[0503] The following abbreviations were used for hematology: CS:
Clotted specimen; LA: Lab accident; NVIM; Not valid due to
improbable result; CLSE: Severe platelet clumping noted; CLSL:
Slight platelet clumping noted; NCLP: No clumping.
[0504] Blood chemistry: TBILI values below the analytical limit
(<0.2) are excluded from calculations.
Globulin (GLOB) was calculated as: total protein-albumin
Albumin/globulin ratio (A/G) was calculated as:
albumin/globulin
[0505] All statistical analyses were carried out separately for
males and females using the individual animal as the basic
experimental unit.
[0506] The following data types were analyzed at each timepoint
separately for Phase 2: body weight; body weight change from
interval to interval; cumulative body weight change from baseline;
food consumption, hematology, and clinical chemistry. The
parameters to analyze were identified as continuous, discrete or
binary. Brincidofovir treated groups were then compared to the
control using the following procedures.
In-Life Study Conduct
[0507] Table 54 below summarizes details regarding the study
conduct, including but not limited to test animals, study
materials, study design, dosing, observations, and results.
TABLE-US-00057 TABLE 54 Experimental Design Study Summary (phase 2)
Species and Strain: Vehicle: 200 mM Sodium Male and Female [Crl: CD
(SD)BR] Phosphate Buffer, pH 8, Variable (Sprague-Dawley derived)
dilution with 5% Dextrose Study Design Group Dose Number (mg/kg)
Sampling Scheme 1 0 Day 1 and Day 7: 3 animals/sex: 2 hours after
the initiation of dose vehicle administration 2 1 Day 1: 3
animals/sex/timepoint: 1, 2, 8, and 24 hours after the initiation
of dose administration Day 7: 3 animals/sex/timepoint: Predose, 2,
8, and 24 hours after the initiation of dose administration 3 15
Same as Group 2
Sampling
[0508] A composite blood sampling design was used such that each
rat was sampled twice per sampling day. Blood samples were
collected according to the scheme shown in Table 54. Blood was
collected via tail vein from unanesthetized animals into tubes
containing anticoagulant and placed on wet ice in an upright
position. Animals were not fasted prior to blood collection. Plasma
was separated by centrifugation (for 10 minutes at approximately
2000 g, at approximately 2-8.degree. C.), and transferred into
individually labeled cryotubes. All cryotubes containing the
collected plasma samples were appropriately labeled as to study
number, animal number, time point, date of sampling and sample
type. All plasma samples were obtained and frozen at approximately
-80.degree. C. (.+-.10.degree. C.) within approximately 2 hours
after collection of each blood sample until analysis.
[0509] All plasma samples were stored frozen [approximately
-80.degree. C. (.+-.10.degree. C.)] and shipped (frozen, on dry
ice) to Pyxant Laboratories for analysis.
Bioanalytical Methods
[0510] Rat plasma bioanalysis was conducted by Pyxant Laboratories.
Plasma samples were analyzed for concentrations of brincidofovir
and cidofovir using a method based upon protein precipitation
extraction followed by LC-MS/MS analysis; calibration ranges for
brincidofovir and cidofovir were 1.00-1500 ng/mL and 5.00-750
ng/mL, respectively, (Pyxant study number 3025) for a 50 .mu.L
aliquot of rat plasma. Sample analysis for ISR was not performed as
part of this non-GLP study.
Data Analysis
Toxicokinetic Analysis
[0511] For the generation of Mean Profiles, all sample
concentrations that were below the limit of quantitation (BLQ) were
set to 0.
[0512] These values are recorded in Table 61 and Table 62, with the
original BLQ result in the column labelled [Concentration] and the
imputed values for calculation of the mean values in the column
labelled [Imputed Concentration].
[0513] The treatment of Mean Profile BLQ values for the purposes of
NCA, are recorded in Table 63 and Table 64, with the original Mean
Profile BLQ result in the column labeled [Mean Concentration] and
the imputed values used for NCA analysis in the column labeled
[Imputed Mean Concentration]. BLQ values at early time points
(prior to T.sub.max) were set to 0, BLQ at time points after
T.sub.max were set to missing.
[0514] All pharmacokinetic parameter estimates were completed using
a Non-Compartmental Model (Model 200-202 for IV infusion
administration). The AUC.sub.last and AUC.sub.inf were calculated
using the linear up log down trapezoidal rule.
[0515] Statistical Analysis was limited to descriptive statistical
analysis including arithmetic mean, standard deviation, % CV of the
arithmetic mean.
[0516] The coefficient of variation (% CV) for mean plasma
concentrations ranged from (9.98 to 173% CV) for brincidofovir and
(4.82 to 173% CV) for cidofovir.
[0517] Peak concentrations for brincidofovir were generally
observed at the end of the 2 hour IV infusion, and decreased
rapidly; brincidofovir concentrations were at or near the lower
limit of quantification (LLOQ, 1 ng/mL) 8 hours after initiation of
the 1 mg/kg dose and 24 hours after the 15 mg/kg dose. Terminal
half-life (t1/2), CL, and Vss were not determinable due to
insufficient data points to characterize the elimination phase. In
general, mean brincidofovir C.sub.max and AUC increased
approximately proportional to the increase in dose from 1 to 15
mg/kg, though AUC was determined on few concentration values after
1 mg/kg dosing. The brincidofovir C.sub.max and AUC.sub.last values
on Day 7 showed a trend toward lower values on Day 7 compared to
Day 1 (Day7/Day1 accumulation ratios (AR) of 0.44 to 0.53).
Further, no sex differences in brincidofovir TK parameters were
observed.
[0518] Overall, exposure to metabolite cidofovir increased less
than proportional to the increase in brincidofovir dose, there were
no apparent sex differences in TK parameters, and no change in TK
parameters after repeat administration.
[0519] Bioanalysis was performed on 108 plasma samples collected
only during Phase 2 to determine the concentrations of
brincidofovir and the cidofovir metabolite, cidofovir. The plasma
samples were collected at 2 hours following the initiation of
dosing on Day 1 and Day 7 (Group 1), or at 1, 2, 8, and 24 hours
following the initiation of dosing on Day 1, and prior to
administration and at 2, 8, and 24 hours following the initiation
of dosing on Day 7 (Groups 2 and 3) and analyzed by LC/MS/MS.
[0520] Toxicokinetic analysis of the plasma concentrations of
brincidofovir and cidofovir was conducted for Phase 2 only.
[0521] On Days 1 and 7, brincidofovir C.sub.max increased in
approximate proportion to dose (for female rats), or less than
proportionally to dose (for male rats). For a 15-fold increase in
dose from 1 mg/kg to 15 mg/kg, C.sub.max increased 17.3- to
18.2-fold for females and 9.2- to 18.6-fold for males. On Days 1
and 7, brincidofovir AUC.sub.last increased in approximate
proportion (for female rats); for a 15-fold increase in dose, the
AUC.sub.last increased 14.1- to 16.1-fold. Due to insufficient data
points in male rats after 1 mg/kg administration, proportionality
of AUC.sub.last could not be determined.
[0522] Between female and male animals, the brincidofovir C.sub.max
on Days 1 and 7 for a 1 or 15 mg/kg dose were similar (difference
within 54%). The brincidofovir AUC.sub.last for a 1 mg/kg dose
could not be compared due to insufficient data points to calculate
AUC in males. The brincidofovir AUC.sub.last on Days 1 and 7, for a
15 mg/kg dose, were similar (difference within 16%) between female
and male animals.
[0523] The brincidofovir C.sub.max and AUC.sub.last values on Day 7
for female animals following twice-weekly dosing of brincidofovir
at 1 mg/kg, and for both female and male animals after 15 mg/kg,
showed a trend toward lower C.sub.max and AUC.sub.last on Day 7
compared to Day 1 (Day7/Day1 accumulation ratios (AR) of 0.44 to
0.53).
TABLE-US-00058 TABLE 55 Brincidofovir TK Parameters (Phase 2)
Following Single and Multiple 2-Hour Intravenous Infusion
Administrations of brincidofovir on Days 1, 3 and 7 to Rats
Brincidofovir Dose (mg/kg) 1 15 Male Female Male Female Period
Period TK Parameter Day 1 Day 7 Day 1 Day 7 Day 1 Day 7 Day 1 Day 7
T.sub.max (h) 2 2 2 2 1 2 1 2 T.sub.last (h) 2 2 8 8 24 24 24 24
C.sub.max (ng/mL) 235 208 191 95.4 4370 1910 3470 1660
C.sub.max/Dose 235 208 191 95.4 291 127 231 110 [(ng/mL)/(mg/kg)]
AUC.sub.last NR NR 459 244 8780 4090 7360 3420 (h*ng/mL)
AUC.sub.last/Dose NR NR 459 244 585 273 491 228 [(h*ng/mL)/(mg/kg)]
AR: C.sub.max NA NR NA 0.50 NA 0.44 NA 0.48 Day 7/Day 1 AR:
AUC.sub.last NA NR NA 0.53 NA 0.47 NA 0.46 Day 7/Day 1 NA, not
applicable; NR, not reportable due to insufficient data points
[0524] As set forth in Tables 55 and 56, on Days 1 and 7, cidofovir
C.sub.max generally increased less than proportional to dose. For a
15-fold increase in brincidofovir dose from 1 mg/kg to 15 mg/kg,
cidofovir C.sub.max increased 2.9- to 4.1-fold for female animals
and 5.6- to 16.8-fold for males. On Days 1 and 7, cidofovir
AUC.sub.last increased less than proportionally to dose for female
animals; for a 15-fold increase in brincidofovir dose, cidofovir
AUC.sub.last increased 9.4- to 16.2-fold. Due to insufficient data
points in male rats after 1 mg/kg administration, proportionality
of AUC.sub.last could not be determined.
[0525] The cidofovir C.sub.max and AUC.sub.last on Days 1 and 7,
after a 15 mg/kg brincidofovir dose, were similar (difference
within 33%) between female and male animals.
[0526] The cidofovir C.sub.max and AUC.sub.last on Day 7 for female
animals following twice-weekly dosing of brincidofovir at 1 mg/kg
showed a trend toward higher C.sub.max and AUC.sub.last on Day 7
compared to Day 1 (Day7/Day1 AR ranged from 1.6 to 1.9). However,
cidofovir C.sub.max and AUC.sub.last for both female and male
animals administered 15 mg/kg (3 doses), were similar (Day7/Day1 AR
ranged from 1.0 to 1.1).
[0527] The metabolite-to-parent (M/P) ratio, calculated on a molar
basis, of AUC.sub.last on Day 1 for female animals were 0.190 and
0.661 for doses of 1 mg/kg and 15 mg/kg of brincidofovir,
respectively. The M/P ratios on Day 7 for female animals were 0.193
and 0.444 for doses of 1 mg/kg and 15 mg/kg of brincidofovir,
respectively.
[0528] Due to insufficient data points in male rats after 1 mg/kg
administration, the M/P ratio for AUC.sub.last could not be
determined. The M/P ratios on Day 1 and 7 after 15 mg/kg
administration of brincidofovir were 0.126 and 0.307,
respectively.
TABLE-US-00059 TABLE 56 Cidofovir TK Parameters Following Single
and Multiple 2-Hour Intravenous Infusion Administrations of
brincidofovir on Days 1, 3 and 7 to Rats Brincidofovir Dose (mg/kg)
1 15 Male Female Male Female Period Period TK Parameter Day 1 Day 7
Day 1 Day 7 Day 1 Day 7 Day 1 Day 7 T.sub.max (h) 8 8 2 2 8 8 8 8
T.sub.last (h) 8 8 8 8 24 24 24 24 C.sub.max (ng/mL) 1.95 5.73 9.35
15 32.8 32.8 38.6 43.6 C.sub.max/Dose 1.95 5.73 9.35 15 2.18 2.18
2.57 2.91 [(ng/mL)/(mg/kg)] AUC.sub.last NR NR 43.4 80.1 548 624
705 756 (h*ng/mL) AUC.sub.last/Dose NR NR 43.4 80.1 36.5 41.6 47
50.4 [(h*ng/mL)/(mg/kg)] AR: C.sub.max NA 2.94 NA 1.60 NA 1.00 NA
1.13 Day 7/Day 1 AR: AUC.sub.last NA NR NA 1.85 NA 1.14 NA 1.07 Day
7/Day 1 M/P Ratio: C.sub.max 0.017 0.056 0.099 0.316 0.035 0.015
0.022 0.053 (molar equivalents) M/P Ratio: AUC.sub.last NR NR 0.190
0.661 0.126 0.307 0.193 0.444 (molar equivalents)
[0529] For Phase 1 and Phase 2, all animals survived until
scheduled termination.
Clinical Observations
Phase 1
[0530] Dose 1 (2 mg/kg): Clinical signs in both male animals
consisted of rapid breath and hunched posture, with and without
piloerection, partially closed eyes, and decreased activity.
[0531] Dose 2 (4 mg/kg); Clinical signs in both males and one
female animal consisted of irregular breathing.
[0532] Dose 3 (10 mg/kg): No clinical signs were observed in either
male or female animals.
[0533] Dose 4 (15 mg/kg): Clinical signs in both male and one
female animal included decreased activity with or without irregular
breathing, piloerection, and bilateral partially closed eyes.
[0534] All clinical signs were reversible and resolved shortly
after the end of the infusion.
Phase 2
[0535] Dose administration of brincidofovir at 1 mg/kg/day or 15
mg/kg/day did not result in any brincidofovir-related clinical
signs in either male or female animals. There were no effects on
body weights (only Phase 2 evaluated) or food consumption related
to treatment with brincidofovir.
Clinical Pathology
[0536] Brincidofovir-related hematologic findings were limited to
slight decreases in reticulocytes in males and females at 15 mg/kg
(-33% and -30% controls, respectively; statistically significant in
males only), associated with increases in mean cell hemoglobin
concentration (MCHC) in males (+2.1% controls, statistically
significant). Decreases in red cell mass (hemoglobin, hematocrit,
red blood cells) were negligible (to -4.2% controls) at the end of
the 7-day dosing phase. The minimal effect of reticulocyte
decreases on red cell mass was attributed to the long lifespan of
red blood cells in rats (.about.45-68 days) relative to
reticulocytes (2-5 days). Red cell mass decreases would be expected
to be more pronounced with continued dosing. Decreases in
reticulocytes were indicative of decreased erythropoiesis in
hematopoietic tissues. In the absence of decreases in food
consumption or body weight or poor clinical condition, they were
likely to be due to brincidofovir-related suppression of
erythropoiesis, which is not unexpected with this class of drug
(nucleotide analog).
[0537] There were minimal brincidofovir-related increases in total
calcium in males at 15 mg/kg (+3.8% controls; not statistically
significant) and females at >1 mg/kg (+4.9% controls;
statistically significant) and minimal increases in urea (BUN) in
females at 15 mg/kg (+29% controls; statistically significant).
Both findings can be observed with altered kidney function
(decreased urinary excretion). In addition, without wishing to be
bound by theory, increases in total calcium may reflect increased
mobilization from bone or increased intestinal absorption.
[0538] Changes in Animal No. 3048 (slight increases in BUN,
creatinine and phosphorus) were not considered to be
brincidofovir-related and secondary to decreased glomerular
filtration rate due to absence of a left kidney (noted
macroscopically).
[0539] All other differences from controls, statistically
significant or otherwise, were not considered to be
brincidofovir-related due to their direction, small magnitude,
infrequent occurrence, lack of relation to dose, lack of
concordance with related endpoints, and/or because values were
comparable to the study control range.
Pathology
[0540] Macroscopic findings were few, occurred sporadically, and
were not considered to be brincidofovir-related as they occurred at
a similar incidence in control animals, lacked a dose relationship,
or were considered congenital anomalies (Animal No. 3048). Male
Animal No. 3048 did not have a left seminal vesicle or a left
kidney; the absence of the left kidney correlated with elevated
blood urea nitrogen (BUN), creatinine, and phosphorus in this
animal.
[0541] For Phase 1, under the conditions of the study, all doses
were well tolerated when administered via a single 2-hour IV
infusion. Therefore, the maximally tolerated dose of brincidofovir
was not determined. However, 15 mg/kg delivered via 2-hour IV
infusion of a 0.75 mg/mL solution of brincidofovir represents the
maximum feasible dose based on in vitro evidence of hemolysis at
brincidofovir concentrations above 0.75 mg/mL.
[0542] For Phase 2, peak concentrations for brincidofovir were
generally observed at the end of the 2-hour IV infusion, and
decreased rapidly.
[0543] In general, mean brincidofovir C.sub.max and AUC increased
approximately proportional to the increase in dose from 1 to 15
mg/kg, though AUC was determined on few concentration values after
1 mg/kg dosing. The brincidofovir C.sub.max and AUC.sub.last values
on Day 7 showed a trend toward lower values on Day 7 compared to
Day 1 (Day7/Day1 accumulation ratios (AR) of 0.44 to 0.53).
Further, no sex differences in brincidofovir TK parameters were
observed. Overall, exposure to metabolite cidofovir increased less
than proportional to the increase in brincidofovir dose, there were
no apparent sex differences in TK parameters, and no change in TK
parameters after repeat administration Under the conditions of the
study, single dose administration of 1 or 15 mg/kg brincidofovir
via 2-hour IV infusion on Days 1, 4 and 7 were well tolerated.
Concentrations of Brincidofovir in Plasma
[0544] The concentration-time data for brincidofovir following
single and repeat administration of 1 mg/kg and 15 mg/kg are
summarized in Table 59 and Table 60.
[0545] The concentrations of brincidofovir and cidofovir in all
control plasma samples were BLQ for each analyte (<1.0 ng/mL and
<5.0 ng/mL, respectively).
[0546] Following single IV infusion administration of brincidofovir
over a 2 hour period, plasma concentrations of brincidofovir were
detected in the first sample (1 hour) at both dose levels (1 mg/kg
and 15 mg/kg). Following twice-weekly administration (3 doses on
Days 1, 3 and 7), brincidofovir was BLQ in predose samples, but was
detected in the first postdose sample (2 hour, end of the infusion)
on Day 7. The median time of peak concentration (T.sub.max) of
brincidofovir occurred at 2 hour following single and twice weekly
1 mg/kg dosing, and at 1 hour following single 15 mg/kg dosing and
at 2 hours following twice weekly 15 mg/kg dosing. Concentrations
of brincidofovir reached BLQ in all males and some females 8 hours
following administration of 1 mg/kg and were at or near the LLOQ by
24 hours following administration of 15 mg/kg. The coefficient of
variation (% CV) for mean plasma concentrations for brincidofovir
ranged from 13.0 to 173% for female animals and from 9.98 to 28.1%
for male animals. In general, the highest % CV was observed at
later time points when concentrations were at or near the LLOQ.
Toxicokinetics of Brincidofovir in Plasma
[0547] Mean plasma brincidofovir concentration-time information
used in the input file for Phoeneix WinNonlin analysis are
contained in Table 63. TK parameters for brincidofovir following
single and twice weekly administration of brincidofovir as an
intravenous infusion to rats are summarized in Table 56. Terminal
half-life (t1/2), CL, and V.sub.ss were not determinable due to
insufficient data points to characterize the elimination phase.
[0548] On Days 1 and 7, brincidofovir C.sub.max increased in
approximate proportion to dose (for female rats), or less than
proportionally to dose (for male rats). For a 15-fold increase in
dose from 1 mg/kg to 15 mg/kg, C.sub.max increased 17.3- to
18.2-fold for females and 9.2- to 18.6-fold for males. On Days 1
and 7, brincidofovir AUC.sub.last increased in approximate
proportion (for female rats); for a 15-fold increase in dose, the
AUC.sub.last increased 14.1- to 16.1-fold. Due to insufficient data
points in male rats after 1 mg/kg administration, proportionality
of AUC.sub.last could not be determined.
[0549] Between female and male animals, the brincidofovir C.sub.max
on Days 1 and 7 for a 1 or 15 mg/kg dose were similar (difference
within 54%). The brincidofovir AUC.sub.last for a 1 mg/kg dose
could not be compared due to insufficient data points to calculate
AUC in males. The brincidofovir AUC.sub.last on Days 1 and 7, for a
15 mg/kg dose, were similar (difference 16%) between female and
male animals.
[0550] The brincidofovir C.sub.max and AUC.sub.last values on Day 7
for female animals following twice-weekly dosing of brincidofovir
at 1 mg/kg, and for both female and male animals after 15 mg/kg,
showed a trend toward lower C.sub.max and AUC.sub.last on Day 7
compared to Day 1 [Day7/Day1 accumulation ratios (AR) of 0.44 to
0.53].
TABLE-US-00060 TABLE 57 Brincidofovir TK Parameters Following
Single and Multiple 2-Hour Intravenous Infusion Administrations of
brincidofovir on Days 1, 3 and 7 to Rats Brincidofovir Dose (mg/kg)
1 15 Male Female Male Female Period Period TK Parameter Day 1 Day 7
Day 1 Day 7 Day 1 Day 7 Day 1 Day 7 T.sub.max (h) 2 2 2 2 1 2 1 2
T.sub.last (h) 2 2 8 8 24 24 24 24 C.sub.max (ng/mL) 235 208 191
95.4 4370 1910 3470 1660 C.sub.max/Dose 235 208 191 95.4 291 127
231 110 [(ng/mL)/(mg/kg)] AUC.sub.last (h*ng/mL) NR NR 459 244 8780
4090 7360 3420 AUC.sub.last/Dose NR NR 459 244 585 273 491 228
[(h*ng/mL)/(mg/kg)] AR: C.sub.max NA NR NA 0.50 NA 0.44 NA 0.48 Day
7/Day 1 AR: AUC.sub.last NA NR NA 0.53 NA 0.47 NA 0.46 Day 7/Day
1
Concentrations of Cidofovir in Plasma
[0551] The concentration-time data for cidofovir following single
and repeat daily administration of 1 mg/kg and 15 mg/kg are
summarized in Table 59 through Table 60.
[0552] In female animals following single IV infusion
administration of brincidofovir, plasma concentrations of cidofovir
were detected 2 hours following administration of 1 mg/kg (second
time point) or 1 hour following administration of 15 mg/kg (first
time point). Following twice-weekly administration of
brincidofovir, plasma concentrations of cidofovir were BLQ in
predose samples, but were detected in all of the 2 hour samples
(first postdose time point) on Day 7. The median T.sub.max of
cidofovir occurred 2 hours following single and twice-weekly 1
mg/kg brincidofovir dosing, and at 8 hours following single and
twice-weekly 15 mg/kg brincidofovir dosing.
[0553] In male animals following single IV infusion administration
of brincidofovir, detectable plasma concentrations of cidofovir
were observed only in a single time point 8 hours following
administration of 1 mg/kg. However, cidofovir was detected in the
first sample (1 hour) following administration of 15 mg/kg.
Following twice-weekly administration of brincidofovir, plasma
concentrations of cidofovir were BLQ in the predose samples and in
the first postdose sample (2 hours), but were detected in the 8
hour samples on Day 7 following administration of 1 mg/kg, and in
the 2 hour samples (first postdose sample) on Day 7 following
administration of 15 mg/kg. The median T.sub.max of cidofovir
occurred at 8 hours following single or twice-weekly 1 mg/kg or 15
mg/kg brincidofovir dosing. In both male and female animals,
concentrations of cidofovir reached BLQ by 24 hours following
single and twice-weekly administration 1 mg/kg brincidofovir and
did not reach BLQ by 24 hours (last time point) following single or
twice-weekly administration of 15 mg/kg brincidofovir. The % CV for
mean concentrations of cidofovir ranged from 4.82 to 88.4% for
female animals and 5.49 to 173% for male animals. In general, the
highest % CV was observed at early or later time points when
cidofovir concentrations were at or near the LLOQ.
Toxicokinetics of Cidofovir in Plasma
[0554] Mean plasma cidofovir concentration-time information used in
the input file for Phoenix WinNonlin analysis are contained in
Table 64. TK parameters for cidofovir following single and twice
weekly administration of brincidofovir as an intravenous infusion
to rats are summarized in Table 58.
[0555] On Days 1 and 7, cidofovir C.sub.max generally increased
less than proportional to dose. For a 15-fold increase in
brincidofovir dose from 1 mg/kg to 15 mg/kg, cidofovir C.sub.max
increased 2.9- to 4.1-fold for female animals and 5.7- to 16.8-fold
for males. On Days 1 and 7, cidofovir AUC.sub.last increased less
than proportionally to dose for female animals; for a 15-fold
increase in brincidofovir dose, cidofovir AUC.sub.last increased
9.4- to 16.2-fold. Due to insufficient data points in male rats
after 1 mg/kg administration, proportionality of AUC.sub.last could
not be determined.
[0556] The cidofovir C.sub.max and AUC.sub.last on Days 1 and 7,
after a 15 mg/kg brincidofovir dose, were similar (difference
within 33%) between female and male animals.
[0557] The cidofovir C.sub.max and AUC.sub.last on Day 7 for female
animals following twice-weekly dosing of brincidofovir at 1 mg/kg
showed a trend toward higher C.sub.max and AUC.sub.last on Day 7
compared to Day 1 (Day7/Day1 AR ranged from 1.6 to 1.9). However,
cidofovir C.sub.max and AUC.sub.last for both female and male
animals administered 15 mg/kg (3 doses), were similar (Day7/Day1 AR
ranged from 1.0 to 1.1).
[0558] The metabolite-to-parent (M/P) ratio of AUC.sub.last on Day
1 for female animals were 0.190 and 0.661 for doses of 1 mg/kg and
15 mg/kg of brincidofovir, respectively. The M/P ratios on Day 7
for female animals were 0.193 and 0.444 for doses of 1 mg/kg and 15
mg/kg of brincidofovir, respectively.
[0559] Due to insufficient data points in male rats after 1 mg/kg
administration, the M/P ratio for AUC.sub.last could not be
determined. The M/P ratios on Day 1 and 7 after 15 mg/kg
administration of brincidofovir were 0.126 and 0.307,
respectively.
TABLE-US-00061 TABLE 58 cidofovir TK Parameters Following Single
and Multiple 2-Hour Intravenous Infusion Administrations of
brincidofovir on Days 1, 3 and 7 to Rats Brincidofovir Dose (mg/kg)
1 15 Male Female Male Female Period Period TK Parameter Day 1 Day 7
Day 1 Day 7 Day 1 Day 7 Day 1 Day 7 T.sub.max (h) 8 8 2 2 8 8 8 8
T.sub.last (h) 8 8 8 8 24 24 24 24 C.sub.max (ng/mL) 1.95 5.73 9.35
15 32.8 32.8 38.6 43.6 C.sub.max/Dose 1.95 5.73 9.35 15 2.18 2.18
2.57 2.91 [(ng/mL)/(mg/kg)] AUC.sub.last (h*ng/mL) NR NR 43.4 80.1
548 624 705 756 AUC.sub.last/Dose NR NR 43.4 80.1 36.5 41.6 47 50.4
[(h*ng/mL)/(mg/kg)] AR: C.sub.max NA 2.94 NA 1.60 NA 1.00 NA 1.13
Day 7/Day 1 AR: AUC.sub.last NA NR NA 1.85 NA 1.14 NA 1.07 Day
7/Day 1 M/P Ratio: C.sub.max 0.017 0.056 0.099 0.316 0.035 0.015
0.022 0.053 (molar equivalents) M/P Ratio: AUC.sub.last NR NR 0.190
0.661 0.126 0.307 0.193 0.444 (molar equivalents)
TABLE-US-00062 TABLE 59 Plasma Concentrations of brincidofovir and
cidofovir Following Single and Multiple 2-Hour Intravenous Infusion
Administrations on Days 1, 3 and 7 at a Nominal brincidofovir Dose
of 1 mg/kg to Female and Male Rats Sex Male Female Dose Time BCV
cidofovir BCV cidofovir (mg/kg) Period (h) Subject Imputed
Concentration.sup.a (ng/mL) 1 Day 1 1 2031 156 0.00 NS NS 2033 244
0.00 NS NS 2035 280 0.00 NS NS 2542 NS NS 109 0.00 2544 NS NS 126
0.00 2546 NS NS 177 0.00 Mean 227 0.00 137 0.00 SD 63.8 0.00 35.4
0.00 % CV 28.1 NC 25.8 NC N 3 3 3 3 1 Day 1 2 2032 220 0.00 NS NS
2034 283 0.00 NS NS 2036 203 0.00 NS NS 2543 NS NS 253 9.62 2547 NS
NS 164 6.22 2572 NS NS 155 12.2 Mean 235 0.00 191 9.35 SD 42.1 0.00
54.2 3.00 % CV 17.9 NC 28.4 32.1 N 3 3 3 3 1 Day 1 8 2031 0.00 5.85
NS NS 2033 0.00 0.00 NS NS 2035 0.00 0.00 NS NS 2542 NS NS 2.30
7.12 2544 NS NS 1.45 5.60 2546 NS NS 0.00 0.00 Mean 0.00 1.95 1.25
4.24 SD 0.00 3.38 1.16 3.75 % CV NC 173 93.0 88.4 N 3 3 3 3 1 Day 1
24 2032 0.00 0.00 NS NS 2034 0.00 0.00 NS NS 2036 0.00 0.00 NS NS
2543 NS NS 0.00 0.00 2547 NS NS 0.00 0.00 2572 NS NS 0.00 0.00 Mean
0.00 0.00 0.00 0.00 SD 0.00 0.00 0.00 0.00 % CV NC NC NC NC N 3 3 3
3 1 Day 7 0 2031 0.00 0.00 NS NS 2033 0.00 0.00 NS NS 2035 0.00
0.00 NS NS 2542 NS NS 0.00 0.00 2544 NS NS 0.00 0.00 2546 NS NS
0.00 0.00 Mean 0.00 0.00 0.00 0.00 SD 0.00 0.00 0.00 0.00 % CV NC
NC NC NC N 3 3 3 3 1 Day 7 2 2032 255 0.00 NS NS 2034 189 0.00 NS
NS 2036 179 0.00 NS NS 2543 NS NS 115 13.5 2547 NS NS 82.1 13.8
2572 NS NS 89.2 17.6 Mean 208 0.00 95.4 15.0 SD 41.3 0.00 17.3 2.29
% CV 19.9 NC 18.1 15.3 N 3 3 3 3 1 Day 7 8 2031 0.00 5.67 NS NS
2033 0.00 5.45 NS NS 2035 0.00 6.07 NS NS 2542 NS NS 6.58 9.51 2544
NS NS 0.00 7.84 2546 NS NS 0.00 5.36 Mean 0.00 5.73 2.19 7.57 SD
0.00 0.314 3.80 2.09 % CV NC 5.49 173 27.6 N 3 3 3 3 1 Day 7 24
2032 0.00 0.00 NS NS 2034 0.00 0.00 NS NS 2036 0.00 0.00 NS NS 2543
NS NS 0.00 0.00 2547 NS NS 0.00 0.00 2572 NS NS 0.00 0.00 Mean 0.00
0.00 0.00 0.00 SD 0.00 0.00 0.00 0.00 % CV NC NC NC NC N 3 3 3 3
.sup.aImputations of zero were made for samples with bioanalytical
result of BLQ.
TABLE-US-00063 TABLE 60 Plasma Concentrations of brincidofovir and
cidofovir Following Single and Multiple 2-Hour Intravenous Infusion
Administrations on Days 1, 3 and 7 at a Nominal brincidofovir Dose
of 15 mg/kg to Female and Male Rats Sex Male Female Dose Time BCV
cidofovir BCV cidofovir (mg/kg) Period (h) Subject Imputed
Concentration.sup.a 15 Day 1 1 3053 4020 8.97 NS NS 3055 4030 0.00
NS NS 3057 5060 7.08 NS NS 3564 NS NS 3030 14.5 3566 NS NS 3930
20.5 3568 NS NS 3450 10.3 Mean 4370 5.35 3470 15.1 SD 598 4.73 450
5.13 % CV 13.7 88.4 13.0 33.9 N 3 3 3 3 15 Day 1 2 3054 2510 25.9
NS NS 3058 2990 13.1 NS NS 3074 3010 15.1 NS NS 3565 NS NS 2460
10.9 3567 NS NS 1860 41.2 3569 NS NS 3380 34.1 Mean 2840 18.0 2570
28.7 SD 283 6.89 766 15.8 % CV 9.98 38.2 29.8 55.2 N 3 3 3 3 15 Day
1 8 3053 10.7 35.8 NS NS 3055 8.23 29.5 NS NS 3057 9.85 33.0 NS NS
3564 NS NS 5.05 37.3 3566 NS NS 7.97 37.7 3568 NS NS 6.57 40.7 Mean
9.59 32.8 6.53 38.6 SD 1.25 3.16 1.46 1.86 % CV 13.1 9.63 22.4 4.82
N 3 3 3 3 15 Day 1 24 3054 1.20 20.5 NS NS 3058 1.10 14.0 NS NS
3074 1.36 15.5 NS NS 3565 NS NS 2.32 24.6 3567 NS NS 1.25 23.2 3569
NS NS 2.48 18.6 Mean 1.22 16.7 2.02 22.1 SD 0.131 3.40 0.669 3.14 %
CV 10.7 20.4 33.2 14.2 N 3 3 3 3 15 Day 7 0 3053 0.00 0.00 NS NS
3055 0.00 0.00 NS NS 3057 0.00 0.00 NS NS 3564 NS NS 0.00 0.00 3566
NS NS 0.00 0.00 3568 NS NS 0.00 0.00 Mean 0.00 0.00 0.00 0.00 SD
0.00 0.00 0.00 0.00 % CV NC NC NC NC N 3 3 3 3 15 Day 7 2 3054 1640
38.8 NS NS 3058 1940 15.0 NS NS 3074 2150 18.3 NS NS 3565 NS NS
2220 20.2 3567 NS NS 995 49.1 3569 NS NS 1750 34.2 Mean 1910 24.0
1660 34.5 SD 256 12.9 618 14.5 % CV 13.4 53.7 37.3 41.9 N 3 3 3 3
15 Day 7 8 3053 9.82 38.4 NS NS 3055 7.60 31.1 NS NS 3057 8.73 28.8
NS NS 3564 NS NS 5.32 36.4 3566 NS NS 6.27 54.2 3568 NS NS 4.75
40.2 Mean 8.72 32.8 5.45 43.6 SD 1.11 5.01 0.768 9.37 % CV 12.7
15.3 14.1 21.5 N 3 3 3 3 15 Day 7 24 3054 1.85 27.2 NS NS 3058 1.32
17.9 NS NS 3074 1.17 19.9 NS NS 3565 NS NS 2.37 30.0 3567 NS NS
0.00 7.10 3569 NS NS 0.00 23.8 Mean 1.45 21.7 0.790 20.3 SD 0.357
4.90 1.37 11.8 % CV 24.7 22.6 173 58.3 N 3 3 3 3 .sup.aImputations
of zero were made for samples with bioanalytical result of BLQ.
TABLE-US-00064 TABLE 61 Measured and Imputed brincidofovir
Concentration Data for Mean Generation Period Dose Time Day 1 Day 7
Analyte (mg/kg) (h) Subject Sex C (ng/mL) IC (ng/mL) C (ng/mL) IC
(ng/mL) BCV 0 2 1015 Male BLQ 0.00 BLQ 0.00 0 2 1016 Male BLQ 0.00
BLQ 0.00 0 2 1017 Male BLQ 0.00 BLQ 0.00 0 2 1523 Female BLQ 0.00
BLQ 0.00 0 2 1524 Female BLQ 0.00 BLQ 0.00 0 2 1525 Female BLQ 0.00
BLQ 0.00 1 0 2031 Male NS NS BLQ 0.00 1 0 2033 Male NS NS BLQ 0.00
1 0 2035 Male NS NS BLQ 0.00 1 0 2542 Female NS NS BLQ 0.00 1 0
2544 Female NS NS BLQ 0.00 1 0 2546 Female NS NS BLQ 0.00 1 1 2031
Male 156 156 NS NS 1 1 2033 Male 244 244 NS NS 1 1 2035 Male 280
280 NS NS 1 1 2542 Female 109 109 NS NS 1 1 2544 Female 126 126 NS
NS 1 1 2546 Female 177 177 NS NS 1 2 2032 Male 220 220 255 255 1 2
2034 Male 283 283 189 189 1 2 2036 Male 203 203 179 179 1 2 2543
Female 253 253 115 115 1 2 2547 Female 164 164 82.1 82.1 1 2 2572
Female 155 155 89.2 89.2 1 8 2031 Male BLQ 0.00 BLQ 0.00 1 8 2033
Male BLQ 0.00 BLQ 0.00 1 8 2035 Male BLQ 0.00 BLQ 0.00 1 8 2542
Female 2.30 2.30 6.58 6.58 1 8 2544 Female 1.45 1.45 BLQ 0.00 1 8
2546 Female BLQ 0.00 BLQ 0.00 1 24 2032 Male BLQ 0.00 BLQ 0.00 1 24
2034 Male BLQ 0.00 BLQ 0.00 1 24 2036 Male BLQ 0.00 BLQ 0.00 1 24
2543 Female BLQ 0.00 BLQ 0.00 1 24 2547 Female BLQ 0.00 BLQ 0.00 1
24 2572 Female BLQ 0.00 BLQ 0.00 15 0 3053 Male NS NS BLQ 0.00 15 0
3055 Male NS NS BLQ 0.00 15 0 3057 Male NS NS BLQ 0.00 15 0 3564
Female NS NS BLQ 0.00 15 0 3566 Female NS NS BLQ 0.00 15 0 3568
Female NS NS BLQ 0.00 15 1 3053 Male 4020 4020 NS NS 15 1 3055 Male
4030 4030 NS NS 15 1 3057 Male 5060 5060 NS NS 15 1 3564 Female
3030 3030 NS NS 15 1 3566 Female 3930 3930 NS NS 15 1 3568 Female
3450 3450 NS NS 15 2 3054 Male 2510 2510 1640 1640 15 2 3058 Male
2990 2990 1940 1940 15 2 3074 Male 3010 3010 2150 2150 15 2 3565
Female 2460 2460 2220 2220 15 2 3567 Female 1860 1860 995 995 15 2
3569 Female 3380 3380 1750 1750 15 8 3053 Male 10.7 10.7 9.82 9.82
15 8 3055 Male 8.23 8.23 7.60 7.60 15 8 3057 Male 9.85 9.85 8.73
8.73 15 8 3564 Female 5.05 5.05 5.32 5.32 15 8 3566 Female 7.97
7.97 6.27 6.27 15 8 3568 Female 6.57 6.57 4.75 4.75 15 24 3054 Male
1.20 1.20 1.85 1.85 15 24 3058 Male 1.10 1.10 1.32 1.32 15 24 3074
Male 1.36 1.36 1.17 1.17 15 24 3565 Female 2.32 2.32 2.37 2.37 15
24 3567 Female 1.25 1.25 BLQ 0.00 15 24 3569 Female 2.48 2.48 BLQ
0.00
TABLE-US-00065 TABLE 62 Measured and Imputed Cidofovir
Concentration Data for Mean Generation Period Dose_Level Time Day 1
Day 7 Analyte (mg/kg) (h) Subject Sex C (ng/mL) IC (ng/mL) C
(ng/mL) IC (ng/mL) cidofovir 0 2 1015 Male BLQ 0.00 BLQ 0.00 0 2
1016 Male BLQ 0.00 BLQ 0.00 0 2 1017 Male BLQ 0.00 BLQ 0.00 0 2
1523 Female BLQ 0.00 BLQ 0.00 0 2 1524 Female BLQ 0.00 BLQ 0.00 0 2
1525 Female BLQ 0.00 BLQ 0.00 1 0 2031 Male NS NS BLQ 0.00 1 0 2033
Male NS NS BLQ 0.00 1 0 2035 Male NS NS BLQ 0.00 1 0 2542 Female NS
NS BLQ 0.00 1 0 2544 Female NS NS BLQ 0.00 1 0 2546 Female NS NS
BLQ 0.00 1 1 2031 Male BLQ 0.00 NS NS 1 1 2033 Male BLQ 0.00 NS NS
1 1 2035 Male BLQ 0.00 NS NS 1 1 2542 Female BLQ 0.00 NS NS 1 1
2544 Female BLQ 0.00 NS NS 1 1 2546 Female BLQ 0.00 NS NS 1 2 2032
Male BLQ 0.00 BLQ 0.00 1 2 2034 Male BLQ 0.00 BLQ 0.00 1 2 2036
Male BLQ 0.00 BLQ 0.00 1 2 2543 Female 9.62 9.62 13.5 13.5 1 2 2547
Female 6.22 6.22 13.8 13.8 1 2 2572 Female 12.2 12.2 17.6 17.6 1 8
2031 Male 5.85 5.85 5.67 5.67 1 8 2033 Male BLQ 0.00 5.45 5.45 1 8
2035 Male BLQ 0.00 6.07 6.07 1 8 2542 Female 7.12 7.12 9.51 9.51 1
8 2544 Female 5.60 5.60 7.84 7.84 1 8 2546 Female BLQ 0.00 5.36
5.36 1 24 2032 Male BLQ 0.00 BLQ 0.00 1 24 2034 Male BLQ 0.00 BLQ
0.00 1 24 2036 Male BLQ 0.00 BLQ 0.00 1 24 2543 Female BLQ 0.00 BLQ
0.00 1 24 2547 Female BLQ 0.00 BLQ 0.00 1 24 2572 Female BLQ 0.00
BLQ 0.00 15 0 3053 Male NS NS BLQ 0.00 15 0 3055 Male NS NS BLQ
0.00 15 0 3057 Male NS NS BLQ 0.00 15 0 3564 Female NS NS BLQ 0.00
15 0 3566 Female NS NS BLQ 0.00 15 0 3568 Female NS NS BLQ 0.00 15
1 3053 Male 8.97 8.97 NS NS 15 1 3055 Male BLQ 0.00 NS NS 15 1 3057
Male 7.08 7.08 NS NS 15 1 3564 Female 14.5 14.5 NS NS 15 1 3566
Female 20.5 20.5 NS NS 15 1 3568 Female 10.3 10.3 NS NS 15 2 3054
Male 25.9 25.9 38.8 38.8 15 2 3058 Male 13.1 13.1 15.0 15.0 15 2
3074 Male 15.1 15.1 18.3 18.3 15 2 3565 Female 10.9 10.9 20.2 20.2
15 2 3567 Female 41.2 41.2 49.1 49.1 15 2 3569 Female 34.1 34.1
34.2 34.2 15 8 3053 Male 35.8 35.8 38.4 38.4 15 8 3055 Male 29.5
29.5 31.1 31.1 15 8 3057 Male 33.0 33.0 28.8 28.8 15 8 3564 Female
37.3 37.3 36.4 36.4 15 8 3566 Female 37.7 37.7 54.2 54.2 15 8 3568
Female 40.7 40.7 40.2 40.2 15 24 3054 Male 20.5 20.5 27.2 27.2 15
24 3058 Male 14.0 14.0 17.9 17.9 15 24 3074 Male 15.5 15.5 19.9
19.9 15 24 3565 Female 24.6 24.6 30.0 30.0 15 24 3567 Female 23.2
23.2 7.10 7.10 15 24 3569 Female 18.6 18.6 23.8 23.8
TABLE-US-00066 TABLE 63 Mean and Imputed brincidofovir
Concentration Data for NCA Period Day 1 Day 7 Dose Time MC IMC MC
IMC Analyte (mg/kg) Sex (h) (ng/mL) (ng/mL) (ng/mL) (ng/mL) BCV 1
Female 0 NS NS 0.00 0.00 1 Female 1 137 137 NS NS 1 Female 2 191
191 95.4 95.4 1 Female 8 1.25 1.25 2.19 2.19 1 Female 24 0.00
missing 0.00 missing 1 Male 0 NS NS 0.00 0.00 1 Male 1 227 227 NS
NS 1 Male 2 235 235 208 208 1 Male 8 0.00 missing 0.00 missing 1
Male 24 0.00 missing 0.00 missing 15 Female 0 NS NS 0.00 0.00 15
Female 1 3470 3470 NS NS 15 Female 2 2570 2570 1660 1660 15 Female
8 6.53 6.53 5.45 5.45 15 Female 24 2.02 2.02 0.790 0.790 15 Male 0
NS NS 0.00 0.00 15 Male 1 4370 4370 NS NS 15 Male 2 2840 2840 1910
1910 15 Male 8 9.59 9.59 8.72 8.72 15 Male 24 1.22 1.22 1.45
1.45
TABLE-US-00067 TABLE 64 Mean and Imputed cidofovir Concentration
Data for NCA Period Day 1 Day 7 Dose Time MC IMC MC IMC Analyte
(mg/kg) Sex (h) (ng/mL) (ng/mL) (ng/mL) (ng/mL) cidofovir 1 Female
0 NS NS 0.00 0.00 1 Female 1 0.00 0.00 NS NS 1 Female 2 9.35 9.35
15.0 15.0 1 Female 8 4.24 4.24 7.57 7.57 1 Female 24 0.00 missing
0.00 missing 1 Male 0 NS NS 0.00 0.00 1 Male 1 0.00 0.00 NS NS 1
Male 2 0.00 0.00 0.00 0.00 1 Male 8 1.95 1.95 5.73 5.73 1 Male 24
0.00 missing 0.00 missing 15 Female 0 NS NS 0.00 0.00 15 Female 1
15.1 15.1 NS NS 15 Female 2 28.7 28.7 34.5 34.5 15 Female 8 38.6
38.6 43.6 43.6 15 Female 24 22.1 22.1 20.3 20.3 15 Male 0 NS NS
0.00 0.00 15 Male 1 5.35 5.35 NS NS 15 Male 2 18.0 18.0 24.0 24.0
15 Male 8 32.8 32.8 32.8 32.8 15 Male 24 16.7 16.7 21.7 21.7
Example 9--Mass Balance, Pharmacokinetics and Tissue Distribution
by Quantitative Whole-Body Autoradiography in Rats Following a
Single Oral or Intravenous Dose of [.sup.14C]Brincidofovir
[0560] The objectives of this study were to characterize the tissue
distribution of total radioactivity in male Sprague Dawley (SD) and
Long-Evans (LE) rats following administration of a single
intravenous (IV, 2-h infusion) or oral (PO, gavage) dose of
[.sup.14C]brincidofovir. In addition, the rate and extent of
excretion (mass balance) and pharmacokinetics (PK) of total
radioactivity in male Sprague Dawley rats following a single
intravenous (IV, 2-h infusion) dose of [.sup.14C]brincidofovir was
examined. Residual plasma and excreta collected on this study were
stored at -70.degree. C. for metabolite profile and identification
experiment conducted under a different protocol.
Materials and Methods
[0561] The study utilized 4 groups of male SD rats (albino), and 3
groups of male LE rats (pigmented); a total of 39 rats. All animals
were obtained from Hilltop Lab Animals, Inc. (Scottdale, Pa.). Rats
weighed between 214 and 265 g at dosing. Rats in Groups 1, 2, 3,
and 4 were administered a single 2-hour (h) IV infusion of
[.sup.14C]brincidofovir at a target dose of 15 mg/kg. Rats in Group
5 were administered a single 2-h IV infusion of
[.sup.14C]brincidofovir at a target dose of 2 mg/kg. Rats in Group
6, and 7 were administered a single oral gavage administration of
[.sup.14C]brincidofovir at a target dose of 15 mg/kg. Three dosing
formulations (one for high dose IV, one for low dose IV, and one
for PO dose) were prepared at QPS on the day of dosing. The IV
formulation for Groups 1, 2, 3, and 4 contained a vehicle of 10 mM
sodium phosphate buffer in 5% dextrose solution at pH 8.0(.+-.0.1).
The IV formulation for Group 5 contained a vehicle of 16 mM sodium
phosphate buffer in 5% dextrose solution at pH 8.0(.+-.0.1), and
the PO dosing formulation contained a vehicle of 12.5 mM sodium
phosphate buffer at pH 8.0(.+-.0.1). The IV dose formulation for
Group 5 and the PO dose formulation for Groups 6 and 7 contained
higher concentration of buffer so that the final buffer
concentration was approximately 10 mM in all dose formulations.
Test Animals
[0562] Sixteen adult male SD rats, and 23 adult male LE rats, which
were obtained from Hilltop Lab Animals, Inc. (Scottdale, Pa.), were
placed on this study. Rats in Groups 1-5 were surgically modified
to have indwelling femoral vein cannulas (FVC) for IV infusion. The
body weight range, source, vendor, and receipt date of the animals
was documented in the raw data. Animals placed on study were
assigned a permanent identification number using a permanent marker
on the tail, while unused spare animals were returned to stock
after successful dose administration of animals placed on study.
Randomization was performed by cannula patency and QPS SOP.
[0563] Tables 65 and 66 give summaries of the dosing and sample
collection protocols.
TABLE-US-00068 TABLE 65 In-Life Study Dosing Summary No. of Target
Dose Target Target Dose Target Dose Target Dose Group Strain/Dose
Animals, Level Radioactivity Date Volume Conc. No. Study route Sex
(mg/kg) Level (.mu.Ci/kg) (mL/kg/g).sup.b (mL/kg) (mg/mL) 1 MB
SD/IV.sup.a 3M 15 mg/kg 180 10 20 0.75 mg/mL 2 PK SD/IV.sup.a 7M 15
mg/kg 180 10 20 0.75 mg/mL 3 QWBA SD/IV.sup.a 3M 15 mg/kg 180 10 20
0.75 mg/mL 4 QWBA LE/IV.sup.a 9M 15 mg/kg 180 10 20 0.75 mg/mL 5
QWBA LE/IV.sup.a 4M 2 mg/kg 200 1.33 2.66 0.75 mg/mL 6 QWBA SD/PO
3M 15 mg/kg 200 -- 5 3 mg/mL 7 QWBA LE/PO 10M 15 mg/kg 200 -- 5 3
mg/mL .sup.aFemoral vein-cannulated; .sup.b2-h infusion time
TABLE-US-00069 TABLE 66 In-Life Sample Collection Summary Purpose,
Group ROA, Cage Terminal Blood Number Strain Urine.sup.a
Feces.sup.a Residues Carcass and Plasma 1 MB, IV, SD Pre-dose, 0-8,
Pre-dose, 0- Daily Cage Frozen NA 8-24, and at 24 and at 24 Rinse
following 24 h intervals h intervals beginning at euthanasia and
through 168 h through 168 24 h, and retained for post-dose; h
post-dose; cage wash potential N = 3 N = 3 and wipes at analysis
termination; N = 3 2 PK, IV, SD NA NA NA NA At 0.5, 1, 2 (end of
infusion), 4, 8, 24, and 72 h; N = 1/time point 3 QWBA, NA NA NA At
2 (end of At 2 IV, SD infusion), 24, (end of infusion), and 168 h;
24, and 168 h; N = 1/time point N = 1/time point 4 QWBA, NA NA NA
At 1, 2 (end of At 1 ,2 IV, LE infusion), 4, 8, (end of infusion),
24, 72, 96, 168 4, 8, 24, 72, 96, h and TBD; 168 and 840 h; N =
1/time point N = 1/time point 5 QWBA, NA NA NA At 2 (end of At 2
IV, LE infusion), 8, 24, (end of infusion), and 72 h 8, 24, and 72
h 6 QWBA, NA NA NA At 2, 24, and At 2, 24, and PO, SD 168 h post-
168 h post-dose; dose; N = 1/time N = 1/time point point 7 QWBA, NA
NA NA At 0.5, 1,2, 4, 8, At 0.5, 1, 2, 4, 8, PO, LE 24, 72, 96, 168
24, 72, 96, 168 h and TBD; and 840 h; N = 1/time point N = 1/time
point ROA = Route of Administration NA = Not Applicable TBD = To be
Determined .sup.aSamples collected over dry ice
Dose Administration
[0564] Animal body weights, which were used for determining the
dosing volume, were measured before dosing on the day of dose
administration. Dose assay results, animal body weights, and
targeted dosing parameters were entered into the Debra LIMS and
target dose volumes for each animal were determined by the Debra
LIMS. Animals received a single 2-h IV infusion of
[.sup.14C]brincidofovir at a target oral dose of 15 mg/kg (Groups
1-4) or 2 mg/kg (Group 5), or an oral gavage administration of
[.sup.14C]brincidofovir at a target oral dose of 15 mg/kg (Groups 6
and 7). The actual dose administered to each rat was determined by
the Debra LIMS, which used individual animal body weight data and
by subtracting the weight of the emptied dose syringe/needle or
needle/infusion line after dosing from the weight of the full
syringe/needle or needle/infusion line prior to dosing. The mean
pre-dose radioactivity concentration (dpm/g dosing solution) was
multiplied by the net weight of the administered dosing solution to
calculate the amount of radioactivity administered to each animal.
Actual doses are presented in Table 67.
TABLE-US-00070 TABLE 67 Body Weights and Doses of
[.sup.14C]brincidofovir Administered to Male Sprague Dawley Rats
(Groups 1, 2, 3, and 6), and Male Long-Evans Rats (Groups 4, 5, and
7) Amount of mg Test Animal Animal Formulation .mu.Ci Article mg/kg
No. Weight (g) Administered (g) Administered .mu.Ci/kg
Administered.sup.a,b Administered.sup.a,b Gp. 1, Mass Balance, Male
SD Rat, 2-h IV, 15 mg/kg 1 251.2 5.0069 50.607 201.459 3.729 14.847
2 250.2 5.0375 50.916 203.501 3.752 14.997 3 261.9 5.2909 53.477
204.189 3.941 15.048 MEAN 254.4 5.1120 51.667 203.050 3.807 14.964
SD 6.5 0.1560 1.575 1.420 0.116 0.104 Gp. 2, PK, Male SD Rats, 2-h
IV, 15 mg/kg 4c 251.8 1.3636 13.782 54.736 1.016 4.034 5c 250.0
2.4727 24.992 99.970 1.842 7.367 6 242.3 4.7611 48.122 198.606
3.546 14.636 7 249.9 5.1748 52.304 209.298 3.855 15.424 8 261.6
5.2120 52.680 201.375 3.882 14.840 9 264.6 5.2057 52.616 198.851
3.878 14.654 10 254.0 5.0652 51.196 201.558 3.773 14.854 MEAN 254.5
5.084 51.384 201.938 3.787 14.882 SD 9.0 0.190 1.918 4.338 0.142
0.320 Gp. 3, QWBA, Male SD Rat, 2-h IV, 15 mg/kg 11 247.3 4.9350
49.880 201.698 3.676 14.864 12 256.6 5.0999 51.547 200.883 3.799
14.804 13 248.4 4.8006 48.521 195.336 3.576 14.395 MEAN 250.8 4.945
49.983 199.306 3.684 14.688 SD 5.1 0.150 1.516 3.462 0.112 0.255
Gp. 4, QWBA, Male LE Rats, 2-h IV 15 mg/kg 14c 240.5 2.3933 24.190
100.582 1.783 7.412 15 232.1 4.5816 46.308 199.517 3.413 14.703 16
234.9 4.6662 47.163 200.779 3.476 14.796 17 236.8 4.7293 47.801
201.861 3.523 14.876 18 214.4 4.2683 43.141 201.219 3.179 14.829 19
247.3 4.9154 49.682 200.897 3.661 14.805 20 237.9 4.7154 47.660
200.337 3.512 14.764 21 242.6 4.7892 48.406 199.531 3.567 14.705 22
235.0 4.5292 45.778 194.801 3.374 14.356 MEAN 235.1 4.649 46.992
199.868.sup.d 3.463 14.729 SD 9.7 0.195 1.969 2.198 0.145 0.162 Gp.
5, QWBA, Male LE Rat, 2-h IV 2 mg/kg 23 231.9 0.6395 52.998 228.538
0.495 2.133 24 244.6 0.6611 54.788 223.990 0.511 2.090 25 231.8
0.6303 52.235 225.347 0.487 2.103 26 243.5 0.7172 59.437 244.095
0.555 2.278 MEAN 238.0 0.662 54.865 230.493 0.512 2.151 SD 7.1
0.039 3.231 9.267 0.030 0.087 Gp. 6, QWBA, Male SD Rats, PO, 15
mg/kg 27 250.7 1.2270 51.798 206.613 3.630 14.480 28 231.2 1.1460
48.379 209.250 3.390 14.665 29 254.8 1.3077 55.205 216.659 3.869
15.184 MEAN 245.6 1.227 51.794 210.841 3.630 14.776 SD 12.6 0.081
3.413 5.208 0.240 0.365 Gp. 7, QWBA, Male LE Rats, PO, 15 mg/kg 30
229.4 1.1586 48.910 213.210 3.428 14.942 31 236.1 1.1913 50.291
213.007 3.525 14.928 32 254.6 1.2685 53.550 210.330 3.753 14.740 33
234.1 1.1959 50.485 215.656 3.538 15.114 34 223.1 1.1223 47.378
212.362 3.320 14.883 35 246.6 1.1773 49.700 201.541 3.483 14.124 36
231.3 1.1473 48.433 209.397 3.394 14.675 37 257.7 1.2626 53.301
206.833 3.735 14.495 38 234.7 1.2077 50.983 217.227 3.573 15.224 39
238.5 1.2192 51.469 215.802 3.607 15.124 MEAN 238.6 1.195 50.450
211.537 3.536 14.825 SD 11.1 0.047 1.987 4.729 0.139 0.332
.sup.aSpecific activity of the oral (PO) formulation was 14.2689
.mu.Ci/mg [.sup.14C]brincidofovir. Dose preparation activity of the
PO formulation was 42.2151 .mu.Ci/g .sup.bSpecific activity of the
intravenous (IV) infusion formulations were 13.5694 (at 15 mg/kg)
and 101.5100 (at 2 mg/kg) .mu.Ci/mg [.sup.14C]brincidofovir. Dose
preparation activity of the IV formulations were 10.1074 .mu.Ci/g
(at 15 mg/kg) and 82.8740 .mu.Ci/g (at 2 mg/kg) .sup.cAnimal
terminated during IV infusion for sample collection. These animals
were omitted from the mean dose calculations. .sup.dThe mean for
all animals dosed in Group 4, 188.836 .mu.Ci/kg was used for
dosimetry estimate. Note: SD = Standard deviation; MB = Mass
Balance; PK = pharmacokinetics; QWBA = Quantitative Whole-Body
Autoradiography
Sample Collection
[0565] Urine, feces, blood/plasma, cage residue samples, and
carcasses were collected during this study as described below for
each group
Excreta Collection (Group 1)
[0566] Three male albino rats were assigned to Group 1 (IV
infusion) for excreta collection.
[0567] Urine samples were collected from each animal into
pre-labeled urine collection tubes at pre-dose (overnight) and at
intervals of 0-8 h, 8-24 h, and at every subsequent 24 h interval
until 168 h post-dose. All urine specimens were collected over dry
ice. The total weight of each urine collection was documented, and
the samples were stored frozen at approximately -70.degree. C.
until LSC analysis. Samples were maintained at approximately
-70.degree. C. following analysis, and were saved for further
analysis, to be conducted under a separate protocol.
[0568] Feces samples were collected from each animal into a
pre-labeled feces collection tube at pre-dose (overnight) and at 24
h intervals post-dose until 168 h post-dose. Feces specimens were
collected over dry ice and the total weight of each feces sample
was documented. Feces samples were stored at approximately
-70.degree. C. until homogenization and LSC analysis, were
maintained at approximately -70.degree. C. following analysis, and
were saved for further analysis, to be conducted under a separate
protocol.
[0569] Cage residue specimens were collected. Cages were rinsed
with approximately 30 mL of deionized water following each daily
post-dose excreta collection beginning at 24 h post-dose. The
interior surfaces of the metabolism cage were sprayed with
approximately 90 mL of Windex solution (or equivalent detergent
solution) and wiped with gauze pads following the final excreta
collection. Cage rinse and cage wash specimens were collected into
tared and pre-labeled containers. The total weight of each cage
rinse and wash were documented. Cage rinse, cage wash, and cage
wipe collections were stored at approximately -20.degree. C.
Carcasses of animals in Groups 1 and 2 were retained at
approximately -20.degree. C. Carcasses and cage residues were not
analyzed because the recovery of radioactivity in excreta was
>90%.
Plasma Pharmacokinetic Sample Collection (Group 2)
[0570] Blood was collected from each animal in Group 2 (IV
infusion) for plasma and blood analysis at the times listed
below.
[0571] Terminal blood samples were collected by cardiac puncture
into pre-labeled blood collection tubes that contained K.sub.2EDTA
as an anticoagulant. A terminal sample (N=1 per time point) was
collected at 0.5, 1, 2, 4, 8, 24, and 72 h after initiation of
infusion and the volume was not limited. Triplicate weighed
aliquots of blood (.about.0.100 g) were removed and analyzed for
total radioactivity using combustion followed by LSC. Blood
aliquots were maintained at approximately 4.degree. C. until
analysis. The remaining blood samples were maintained on wet ice
(approximately 4.degree. C.) and then centrifuged to obtain plasma
within 1 h of the blood collection time. Centrifuge settings were
recorded in the study notebook. Duplicate aliquots of plasma (0.050
mL) were analyzed for radioactivity using direct counting by LSC,
and residual plasma was stored at approximately -70.degree. C. and
was saved for possible future analysis under a separate study
protocol. Residual red blood cells and the animal carcasses of
Group 2 rats were discarded as radioactive waste.
QWBA (Groups 3-7)
[0572] Each animal, at their respective termination time points,
was euthanized for QWBA analysis immediately after blood
collection. One rat per time point per Group was euthanized as
listed below.
Group 3 (IV)--2 (end of infusion), 24, and 168 h Group 4 (IV)--1, 2
(end of infusion), 4, 8, 24, 72, 96, 168, and 840 h Group 5 (IV)--2
(end of infusion), 8, 24, and 72 h
Group 6 (PO)--2, 24, and 168 h
Group 7 (PO)--0.5, 1, 2, 4, 8, 24, 72, 96, 168, and 840 h
[0573] Each rat was deeply anesthetized via isoflurane anesthesia,
a blood sample was collected by cardiocentesis (approximately 2 mL)
into tubes that contained K.sub.2EDTA as the anticoagulant, and the
rat was euthanized by being frozen in a hexane/solid carbon dioxide
bath for at least 15 min. The blood samples were maintained on wet
ice (approximately 4.degree. C.) and then centrifuged to obtain
plasma within 1 h of the blood collection time. Centrifuge settings
were recorded in the study notebook. Duplicate aliquots of plasma
(0.050 mL) were analyzed for radioactivity using direct counting by
LSC, and residual plasma was stored at approximately -70.degree. C.
and was saved for possible future analysis under a separate study
protocol. Residual red blood cells were discarded as radioactive
waste.
Sample Storage Conditions
[0574] Carcass and cage residue samples were stored frozen at
-20.degree. C. until sample analysis, and residual plasma and
excreta were stored frozen at -70.degree. C.
Sample Analysis
LSC Analysis of Blood, Plasma, and Excreta
[0575] Feces were homogenized and analyzed for total radioactivity
content. Weighed feces specimens for each rat were homogenized in
approximately 3 volumes of water (approximately 3.times. the weight
of the feces specimen). The total weight of each homogenate was
determined and triplicate weighed aliquots (.about.0.5 g) were
combusted in a Packard Sample Oxidizer, followed by LSC analysis.
The actual weights of the individual aliquots and the amount of
solvent used in homogenization were recorded. Pre-weighed portions
of each feces homogenate were placed into a CombustoCone.RTM. that
contained a CombustoPad.RTM., allowed to dry over-night in a fume
hood, and were burned completely in the sample oxidizer.
[0576] Triplicate weighed aliquots (0.100 g) of blood aliquots
(Group 2 only) were combusted in the Packard Sample Oxidizer,
followed by LSC analysis.
[0577] The .sup.14CO2 liberated in combustion was trapped in a
solution of Carbo-Sorb.RTM., Permafluor.RTM. (scintillation fluid)
was added, and the radioactivity was determined by LSC. Blank cones
that contained a known amount of radioactivity (0.100 mL
SpecChec.RTM.) were oxidized and compared to non-combusted
standards to determine the percent recovery from the combustion
process. These controls were analyzed in triplicate, and
quantitative recovery of .sup.14C from these control samples was
determined on each day of sample combustion. Blank cones (without
added radioactivity) were burned to evaluate the radioactivity
carryover between samples.
[0578] Fecal homogenates were maintained at approximately
-70.degree. C. after analysis and were saved for further analysis
under a separate study protocol.
[0579] Urine (0.300 mL), and plasma (0.050 mL) specimens were
thawed, if necessary, then aliquoted in duplicate by volume and
analyzed by LSC. Volumes or weights of sample aliquots were
documented in the study records. Ultima Gold scintillation fluid (5
mL, PerkinElmer) was added to each urine, and plasma aliquot,
aliquot were mixed thoroughly, and then analyzed by LSC for
radioactivity.
[0580] The radioactivity (counts per minute) in each sample was
converted to disintegrations per minute (dpm) by means of an
external standardization and a quench curve. Radioactivity content
was quantified by a Model 2800TR or Model 2900TR Liquid
Scintillation Analyzer (PerkinElmer). All samples were counted for
at least 5 minutes or at least 100,000 counts per minute (cpm). LSC
results for duplicate samples that differed by more than 10% from
the mean value, were re-aliquoted and re-analyzed, if sufficient
volume was available. If the LSC results for triplicate samples had
a % CV that was >10%, then the sample was re-homogenized and
re-analyzed, if sufficient volume was available. These
specifications were applied to all sample aliquots that had an
average radioactivity greater than 500 dpm, and values that were
under 500 dpm were accepted as is. A lower limit of quantification
(LLOQ) was applied to the data equal to 2 times the counts per
minute (CPM) value of a background sample.
QWBA
[0581] The pinna, distal limbs, hair, and tail were removed from
each frozen carcass and each frozen carcass was embedded in an
aqueous suspension of approximately 2% (w/v) carboxymethylcellulose
and frozen into a block. The blocks were stored at approximately
-20.degree. C. prior to sectioning. Each blocked carcass was
mounted on the object stage of a cryomicrotome (Leica CM3600
Cryomacrocut, Nussloch, Germany and Vibratome 9800, St. Louis, Mo.)
maintained at approximately -20.degree. C. and internal standards
(3), which were plasma fortified with [.sup.14C]-glucose at one
concentration (approximately 0.05 .mu.Ci/mL), were placed into the
frozen blocks, prior to sectioning, and were used for section
thickness quality control. A number of whole-body sections
(approximately 40 .mu.m thick) were taken in the sagittal plane at
various levels of interest using a whole-body cryomicrotome set at
-20.degree. C. Sagittal sections (20-50 .mu.m thick) of frozen
carcasses were exposed to phosphor imaging plates. Regions of
images in tissues were converted to concentrations using standards.
All of the major tissues, organs, and biological fluids were
represented. The sections were collected on adhesive tape (Scotch
8210, 3M Corp., USA) and dehydrated in the cryomicrotome for at
least 48 h prior to removal for mounting and exposure.
[0582] Tissue concentration data was determined for the following
tissues and/or contents: adipose (brown and white), adrenal gland,
bile (in duct), blood (cardiac), bone, bone marrow, brain
(cerebrum, cerebellum, medulla), cecum (and contents), large
intestine (and contents), epididymis, esophagus, eye (uvea and
lens), Harderian gland, heart, kidney (cortex and medulla), liver,
lung, lymph node, pancreas, pituitary gland, prostate gland,
salivary gland, seminal vesicles, skeletal muscle, skin (pigmented
and non-pigmented), small intestine (and contents), stomach
(gastric mucosa and contents), spleen, spinal cord, testis, thymus,
thyroid, and urinary bladder (and contents).
[0583] A set of whole-body sections for each rat was mounted on a
cardboard backing, covered with a thin plastic wrap, and exposed
along with calibration standards, which were .sup.14C-glucose mixed
with blood at 10 different concentrations (0.0009595 to 7.806
.mu.Ci/g), to a .sup.14C-sensitive phosphor imaging plate (Fuji
Biomedical, Stamford, Conn.). The imaging plate and sections were
placed in light-tight exposure cassettes, in a copper-lined lead
safe, for a 4-day exposure at room temperature. The imaging plate
was scanned using the Typhoon 9410 image acquisition system
(GE/Molecular Dynamics, Sunnyvale, Calif., USA) and the resultant
image stored on a dedicated QPS computer server. Quantification was
performed by image densitometry using MCD image analysis software
(v. 7.0, Interfocus Imaging Ltd) and a standard curve constructed
from the integrated response [i.e., Molecular Dynamics Counts per
square millimeter (MDC/mm.sup.2)] and the nominal concentrations of
the .sup.14C-calibration standards. The concentrations of
radioactivity were expressed as the .mu.g equivalents of
[.sup.14C]brincidofovir per gram sample (.mu.g equiv/g). A lower
limit of quantification (LLOQ) was applied to the data (See section
6.7.4).
[0584] Tissue concentrations that fell below the LLOQ were
identified as being below the quantification limit (BQL). Tissue
areas that were not visualized on autoradiographic images were
identified as no sample (NS) and reported as BQL. If no tissues
were visualized on the autoradiograph(s) for an animal, then no
calibration curves were generated and all tissue concentrations for
that animal were reported as BQL.
[0585] The results are based on original, electronic, digital
images that were selected from a complete set of
autoradiographs.
Animal Observations
[0586] No observable abnormalities were observed in the rats on
this study.
Group 1--Mass Balance in Rats after a 2-H Intravenous Infusion
[0587] A summary of the individual animal and mean excretion data
for total radioactivity in Group 1 male SD rats is presented in
Table 68.
TABLE-US-00071 TABLE 68 Excretion of Total Radioactivity Following
an 2-Hour Intravenous Infusion of [.sup.14C]brincidofovir to Male
Sprague Dawley Rats at a Target Dose of 15 mg/kg (Group 1) Percent
Recovery (% Dose) Rat Numbers Sample Time Point 01 02 03 Mean SD
Urine Pre-dose 0.0 0.0 0.0 0.0 0.0 0-8 h 33.0 29.3 30.7 31.0 1.9
8-24 h 10.7 11.5 16.2 12.8 3.0 24-48 h 4.3 3.6 4.5 4.1 0.5 48-72 h
1.6 1.6 1.9 1.7 0.2 72-96 h 0.8 0.8 0.8 0.8 0.0 96-120 h 0.4 0.3
0.5 0.4 0.1 120-144 h 0.2 0.2 0.3 0.2 0.0 144-168 h 0.2 0.1 0.2 0.2
0.0 Subtotal 51.2 47.5 55.1 51.2 3.8 Feces Pre-dose 0.0 0.0 0.0 0.0
0.0 0-24 h 37.3 37.6 35.1 36.7 1.4 24-48 h 3.4 4.6 3.5 3.8 0.7
48-72 h 0.9 1.0 0.8 0.9 0.1 72-96 h 0.4 0.4 0.4 0.4 0.0 96-120 h
0.2 0.2 0.2 0.2 0.0 120-144 h 0.1 0.1 0.1 0.1 0.0 144-168 h 0.1 0.1
0.1 0.1 0.0 Subtotal 42.4 44.2 40.2 42.2 2.0 Total 93.6 91.6 95.2
93.5 1.8 SD = Standard deviation;
[0588] The primary route of elimination of radioactivity after a
2-h IV infusion administration of 15 mg/kg [.sup.14C]brincidofovir
to male rats was in the urine, which accounted for an average of
51.2% of the administered dose over a 168 h period. An average of
42.2% of the administered dose was recovered in feces. The total
recovery of radioactivity in urine and feces combined in Group 1
male rats averaged 93.5% of the dose over the 168 h collection
period. Most of the urinary excretion (43.8% of the dose or 86% of
the radioactivity of the radioactivity excreted in the urine) and
fecal excretion (36.7% of the dose or 87% of the radioactivity
excreted in the feces) of radioactivity occurred in the first 24 h
after dosing.
Group 2--Plasma Pharmacokinetics in Rats after IV Administration of
15 mg/kg
[0589] The plasma total radioactivity concentration versus time
data, PK parameters, and blood to plasma ratios for Group 2 male SD
rats are reported in Table 69.
TABLE-US-00072 TABLE 69 Blood and Plasma Concentration and
Pharmacokinetic Parameters of Male Sprague Dawley Rats After a
Single 2-Hour Intravenous Infusion of 15 mg/kg of
[.sup.14C]brincidofovir (Group 2) Plasma Blood Blood to Time Point
Concentration Concentration Plasma (h) (.mu.g/mL) (.mu.g equiv/mL)
Ratio 0.5 2.557 1.666 0.65 1 4.609 3.017 0.65 2 10.297 6.393 0.62 4
5.363 3.845 0.72 8 1.961 1.584 0.81 24 0.234 0.294 1.26 72 0.045
0.057 1.27 t.sub.1/2 (h) 13.0 14.5 T.sub.max (h) 2 2 C.sub.max
(.mu.g equiv/mL) 10.297 6.393 AUC.sub.last (.mu.g equiv- 64.448
50.836 h/mL) AU.sub.Call (.mu.g equiv- 64.448 50.836 h/mL)
AUC.sub.inf.sub.--.sub.obs (.mu.g 65.292 52.028 equiv-h/mL)
[0590] The C.sub.max of [.sup.14C]brincidofovir total radioactivity
in plasma after a 2-h IV infusion administration to male rats at 15
mg/kg was 10.3 .mu.g equiv/mL at a T.sub.max of 2 h (i.e., end of
infusion), and the concentration decreased to 0.045 .mu.g equiv/mL
at 72 h post-dose. The AUC.sub.last of [.sup.14C]brincidofovir in
plasma was 64.4 .mu.g equiv. h/mL, and the t.sub.1/2 was 13.0
h.
[0591] Blood to plasma ratios for Group 2 SD rats showed that
[.sup.14C]brincidofovir-derived radioactivity initially partitioned
preferentially into plasma versus the cellular portion of blood of
rats at 0. 5 to 8 h (ranged between 0. 62-0.8 l), but then showed
more partitioning into the cellular aspects at 24 and 72 h
post-dose with a ratios of 1.26 and 1.27, respectively.
Groups 3-7--Comparison of blood to plasma concentration ratios in
albino and pigmented rats after IV and PO administration of 15
mg/kg (IV and PO) and 2 mg/kg (IV)
[0592] The blood to plasma concentration ratios for Group 3 to
Group 7 male SD and LE rats are reported in Table 70. A comparison
of blood and plasma concentration versus time profiles obtained
from Groups 3-7 is presented in FIG. 3.
TABLE-US-00073 TABLE 70 Blood to Plasma Ratios in Male Rats After a
Single 2-Hour Intravenous Infusion or Oral Gavage Administration of
[.sup.14C]brincidofovir (Groups 3-7) Blood:Plasma Ratio Group/ Dose
Dose Time After Dose Initiation (h) Strain Route (mg/kg) 0.5 1 2 4
8 24 72 3/SD IV 15 NS NS 0.96 NS NS 1.23 NS 4/LE IV 15 NS 1.10 1.08
0.92 1.39 1.46 3.06 5/LE IV 2 NS NS 0.89 NS 1.03 1.41 1.25 6/SD PO
15 NS NS 0.64 NS NS 0.78 NS 7/LE PO 15 ND 1.13 0.98 0.80 0.76 0.87
ND ND = not determined, blood and/or plasma BQL NS = no sample
collected at this time point
[0593] Similar to Group 2, trends for higher blood:plasma ratios at
later time-points were observed in other groups of animals
administered IV [.sup.14C]brincidofovir (SD rats, Group 3; LE rats,
Groups 4 and 5), though ratios at early (sparse) time-points were
approximately unity. Following oral administration of
[.sup.14C]brincidofovir, blood:plasma ratios ranged between
0.64-1.13, and no trends for higher blood:plasma ratios at later
time-points (up to 24 h after dosing) were observed.
Group 3 and 4--QWBA Tissue Distribution in Albino and Pigmented
Male Rats after IV Administration of 15 mg/kg
[0594] A summary of tissue distribution of [.sup.14C]brincidofovir
in male albino (Group 3; n=3) and pigmented rats (Group 4; n=9) are
presented in Table 71 and Table 72, respectively.
TABLE-US-00074 TABLE 71 Concentrations of Radioactivity in Tissues
of Male Sprague Dawley (Albino, Group 3) Rats After a Single 2-Hour
Intravenous Infusion of [.sup.14C]brincidofovir at a Target Dose of
15 mg/kg Concentration (.mu.g equiv./g) Rat# 11 Rat# 12 Rat# 13
Tissue Type Tissue 2 h 24 h 168 h Vascular/ Plasma (by LSC) 7.130
0.251 BQL Lymphatic Blood (cardiac) 6.865 0.308 BQL Bone Marrow
3.411 1.219 0.196 Lymph Node 4.055 1.004 0.247 Spleen 4.536 2.244
0.694 Thymus 2.373 0.556 0.105 Excretory/ Bile (in duct) NI 1.469
0.058 Metabolic Kidney Cortex 106.513 32.109 0.474 Kidney Medulla
33.444 4.271 0.186 Liver 229.371 13.907 0.453 Urinary Bladder
35.978 2.264 0.132 Urinary Bladder 47.034 21.237 0.121 (contents)
Central Brain (cerebellum) 0.907 0.161 BQL Nervous Brain (cerebrum)
0.803 0.107 BQL System Brain (medulla) 0.864 0.132 BQL Spinal Cord
0.437 0.079 BQL Endocrine Adrenal Gland 4.308 1.680 0.647 Pituitary
Gland 2.793 1.021 0.257 Thyroid 2.751 0.791 0.239 Secretory
Harderian Gland 2.081 1.278 0.954 Pancreas 1.974 0.557 BQL Salivary
Gland 2.049 0.395 0.047 Fatty Adipose (brown) 2.088 0.473 0.391
Adipose (white) 0.386 0.135 BQL Dermal Skin (non-pigmented) 2.277
0.602 0.121 Reproductive Epididymis 2.391 0.566 0.234 Prostate
Gland 2.033 0.254 0.051 Seminal Vesicles 1.809 1.636 0.817 Testis
1.647 0.436 0.078 Skeletal/ Bone 0.449 0.095 BQL Muscular Heart
2.540 0.408 0.057 Skeletal Muscle 0.868 0.099 BQL Respiratory Lung
5.675 1.128 0.296 Alimentary Cecum 3.658 5.981 0.099 Canal Cecum
(contents) 0.085 58.016 0.196 Esophagus 3.567 0.572 0.043 Large
Intestine 3.581 2.019 0.129 Large Intestine BQL 35.809 0.374
(contents) Small Intestine 43.741 6.290 1.704 Small Intestine
95.869 66.664 0.039 (contents) Stomach (gastric 3.777 1.333 0.062
mucosa) Stomach (contents) 3.827 26.408 0.051 Ocular Eye (lens) BQL
BQL BQL Eye (uveal tract) 2.342 0.469 0.095 BQL = Value is below
the LLOQ; NI = Not identified on acceptable sections. LLOQ =
0.00048400 .mu.Ci/g/0.0135694 .mu.Ci/.mu.g = 0.036 .mu.g
equivalent/g tissue ULOQ = 20.97300000 .mu.Ci/g/0.0135694
.mu.Ci/.mu.g = 1545.610 .mu.g equivalent/g tissue
TABLE-US-00075 TABLE 72 Concentrations of Radioactivity in Tissues
of Male Long-Evans (Pigmented, Group 4) Rats After a Single 2-Hour
Intravenous Infusion of [.sup.14C]brincidofovir at a Target Dose of
15 mg/kg Concentration (.mu.g equiv/g tissue) Rat #14 Rat #15 Rat
#16 Rat #17 Rat #18 Rat #19 Rat #20 Rat #21 Rat #22 Tissue Type
Tissue 1 h 2 h 4 h 8 h 24 h 72 h 96 h 168 h 840 h Vascular/ Plasma
(by LSCa) 2.754 4.918 3.080 1.207 0.154 0.017 BQL BQL BQL Lymphatic
Blood (cardiac) 3.040 5.291 2.839 1.679 0.225 0.052 BQL BQL BQL
Bone Marrow 1.464 3.348 2.550 2.045 0.898 0.552 0.280 0.232 0.043
Lymph Node 1.682 4.002 2.244 1.936 0.745 0.710 0.270 0.247 0.062
Spleen 2.081 4.314 3.249 2.728 1.870 1.675 1.106 0.892 0.273 Thymus
1.223 2.125 1.205 1.107 0.403 0.359 0.215 0.100 BQL Excretory/ Bile
(in duct) 243.705 508.358 178.597 BQL 1.010 0.526 0.044 BQL BQL
Metabolic Kidney Cortex 36.713 97.276 76.678 56.528 19.453 4.070
1.041 0.330 BQL Kidney Medulla 8.092 26.987 25.330 22.989 7.117
1.478 0.648 0.138 BQL Liver 126.516 229.669 118.543 53.166 13.918
3.261 1.269 0.451 BQL Urinary Bladder 6.053 5.004 22.781 5.643
1.885 0.336 0.100 BQL BQL Urinary Bladder 19.057 26.805 814.445
82.440 22.258 1.911 0.357 0.104 BQL (contents) Central Brain
(cerebellum) 0.569 1.144 0.422 0.284 0.099 0.073 BQL BQL BQL
Nervous Brain (cerebrum) 0.391 0.887 0.341 0.196 0.070 0.062 BQL
BQL BQL System Brain (medulla) 0.498 0.858 0.376 0.199 0.085 0.059
BQL BQL BQL Spinal Cord 0.462 0.940 0.277 0.116 0.060 BQL BQL BQL
BQL Endocrine Adrenal Gland 2.523 4.802 3.282 3.179 1.253 1.186
0.776 0.490 0.091 Pituitary Gland 2.397 6.343 4.081 1.089 0.431
0.603 0.297 0.189 BQL Thyroid 1.486 2.454 1.884 1.532 0.567 0.376
0.197 0.459 BQL Secretory Harderian Gland 0.897 2.049 1.476 1.284
0.665 1.098 0.536 0.403 BQL Pancreas 0.864 1.896 1.051 0.834 0.315
0.194 0.082 BQL BQL Salivary Gland 1.335 2.508 1.153 0.821 0.314
0.190 0.134 0.056 BQL Fatty Adipose (brown) 0.839 1.680 1.123 0.755
0.306 0.402 0.187 0.132 BQL Adipose (white) 0.369 0.365 0.317 0.124
0.058 0.062 BQL BQL BQL Dermal Skin (non-pigmented) 0.979 2.556
1.925 1.273 0.312 0.443 0.269 0.226 BQL Skin (pigmented) 1.193
2.848 2.118 1.233 0.491 0.337 0.209 0.090 BQL Reproductive
Epididymis 0.828 1.654 1.795 0.821 0.362 0.243 0.204 0.129 BQL
Prostate Gland 0.561 0.901 0.715 0.542 0.157 0.104 0.127 BQL BQL
Seminal Vesicles 0.938 1.834 2.285 1.717 1.127 1.374 1.038 0.361
BQL Testis 0.595 1.321 1.021 0.802 0.285 0.240 0.196 0.098 BQL
Skeletal/ Bone 0.130 0.257 0.183 0.190 BQL 0.036 0.040 BQL BQL
Muscular Heart 1.190 2.401 0.997 0.815 0.226 0.173 0.118 0.062 BQL
Skeletal Muscle 0.427 0.611 0.343 0.152 0.063 0.070 0.040 BQL BQL
Respiratory Lung 3.063 6.350 3.690 2.233 0.585 0.886 0.362 0.178
BQL Alimentary Cecum 1.781 3.052 2.443 6.189 7.077 0.527 0.208
0.080 BQL Canal Cecum (contents) 0.099 1.065 42.166 175.858 30.022
1.987 0.159 0.123 BQL Esophagus 1.750 2.456 1.803 1.747 0.377 0.357
0.098 0.041 BQL Large Intestine 1.455 5.497 2.338 3.071 3.064 0.993
0.545 0.278 BQL Large Intestine BQL 2.223 137.870 235.751 48.798
3.347 0.273 0.103 BQL (contents) Small Intestine 6.622 71.029
42.479 48.384 3.184 4.569 0.911 0.339 BQL Small Intestine 50.503
422.957 55.541 22.421 0.962 0.615 0.120 BQL BQL (contents) Stomach
(gastric 1.446 2.518 1.430 1.223 0.295 0.225 0.110 0.069 BQL
mucosa) Stomach (contents) 1.125 6.178 0.960 10.997 0.170 0.080 BQL
BQL BQL Ocular Eye (lens) 0.039 0.077 BQL BQL BQL BQL BQL BQL BQL
Eye (uveal tract) 0.875 1.411 1.412 0.925 0.452 0.322 0.188 BQL
BQL
Group 3--QWBA Tissue Distribution in Albino Male Rats (Sparse
Sampling)
[0595] [.sup.14C]Brincidofovir-derived radioactivity was well
distributed into most tissues of albino male rats after a 2-h IV
infusion at 15 mg/kg (n=1 animal at 2, 24, and 168 h after
initiation of dosing), and most tissues had concentrations that
were slightly lower than in blood/plasma at the end of the
infusion, but were higher than blood/plasma for later time points.
Excretory and alimentary canal tissue concentrations were much
higher than blood/plasma at all time points. The highest observed
concentration of [.sup.14C]brincidofovir-derived radioactivity in
blood was 6.9 .mu.g equiv/g, which was observed at 2 h (end of
infusion time and the first collection time point for this
sparsely-sampled group). The C.sub.max of
[.sup.14C]brincidofovir-derived radioactivity in most tissues (35
of 37 tissues) were found at 2 h post-dose (first sampled time
point) when most of the tissues had concentrations that were
between 1.0 and 6.0 .mu.g equiv/g.
[0596] The tissues with the highest concentrations (>20.0 .mu.g
equiv/g) at the respective T.sub.max were: liver (229.4 .mu.g
equiv/g), kidney cortex (106.5 .mu.g equiv/g), small intestine
(43.7 .mu.g equiv/g), urinary bladder (36.0 .mu.g equiv/g), and
kidney medulla (33.4 .mu.g equiv/g). The tissues with the lowest
concentrations (<1.0 .mu.g equiv/g) at their respective
T.sub.max were: brain, spinal cord, white adipose, bone, skeletal
muscle, and eye lens.
[0597] The highest overall concentrations determined by QWBA were
observed in the contents of the alimentary canal (C.sub.max ranged
from 26.4 .mu.g equiv/g in stomach at 24 h to 95.9 .mu.g equiv/g in
small intestine at 2 h), and urinary bladder contents (47.0 .mu.g
equiv/g at 2 h), which reflected the routes of elimination for the
[.sup.14C]brincidofovir-drug-derived radioactivity after a 2-h IV
infusion.
[0598] At 168 h post-dose, elimination of drug related material was
not complete in most tissues; however those tissues that did
contain radioactivity had concentrations that were <1.0 .mu.g
equiv/g.
Group 4--QWBA Tissue Distribution in Pigmented Male Rats
[0599] [.sup.14C]Brincidofovir-derived radioactivity was well
distributed into most tissues of pigmented male rats after a 2-h IV
infusion at 15 mg/kg (n=1 animal at 1, 2, 4, 8, 24, 72, 96, 168 and
840 h) after initiation of dosing, and most tissues had
concentrations that were slightly lower than in blood/plasma at
early time-points (up to 4 hours after infusion initiation), but
were higher than blood/plasma for later time-points. Tissue:plasma
(T/P) ratios based on AUC (AUC.sub.tissue:AUC.sub.plasma; Table 78)
show values >1 for most tissues, indicating higher tissue
concentrations relative to plasma. Excretory and alimentary canal
tissue concentrations were much higher than blood/plasma at all
time points. The highest observed concentration of
[.sup.14C]brincidofovir-derived radioactivity in blood was 5.3
.mu.g equiv/g, which was observed at 2 h (end of infusion and the
second collection time point for this intensively-sampled group).
The C.sub.max of [.sup.14C]brincidofovir-derived radioactivity in
most tissues (33 of 38 tissues) were found at 2 h post-dose when
most of the tissues had concentrations that were between 1.0 and
6.0 .mu.g equiv/g.
[0600] The tissues with the highest concentrations (>20.0 .mu.g
equiv/g) at the respective T.sub.max were: liver (229.7 .mu.g
equiv/g), kidney cortex (97.3 .mu.g equiv/g), small intestine (71.0
.mu.g equiv/g), kidney medulla (27.0 .mu.g equiv/g), and urinary
bladder (22.8 .mu.g equiv/g). The tissues with the lowest
concentrations (<0.5 .mu.g equiv/g) at their respective
T.sub.max were: brain (cerebrum and medulla), spinal cord, white
adipose, prostate gland, bone, skeletal muscle, and eye lens.
[0601] The highest overall concentrations determined by QWBA were
observed in the urinary bladder contents (814.4 .mu.g equiv/g at 4
h), bile (508.4 .mu.g equiv/g at 2 h), and contents of the
alimentary canal (C.sub.max ranged from 11.0 .mu.g equiv/g in
stomach at 8 h to 423.0 .mu.g equiv/g in small intestine at 2 h),
which reflected the routes of elimination for the
[.sup.14C]brincidofovir-drug-derived radioactivity after a 2-h IV
infusion. Without wishing to be bound by theory, bile may have been
the source for the high concentration of radioactivity in the
intestinal contents with high concentrations of radioactivity
present in early time points. The high concentration in the stomach
would support this observation, since it is in close proximity to
the entrance of bile into the digestive system, although gastric
secretion may also possible, based on the observation that
radioactivity was present in the gastric mucosa at 168 h.
[0602] Tissue concentrations in most tissues decreased steadily,
but at 168 h post-dose, elimination of drug related material was
not complete in most tissues. In tissues containing radioactivity
the concentrations present were <1.0 .mu.g equiv/g. At 840 h
post-dose (last sampled time point) radioactivity was still present
in bone marrow, lymph node, spleen, and adrenal gland, but was
approaching the LLOQ (0.036 .mu.g equiv/g) in these tissues except
spleen (0.270 .mu.g equiv/g).
[0603] At sparse time-points examined in SD rats (Group 3), no
substantial differences in tissue concentrations were observed
relative to LE rats administered the same dose (Group 4). A
comparison of tissue distribution of radioactivity between
non-pigmented (SD rats Group 3) and pigmented tissues (LE rats
Group 4) showed that concentrations in the pigmented tissues (i.e.,
eye uveal tract and pigmented skin) were similar to those
concentrations observed in the non-pigmented tissues, which
suggested that there was no specific association of
[.sup.14C]brincidofovir-drug-derived radioactivity with
melanin.
Group 5--QWBA Tissue Distribution in Pigmented Male Rats after IV
Dosing at 2 mg/kg (Sparse Sampling)
[0604] A summary of tissue distribution of [.sup.14C]brincidofovir
in male pigmented rats administered a single 2-h IV infusion at 2
mg/kg (n=1 animal at 2, 8, 24, and 72 h after initiation of dosing)
is presented in Table 73.
TABLE-US-00076 TABLE 73 Concentrations of Radioactivity in Tissues
of Male Long-Evans (Pigmented, Group 5) Rats After a Single 2-Hour
Intravenous Infusion of [.sup.14C] brincidofovir at a Target Dose
of 2 mg/kg Concentration (.mu.g equiv/g tissue) Rat# Rat# Rat# Rat#
23 24 25 26 Tissue Type Tissue 2 h 8 h 24 h 72 h Vascular/ Plasma
(by LSC) 0.863 0.173 0.029 0.008 Lymphatic Blood (cardiac) 0.771
0.178 0.041 0.010 Bone Marrow 0.596 0.225 0.163 0.074 Lymph Node
0.453 0.219 0.149 0.085 Spleen 0.821 0.561 0.358 0.284 Thymus 0.331
0.136 0.078 0.050 Excretory/ Bile (in duct) 32.663 3.781 0.097
0.069 Metabolic Kidney Cortex 11.047 8.188 3.570 0.772 Kidney
Medulla 3.359 1.362 0.854 0.257 Liver 34.158 10.631 4.037 1.393
Urinary Bladder 0.474 0.519 0.253 0.117 Urinary Bladder 22.905
3.544 1.348 0.249 (contents) Central Brain (cerebellum) 0.172 0.036
0.020 0.012 Nervous Brain (cerebrum) 0.150 0.030 0.016 0.006 System
Brain (medulla) 0.101 0.027 0.019 0.006 Spinal Cord 0.151 0.024
0.011 0.006 Endocrine Adrenal Gland 0.685 0.440 0.245 0.167
Pituitary Gland 0.899 0.367 0.089 0.055 Thyroid 0.348 0.157 0.105
0.056 Secretory Harderian Gland 0.300 0.162 0.116 0.100 Pancreas
0.406 0.089 0.052 0.022 Salivary Gland 0.494 0.111 0.071 0.030
Fatty Adipose (brown) 0.356 0.099 0.050 0.039 Adipose (white) 0.120
0.015 0.033 0.005 Dermal Skin (non-pigmented) 0.288 0.268 0.108
0.068 Skin (pigmented) 0.460 0.186 0.116 0.047 Reproductive
Epididymis 0.328 0.088 0.089 0.037 Prostate Gland 0.166 0.039 0.035
0.015 Seminal Vesicles 0.364 0.304 0.327 0.172 Testis 0.231 0.091
0.076 0.041 Skeletal/ Bone 0.022 0.017 0.019 0.010 Muscular Heart
0.373 0.109 0.040 0.025 Skeletal Muscle 0.167 0.023 0.017 0.007
Respiratory Lung 0.997 0.283 0.195 0.115 Alimentary Cecum 0.507
0.696 0.145 0.090 Canal Cecum (contents) 0.051 30.663 3.948 0.191
Esophagus 0.425 0.346 0.134 0.036 Large Intestine 0.545 1.028 0.504
0.128 Large Intestine 0.059 48.477 4.244 0.335 (contents) Small
Intestine 1.710 2.462 0.704 0.076 Small Intestine 63.303 1.982
0.207 0.032 (contents) Stomach (gastric 0.553 0.122 0.077 0.031
mucosa) Stomach (contents) 0.014 0.051 0.006 0.008 Ocular Eye
(lens) BQL BQL BQL BQL Eye (uveal tract) 0.352 0.144 0.092 0.061
BQL = Value is below the LLOQ LLOQ = 0.00048400 .mu.Ci/g/0.10151
.mu.Ci/.mu.g = 0.005 .mu.g equivalent/g tissue ULOQ = 20.97300000
.mu.Ci/g/0.10151 .mu.Ci/.mu.g = 206.610 .mu.g equivalent/g
tissue
[0605] [.sup.14C]Brincidofovir-derived radioactivity was well
distributed into most tissues of pigmented male rats after a 2-h IV
infusion at 2 mg/kg, and most tissues had concentrations that were
slightly lower than in blood/plasma at the end of infusion, but
were higher than blood/plasma for later time points. Excretory and
alimentary canal tissues were much higher than blood/plasma at all
time points. The highest observed concentration of [.sup.14C]
brincidofovir-derived radioactivity in blood was 0.771 .mu.g
equiv/g, which was observed at 2 h (end of infusion and the first
collection time point for this sparsely-sampled group) and was
7-fold lower than observed after a 15 mg/kg dose. The C.sub.max of
[.sup.14C]brincidofovir-derived radioactivity in most tissues (35
of 38 tissues) were found at 2 h post-dose (first sampled time
point) when most of the tissues had concentrations that were
between 0.2 and 0.7 .mu.g equiv/g, which was about 10-fold lower
than that observed in rats administered 15 mg/kg. Mean tissue
concentration ratios after IV administration of 2 mg/kg (Group 5)
compared to 15 mg/kg (Group 4) indicated that the increase in
tissue concentrations were approximately dose proportional (i.e.,
with increase in dose of 7.5, a tissue concentration ratio between
4-15) in the vascular lymphatic, excretory/metabolic, central
nervous, endocrine, secretory, fatty, dermal, reproductive,
skeletal/muscular, and respiratory (lung) tissues (Table 74). Mean
tissue concentration ratios were generally greater in the small
intestine, indicating a greater than proportional increase in small
intestine concentrations with an increase in dose from 2 to 15
mg/kg.
TABLE-US-00077 TABLE 74 [.sup.14C]Brincidofovir Ratio of Individual
Tissue Concentration of Group 4 (15 mg/kg) to Group 5 (2 mg/kg)
Concentration (.mu.g equiv/g tissue) G5 (2 G 4 (15 G 5 (2 G4 (15 G5
(2 G4 (15 G 5 (2 G 4 (15 mg/kg) mg/kg) mg/kg) mg/kg) mg/kg) mg/kg)
mg/kg) mg/kg) Rat #23 Rat #15 Ratio Rat #24 Rat #17 Ratio Rat #25
Rat #18 Ratio Rat #26 Rat #19 Ratio Tissue Type Tissue 2 h 2 h 2 h
8 h Lo 8 h Hi 8 h 24 h Lo 24 h Hi 24 h 72 h Lo 72 h Hi 72 h
Vascular/ Blood 0.771 5.291 7 0.178 1.679 9 0.041 0.225 5 0.010
0.052 5 Lymphatic (cardiac) Vascular/ Bone 0.596 3.348 6 0.225
2.045 9 0.163 0.898 6 0.074 0.552 7 Lymphatic Marrow Vascular/
Lymph 0.453 4.002 9 0.219 1.936 9 0.149 0.745 5 0.085 0.710 8
Lymphatic Node Vascular/ Spleen 0.821 4.314 5 0.561 2.728 5 0.358
1.870 5 0.284 1.675 6 Lymphatic Vascular/ Thymus 0.331 2.125 6
0.136 1.107 8 0.078 0.403 5 0.050 0.359 7 Lymphatic Excretory/
Kidney 11.047 97.276 9 8.188 56.528 7 3.570 19.453 5 0.772 4.070 5
Metabolic Cortex Excretory/ Kidney 3.359 26.987 8 1.362 22.989 17
0.854 7.117 8 0.257 1.478 6 Metabolic Medulla Excretory/ Liver
34.158 229.669 7 10.631 53.166 5 4.037 13.918 3 1.393 3.261 2
Metabolic Excretory/ Urinary 0.474 5.004 11 0.519 5.643 11 0.253
1.885 7 0.117 0.336 3 Metabolic Bladder Central Brain 0.172 1.144 7
0.036 0.284 8 0.020 0.099 5 0.012 0.073 6 Nervous (cerebellum)
System Central Brain 0.150 0.887 6 0.030 0.196 7 0.016 0.070 4
0.006 0.062 10 Nervous (cerebrum) System Central Brain 0.101 0.858
8 0.027 0.199 7 0.019 0.085 4 0.006 0.059 10 Nervous (medulla)
System Central Spinal Cord 0.151 0.940 6 0.024 0.116 5 0.011 0.060
5 0.006 BQL NC Nervous System Endocrine Adrenal 0.685 4.802 7 0.440
3.179 7 0.245 1.253 5 0.167 1.186 7 Gland Endocrine Pituitary 0.899
6.343 7 0.367 1.089 3 0.089 0.431 5 0.055 0.603 11 Gland Endocrine
Thyroid 0.348 2.454 7 0.157 1.532 10 0.105 0.567 5 0.056 0.376 7
Secretory Harderian 0.300 2.049 7 0.162 1.284 8 0.116 0.665 6 0.100
1.098 11 Gland Secretory Pancreas 0.406 1.896 5 0.089 0.834 9 0.052
0.315 6 0.022 0.194 9 Secretory Salivary 0.494 2.508 5 0.111 0.821
7 0.071 0.314 4 0.030 0.190 6 Gland Fatty Adipose 0.356 1.680 5
0.099 0.755 8 0.050 0.306 6 0.039 0.402 10 (brown) Fatty Adipose
0.120 0.365 3 0.015 0.124 8 0.033 0.058 2 0.005 0.062 12 (white)
Dermal Skin (non- 0.288 2.556 9 0.268 1.273 5 0.108 0.312 3 0.068
0.443 7 pigmented) Dermal Skin 0.460 2.848 6 0.186 1.233 7 0.116
0.491 4 0.047 0.337 7 (pigmented) Reproductive Epididymis 0.328
1.654 5 0.088 0.821 9 0.089 0.362 4 0.037 0.243 7 Reproductive
Prostate 0.166 0.901 5 0.039 0.542 14 0.035 0.157 4 0.015 0.104 7
Gland Reproductive Seminal 0.364 1.834 5 0.304 1.717 6 0.327 1.127
3 0.172 1.374 8 Vesicles Reproductive Testis 0.231 1.321 6 0.091
0.802 9 0.076 0.285 4 0.041 0.240 6 Skeletal/ Bone 0.022 0.257 12
0.017 0.190 11 0.019 BQL NC 0.010 0.036 NC Muscular Skeletal/ Heart
0.373 2.401 6 0.109 0.815 7 0.040 0.226 6 0.025 0.173 7 Muscular
Skeletal/ Skeletal 0.167 0.611 4 0.023 0.152 7 0.017 0.063 4 0.007
0.070 10 Muscular Muscle Respiratory Lung 0.997 6.350 6 0.283 2.233
8 0.195 0.585 3 0.115 0.886 8 Tract Alimentary Cecum 0.507 3.052 6
0.696 6.189 9 0.145 7.077 49 0.090 0.527 6 Canal Alimentary
Esophagus 0.425 2.456 6 0.346 1.747 5 0.134 0.377 3 0.036 0.357 10
Canal Alimentary Large 0.545 5.497 10 1.028 3.071 3 0.504 3.064 6
0.128 0.993 8 Canal Intestine Alimentary Small 1.710 71.029 42
2.462 48.384 20 0.704 3.184 5 0.076 4.569 60 Canal Intestine
Alimentary Stomach 0.553 2.518 5 0.122 1.223 10 0.077 0.295 4 0.031
0.225 7 Canal (gastric mucosa) Ocular Eye (lens) BQL 0.077 NC BQL
BQL NC BQL BQL NC BQL BQL NC Ocular Eye 0.352 1.411 4 0.144 0.925 6
0.092 0.452 5 0.061 0.322 5 (uveal tract) NC = Not Calculated NI =
Tissue not collected during sectioning NS = Not Sampled, tissues
that could not be visualized on autoradioluminograph due to BQL
radioactivity BQL = Value is below the LLOQ LLOQ = 0.00048400
.mu.Ci/g / 0.10151 .mu.Ci/.mu.g = 0.005 .mu.g equivalent / g tissue
ULOQ = 20.97300000 .mu.Ci/g / 0.10151 .mu.Ci/.mu.g = 206.610 .mu.g
equivalent / g tissue
[0606] The tissues with the highest concentrations >1.0 .mu.g
equiv/g at the respective T.sub.max were: liver (34.2 .mu.g
equiv/g), kidney cortex (11.0 .mu.g equiv/g), kidney medulla (3.4
.mu.g equiv/g), small intestine (2.5 .mu.g equiv/g), and large
intestine (1.0 .mu.g equiv/g). The tissues with the lowest
concentrations (<0.20 .mu.g equiv/g) at their respective
T.sub.max were: brain, spinal cord, white adipose, prostate gland,
bone, skeletal muscle, and eye lens.
[0607] The highest overall non-tissue concentrations determined by
QWBA were observed in the contents of the alimentary canal
(C.sub.max ranged from 0.051 .mu.g equiv/g in stomach at 8 h to
63.3 .mu.g equiv/g in small intestine at 2 h), bile (32.7 .mu.g
equiv/g at 2 h), and urinary bladder contents (22.9 .mu.g equiv/g
at 2 h), which reflected the routes of elimination for the
[.sup.14C]brincidofovir-drug-derived radioactivity after a 2-h IV
infusion at 2 mg/kg.
[0608] Tissue concentrations in most tissues decreased steadily,
but at 72 h post-dose (last time point analyzed for this group),
drug related material was detected in all tissues, but most
tissues, not associated with organs of clearance, had
concentrations that were <0.10 .mu.g equiv/g.
Group 6 and 7--QWBA Tissue Distribution in Albino and Pigmented
Male Rats after PO Dosing at 15 mg/kg
[0609] A summary of tissue distribution of [.sup.14C]brincidofovir
in male albino (Group 6; n=3) and pigmented rats (Group 7; n=10)
are presented in Table 75 and Table 76, respectively.
Pharmacokinetic parameters are given in Table 77.
TABLE-US-00078 TABLE 75 Concentrations of Radioactivity in Tissues
of Male Sprague Dawley (Albino, Group 6) Rats After a Single Oral
Dose of [.sup.14C]brincidofovir at a Target Dose of 15 mg/kg
Concentration (.mu.g equiv./g) Rat# 27 Rat# 28 Rat# 29 Tissue Type
Tissue 2 h 24 h 168 h Vascular/ Plasma (by LSC) 0.313 0.253 BQL
Lymphatic Blood (cardiac) 0.200 0.197 BQL Bone Marrow 0.080 0.407
0.075 Lymph Node 0.095 0.329 0.124 Spleen 0.116 0.831 0.239 Thymus
0.045 0.142 BQL Excretory/ Bile (in duct) 9.639 0.465 0.045
Metabolic Kidney Cortex 2.752 18.379 0.588 Kidney Medulla 2.189
7.926 0.274 Liver 7.260 20.567 0.450 Urinary Bladder 4.367 17.321
0.087 Urinary Bladder 9.808 39.903 0.549 (contents) Central Brain
(cerebellum) 0.035 BQL BQL Nervous Brain (cerebrum) BQL BQL BQL
System Brain (medulla) BQL BQL BQL Spinal Cord BQL BQL BQL
Endocrine Adrenal Gland 0.114 0.927 0.035 Pituitary Gland BQL 0.257
0.071 Thyroid 0.084 0.291 0.036 Secretory Harderian Gland 0.062
0.315 0.100 Pancreas 0.092 0.164 0.034 Salivary Gland 0.067 0.103
BQL Fatty Adipose (brown) 0.053 0.212 BQL Adipose (white) BQL 0.079
BQL Dermal Skin (non-pigmented) 0.088 0.189 0.063 Reproductive
Epididymis 0.073 0.091 0.075 Prostate Gland 0.069 0.217 BQL Seminal
Vesicles 0.048 0.333 0.097 Testis 0.039 0.118 BQL Skeletal/ Bone
BQL 0.047 BQL Muscular Heart 0.080 0.116 BQL Skeletal Muscle 0.039
BQL BQL Respiratory Lung 0.184 0.296 0.078 Alimentary Cecum 0.109
7.533 0.064 Canal Cecum (contents) BQL 28.579 0.170 Esophagus 0.593
0.271 BQL Large Intestine 0.084 2.194 0.076 Large Intestine BQL
34.450 0.168 (contents) Small Intestine 236.279 84.063 2.680 Small
Intestine 321.878 11.948 0.054 (contents) Stomach (gastric 11.183
1.075 0.041 mucosa) Stomach (contents) 877.490 5.664 BQL Ocular Eye
(lens) BQL BQL BQL Eye (uveal tract) 0.145 0.290 BQL BQL = Value is
below the LLOQ LLOQ = 0.00048400 .mu.Ci/g/0.01427 .mu.Ci/.mu.g =
0.034 .mu.g equivalent/g tissue ULOQ = 20.97300000 .mu.Ci/g/0.01427
.mu.Ci/.mu.g = 1469.727 .mu.g equivalent/g tissue
TABLE-US-00079 TABLE 76 Concentrations of Radioactivity in Tissues
of Male Long-Evans (Pigmented, Group 7) Rats After a Single Oral
Dose of [.sup.14C]brincidofovir at a Target Dose of 15 mg/kg
Concentration (.mu.g equiv/g tissue) Rat #30 Rat #31 Rat #32 Rat
#33 Rat #34 Rat #35 Rat #36 Rat #37 Rat #38 Rat #39 Tissue Type
Tissue 0.5 h 1 h 2 h 4 h 8 h 24 h 72 h 96 h 168 h 840 h Vascular/
Plasma 0.040 0.248 0.446 1.216 1.499 0.184 0.017 BQL BQL BQL (by
LSC) Lymphatic Blood (cardiac) BQL 0.280 0.435 0.978 1.132 0.160
BQL 0.035 BQL BQL Bone Marrow BQL 0.085 0.117 0.254 0.412 0.391
0.167 0.211 0.094 BQL Lymph Node 0.050 0.153 0.097 0.380 0.421
0.389 0.339 0.141 0.129 BQL Spleen 0.267 0.111 0.166 0.319 0.533
0.759 0.469 0.466 0.274 BQL Thymus 0.083 0.065 0.104 0.154 0.224
0.116 0.040 0.083 BQL BQL Excretory/ Bile (in duct) 0.572 8.660
20.537 36.265 17.878 0.614 2.425 0.317 0.140 BQL Metabolic Kidney
Cortex 0.146 1.452 3.976 10.662 17.789 8.640 2.368 1.696 0.342
0.051 Kidney 0.096 0.602 3.593 5.104 9.070 2.724 1.023 0.653 0.201
BQL Medulla Liver 0.432 7.701 12.037 36.137 34.744 18.184 2.183
2.680 0.393 0.034 Urinary Bladder BQL 1.146 0.214 1.588 3.728 0.659
0.177 0.256 0.076 BQL Urinary Bladder 0.200 11.376 7.593 17.825
110.910 11.541 0.205 1.292 0.278 BQL (contents) Central Brain BQL
BQL BQL 0.037 0.075 BQL BQL BQL BQL BQL (cerebellum) Nervous Brain
BQL BQL BQL 0.035 0.066 BQL BQL BQL BQL BQL (cerebrum) System Brain
BQL BQL BQL 0.049 0.043 BQL BQL BQL BQL BQL (medulla) Spinal Cord
BQL BQL BQL 0.037 BQL BQL BQL BQL BQL BQL Endocrine Adrenal Gland
BQL 0.180 0.372 0.749 1.331 0.578 0.278 0.172 0.058 BQL Pituitary
Gland BQL 0.045 0.085 0.330 0.266 0.151 0.076 0.073 0.042 BQL
Thyroid BQL 0.118 0.168 0.317 0.298 0.139 0.117 0.089 0.038 BQL
Secretory Harderian BQL 0.046 0.090 0.181 0.244 0.150 0.155 0.194
0.110 BQL Gland Pancreas 0.066 0.166 0.193 0.255 0.214 0.096 0.051
0.044 BQL BQL Salivary Gland BQL 0.087 0.112 0.212 0.234 0.109 BQL
0.052 BQL BQL Fatty Adipose 0.040 0.099 0.084 0.302 0.240 0.099
0.050 0.044 0.037 BQL (brown) Adipose (white) BQL BQL 0.063 0.089
0.076 BQL BQL BQL BQL BQL Dermal Skin (non- BQL 0.102 0.100 0.261
0.429 0.107 0.058 0.067 BQL BQL pigmented) Skin BQL 0.086 0.156
0.416 0.429 0.157 0.069 0.077 BQL BQL Reproductive Epididymis BQL
0.084 0.064 0.294 0.229 0.146 0.064 0.108 0.042 BQL Prostate Gland
BQL 0.077 0.120 0.148 0.128 0.100 BQL 0.052 BQL BQL Seminal BQL
1.018 0.109 0.221 0.244 0.259 0.156 0.185 0.063 BQL Vesicles Testis
BQL 0.042 0.051 0.142 0.223 0.152 0.078 0.098 0.041 BQL Skeletal/
Bone BQL BQL BQL 0.099 0.043 0.035 BQL BQL BQL BQL Muscular Heart
BQL 0.083 0.182 0.247 0.358 0.121 0.061 0.077 0.045 BQL Skeletal
BQL 0.046 0.060 0.074 0.098 BQL BQL BQL BQL BQL Muscle Respiratory
Lung BQL 0.226 0.386 0.768 1.025 0.178 0.136 0.105 0.065 BQL
Alimentary Cecum BQL 0.127 0.289 0.931 7.561 0.688 0.189 0.327
0.137 BQL Canal Cecum BQL BQL 0.050 38.957 256.303 35.629 1.035
0.698 0.071 BQL (contents) Esophagus BQL BQL 0.185 4.636 0.458
0.179 BQL 0.071 BQL BQL Large Intestine BQL 0.107 0.136 0.265 3.338
1.681 0.276 0.413 0.060 BQL Large Intestine BQL BQL BQL BQL 378.695
69.825 1.236 1.252 0.108 BQL (contents) Small Intestine 72.672
374.71 295.445 458.485 178.32 74.587 6.889 16.979 5.867 0.070 Small
Intestine 53.944 82.546 72.107 187.589 98.195 69.109 0.215 9.654
BQL BQL (contents) Stomach 2.769 7.730 11.955 0.833 0.880 0.937
0.108 0.101 0.042 BQL (gastric mucosa) Stomach 670.358 1004.619
328.988 274.917 17.792 0.056 BQL BQL BQL BQL (contents) Ocular Eye
(lens) BQL BQL BQL BQL BQL BQL BQL BQL BQL BQL Eye (uveal BQL BQL
0.193 0.227 0.601 0.178 BQL 0.075 BQL BQL tract) BQL = Value is
below the LLOQ LLOQ = 0.00048400 .mu.Ci/.mu.g = 0.034 .mu.g
equivalent/g tissue ULOQ = 20.97300000 .mu.Ci/g/0.01427
.mu.Ci/.mu.g = 1469.727 .mu.g equivalent/g tissue
TABLE-US-00080 TABLE 77 Rat Tissue Pharmacokinetic Parameters After
a Single Oral Dose of [.sup.14C]brincidofovir at a Target Dose of
15 mg/kg Rat PK No. of AUC.sub.all AUC.sub.inf_obs C.sub.max (.mu.g
points (.mu.g (.mu.g Organ/Tissue (equiv/g) t.sub.1/2 (h) in
t.sub.1/2 r.sup.2 T.sub.max (h) equiv h/g) equiv h/g) Adipose
(brown) 0.302 233.5 3 0.96 4 24.3703 24.4043 Adipose (white) .sup.a
0.089 17.6 2 1.00 4 1.1215 2.4387 Adrenal Gland 1.331 43.2 3 1.00 8
74.5860 58.7136 Blood (cardiac) .sup.a 1.132 32.8 2 1.00 8 21.9165
22.3146 Bone .sup.a 0.099 53.9 2 1.00 4 1.8470 3.7274 Bone Marrow a
0.412 96.3 3 0.75 8 68.7413 50.2191 Brain (cerebellum) 0.075 ND 0
ND 8 0.8610 ND Brain (cerebrum) 0.066 ND 0 ND 8 0.7650 ND Brain
(medulla) 0.049 ND 0 ND 4 0.5770 ND Cecum .sup.a 7.561 69.9 4 0.71
8 174.4118 142.1970 Epididymis .sup.a 0.294 89.0 4 0.72 4 31.1150
22.3936 Esophagus 4.636 37.2 3 0.88 4 27.9015 29.1559 Eye (lens) 0
ND ND ND ND ND ND Eye (uveal tract) .sup.a 0.601 57.7 2 1.00 8
16.2765 19.8244 Harderian Gland .sup.a 0.244 151.8 3 0.64 8 63.7645
50.8931 Heart .sup.a 0.358 110.8 4 0.81 8 31.1603 23.2365 Kidney
Cortex .sup.a 17.789 244.8 2 1.00 8 804.4980 822.5076 Kidney
Medulla 9.070 41.2 3 1.00 8 342.0130 286.4307 Large Intestine
.sup.a 3.338 37.6 3 0.82 8 140.3313 123.4302 Liver .sup.a 36.137
190.3 2 1.00 4 1426.6340 1435.9700 Lung 1.025 93.0 3 0.99 8 53.1145
39.9962 Lymph Node .sup.a 0.421 86.4 3 0.56 8 85.04325 57.7701
Pancreas.sup.b 0.255 62.1 3 0.98 4 10.3720 12.7271 Pituitary Gland
0.330 106.2 3 0.97 4 30.5073 22.8331 Plasma (LSC) 1.499 ND ND ND 8
22.3885 ND Prostate Gland 0.148 70.1 3 0.99 4 7.6578 11.0471
Salivary Gland a 0.234 67.4 2 1.00 8 9.1933 12.3800 Seminal
Vesicles 1.018 64.7 3 0.86 1 50.2500 34.9600 Skeletal Muscle 0.098
ND 0 ND 8 1.3265 ND Skin (non-pigmented) .sup.a 0.429 93.7 3 0.72 8
14.0275 20.6749 Skin (pigmented) .sup.a 0.429 63.4 3 0.81 8 17.0405
21.3144 Small Intestine 458.485 98.1 3 0.99 4 9575.1810 9585.0880
Spinal Cord 0.037 ND 0 ND 4 0.1110 ND Spleen 0.759 115.4 3 0.95 24
172.2208 125.7566 Stomach (mucosa) 11.955 66.8 3 0.97 2 90.7575
80.6917 Testis .sup.a 0.223 87.2 3 0.77 8 30.3920 21.7757 Thymus
.sup.a 0.224 96.9 3 0.23 8 12.0843 20.6983 Thyroid 0.317 59.0 3
1.00 4 31.3395 21.8086 Urinary Bladder 3.728 49.7 4 0.87 8 111.2445
91.1627 ND = Not determined; unable to determine value from the
data. .sup.a t.sub.1/2 values for these tissue/organs are obtained
from insufficient data points and/or the resulting r.sup.2 values
fall outside acceptance criteria (.gtoreq.0.85), thus, these values
should be considered estimates.
Group 6--QWBA Tissue Distribution in Albino Male Rats after Dosing
(Sparse Sampling)
[0610] [.sup.14C]Brincidofovir-derived radioactivity was well
distributed into most tissues of albino male rats after a single
oral dose at 15 mg/kg (n=1 at 2, 24, and 168 h after dosing), and
most tissues had concentrations that were slightly lower than in
blood/plasma at early time-point (2 hours after dosing), but were
higher than blood/plasma for later time points. Excretory and
alimentary canal tissue concentrations were much higher than
blood/plasma at all time points. The highest observed concentration
of [.sup.14C]brincidofovir-derived radioactivity in blood was 0.200
.mu.g equiv/g, which was observed at 2 h (the first collection time
point for this sparsely-sampled group). The C.sub.max of
[.sup.14C]brincidofovir-derived radioactivity in most tissues (31
of 37 tissues) were found at 24 h post-dose (second sampled time
point) when most of the tissues had concentrations that were
between 0.10 and 0.50 .mu.g equiv/g.
[0611] The tissues with the highest concentrations (>7.0 .mu.g
equiv/g) at the respective T.sub.max were: small intestine (236.3
.mu.g equiv/g), liver (20.6 .mu.g equiv/g), kidney cortex (18.4
.mu.g equiv/g), urinary bladder (17.3 .mu.g equiv/g), stomach (11.2
.mu.g equiv/g), kidney medulla (7.9 .mu.g equiv/g), and cecum (7.5
.mu.g equiv/g). The tissues with the lowest concentrations
(<0.10 .mu.g equiv/g) at their respective T.sub.max were: brain,
spinal cord, white adipose, bone, skeletal muscle, and eye
lens.
[0612] The highest overall concentrations determined by QWBA were
observed in the contents of the alimentary canal (C.sub.max ranged
from 28.6 .mu.g equiv/g in cecum at 24 h to 877.5 .mu.g equiv/g in
stomach at 2 h), urinary bladder contents (39.9 .mu.g equiv/g at 24
h), and bile (9.7 .mu.g equiv/g at 2 h), which reflected the route
of dose administration and/or the routes of elimination for the
[.sup.14C]brincidofovir-drug-derived radioactivity after a single
oral dose at 15 mg/kg.
[0613] At 168 h post-dose, elimination of drug related material was
not complete, with detectable concentrations (<0.60 .mu.g
equiv/g) in approximately half of the tissues. In small intestine,
a higher residual concentration (2.7 .mu.g equiv/g) was observed
relative to other tissues at 168 h.
Group 7--QWBA Tissue Distribution in Pigmented Male Rats after PO
Dosing
[0614] [.sup.14C]Brincidofovir-derived radioactivity was well
distributed into most tissues of pigmented male rats after a single
oral dose at 15 mg/kg (n=1 animal at 0.5, 1, 2, 4, 8, 24, 72, 96,
168 and 840 h after dosing), and most tissues had concentrations
that were slightly lower than in blood/plasma at time points up to
24 hours after dosing, but were higher than blood/plasma for later
time points. Excretory and alimentary canal tissue concentrations
were much higher than blood/plasma at all time points.
Tissue:plasma (T/P) ratios based on AUC (Table 78) show values
>1 for most tissues, indicating higher tissue concentration
relative to plasma. The highest observed concentration of
[.sup.14C]brincidofovir-derived radioactivity in blood was 1.1
.mu.g equiv/g, which was observed at 8 h. The C.sub.max of
[.sup.14C]brincidofovir-derived radioactivity in most tissues (20
of 38 tissues) were found at 8 h post-dose when most of the tissues
had concentrations that were between 0.20 and 0.40 .mu.g
equiv/g.
TABLE-US-00081 TABLE 78 Tissue Distribution of Total Radioactivity
in Male Long-Evans Rats Following a Single Two Hour Intravenous
Infusion (Group 4) or Oral Dose (Group 7) of
[.sup.14C]brincidofovir at a Target Dose of 15 mg/kg Tissue/Plasma
Ratio Tissue C.sub.max AUC.sub.all AUC.sub.all Tissue/AUC.sub.all
Concentration (.mu.g equiv/g) (.mu.g equiv h/g) Plasma .mu.g
equiv/g (168 h) IV PO Ratio IV PO Ratio IV PO IV PO Tissue (Group
4) (Group 7) (PO/IV) (Group 4) (Group 7) (PO/IV) (Group 4) (Group
7) (Group 4) (Group 7) Plasma (by 4.918 1.499 0.30 36.3690 22.3885
0.62 1.0 1.0 BQL BQL LSC) Blood 5.291 1.132 0.21 45.3555 21.9165
0.48 1.2 1.0 BQL BQL (cardiac) Adipose 1.68 0.302 0.18 96.6220
24.3703 0.25 2.7 1.1 0.132 0.037 (brown) Adipose 0.369 0.089 0.24
7.1955 1.1215 0.16 0.2 0.1 BQL BQL (white) Adrenal 4.802 1.331 0.28
384.2580 74.5860 0.19 10.6 3.3 0.49 0.058 Gland Bone 0.257 0.099
0.39 6.1805 1.8470 0.30 0.2 0.1 BQL BQL Bone Marrow 3.348 0.412
0.12 197.3860 68.7413 0.35 5.4 3.1 0.232 0.094 Brain 1.144 0.075
0.07 12.1870 0.8610 0.07 0.3 0.04 BQL BQL (cerebellum) Brain 0.887
0.066 0.07 9.1765 0.7650 0.08 0.3 0.03 BQL BQL (cerebrum) Brain
0.858 0.049 0.06 9.7470 0.5770 0.06 0.3 0.0 BQL BQL (medulla) Cecum
7.077 7.561 1.07 360.7580 174.4118 0.48 9.9 7.8 0.08 0.137
Epididymis 1.795 0.294 0.16 95.0160 31.1150 0.33 2.6 1.4 0.129
0.042 Esophagus 2.456 4.636 1.89 73.1850 27.9015 0.38 2.0 1.2 0.041
BQL Eye (lens) 0.077 ND ND 0.1545 ND ND 0.0042 ND BQL BQL Eye
(uveal 1.412 0.601 0.43 51.5575 16.2765 0.32 1.4 0.7 BQL BQL tract)
Harderian 2.049 0.244 0.12 257.6905 63.7645 0.25 7.1 2.8 0.403 0.11
Gland Heart 2.401 0.358 0.15 58.1205 31.1603 0.54 1.6 1.4 0.062
0.045 Kidney 97.276 17.789 0.18 1919.6850 804.4980 0.42 52.8 35.9
0.33 0.342 Cortex Kidney 26.987 9.07 0.34 717.8445 342.0130 0.48
19.7 15.3 0.138 0.201 Medulla Large 5.497 3.338 0.61 310.7965
140.3313 0.45 8.5 6.3 0.103 0.06 Intestine Liver 229.669 36.137
0.16 2149.7645 1426.6340 0.66 59.1 63.7 0.451 0.393 Lung 6.35 1.025
0.16 180.1960 53.1145 0.29 5.0 2.4 0.178 0.065 Lymph Node 4.002
0.421 0.11 208.8530 85.04325 0.41 5.7 3.8 0.247 0.129 Pancreas
1.896 0.255 0.13 36.2010 10.3720 0.29 1.0 0.5 BQL BQL Pituitary
6.343 0.33 0.05 155.1085 30.5073 0.20 4.3 1.4 0.189 0.042 Gland
Prostate 0.901 0.148 0.30 24.3415 7.6578 0.62 0.7 0.3 BQL BQL Gland
Salivary 2.508 0.234 0.09 60.9180 9.1933 0.15 1.7 0.4 0.056 BQL
Gland Seminal 2.285 1.018 0.45 297.3580 50.2500 0.17 8.2 2.2 0.361
0.063 Vesicles Skeletal 0.611 0.098 0.16 10.3485 1.3265 0.13 0.3
0.1 BQL BQL Muscle Skin (non- 2.556 0.429 0.17 146.2340 14.0275
0.10 4.0 0.6 0.226 BQL pigmented) Skin 2.848 0.429 0.15 95.5050
17.0405 0.18 2.6 0.8 0.09 BQL (pigmented) Small 71.029 458.485 6.45
1160.6505 9575.1810 8.25 31.9 427.7 0.339 5.867 Intestine Spinal
Cord 0.94 0.037 0.04 5.7830 0.1110 0.02 0.2 0.005 BQL BQL Spleen
4.314 0.759 0.18 642.3590 172.2208 0.27 17.7 7.7 0.892 0.274
Stomach 2.518 11.955 4.75 70.2310 90.7575 1.29 1.9 4.1 0.069 0.042
(mucosa) Testis 1.321 0.223 0.17 77.2835 30.3920 0.39 2.1 1.4 0.098
0.041 Thymus 2.125 0.224 0.11 92.4355 12.0843 0.13 2.5 0.5 0.1 BQL
Thyroid 2.454 0.317 0.13 238.0230 31.3395 0.13 6.5 1.4 0.459 0.038
Urinary 22.781 3.728 0.16 215.5480 111.2445 0.52 5.9 5.0 BQL 0.076
Bladder BQL = Below quantification limit LLOQ = 0.036 ug equiv/g
(Group 4 IV) LLOQ = 0.034 ug equiv/g (Group 7 PO) ND = Not
determined, insufficient data
[0615] FIG. 4 and FIG. 5 show plots of the concentration of
brincidofovir in various tissue as a function of time and route of
administration. FIG. 4 compares the concentration of brincidofovir
in the small intestine for oral and IV dosing as a function of
time. FIG. 5 compares the concentration of brincidofovir in the
kidney cortex for oral and IV dosing as a function of time.
[0616] The tissues with the highest concentrations (>2.0 .mu.g
equiv/g) at the respective T.sub.max were: small intestine (458.5
.mu.g equiv/g), liver (36.1 .mu.g equiv/g), kidney cortex (17.8
.mu.g equiv/g), stomach (12.0 .mu.g equiv/g), kidney medulla (9.1
.mu.g equiv/g), cecum (7.6 .mu.g equiv/g), urinary bladder (3.7
.mu.g equiv/g), and large intestine (3.3 .mu.g equiv/g). The
tissues with the lowest concentrations (<0.10 .mu.g equiv/g) at
their respective T.sub.max were: brain, spinal cord, white adipose,
bone, skeletal muscle, and eye lens.
[0617] The highest overall concentrations determined by QWBA were
observed in the contents of the alimentary canal (C.sub.max ranged
from 187.6 .mu.g equiv/g in small intestine at 4 h to 1004.6 .mu.g
equiv/g in stomach at 1 h), urinary bladder contents (110.9 .mu.g
equiv/g at 8 h), and bile (36.3 .mu.g equiv/g at 4 h), which
reflected route of dose administration and/or the routes of
elimination for the [.sup.14C]brincidofovir drug-derived
radioactivity after a single oral dose.
[0618] Tissue concentrations in most tissues decreased steadily,
but at 168 h post-dose, drug related material was detected in
approximately half of the tissues, with most concentrations
<0.40 .mu.g equiv/g, except for small intestine (5.9 .mu.g
equiv/g). At 840 h post-dose (last sampled time point)
radioactivity was still present in small intestine, kidney cortex,
and liver, but concentration were near the LLOQ (0.034 .mu.g
equiv/g) in all tissues.
[0619] The primary route of elimination of radioactivity after a
2-h IV infusion of [.sup.14C]brincidofovir at 15 mg/kg in male SD
rats was in urine, which accounted for an average of 51.2% of the
administered dose over the 168 h collection period. An average of
42.2% of the administered dose was recovered in feces. Most
(.about.86%) of the urinary and fecal excretion of radioactivity
occurred in the first 24 h after dosing.
[0620] Following a 2-h IV infusion, radioactivity in most tissues
(except small intestine) increased in proportion to dose (from 2 to
15 mg/kg) across each time point. In small intestinal tissue, the
increase was greater than proportional to dose. Following oral
administration radioactivity was readily absorbed. Radioactivity
was well distributed with peak concentrations of radioactivity in
most tissues (34 of 38) occurring at 4 to 8 h after oral
administration, and at the end of the IV infusion (2-h). The
qualitative distribution patterns of the radioactivity were similar
after IV or oral administration; quantitatively, tissue radioactive
exposure, adjusted for the radioactive bioavailability
(approximately 50%), generally was greater after IV administration
than after oral gavage. Tissues with highest concentrations of
radioactivity were associated with excretory organs (i.e., liver,
kidney, and intestine). The tissue to plasma ratios were high
(>30) for each of these tissues, and in kidney cortex and liver
the ratios were similar between the IV and oral routes of
administration. Small intestinal tissue to plasma ratio was highest
after oral administration (T/P=428) as expected based on the route
of administration. However, notable radioactivity was associated
with the small intestine (tissue to plasma ratio 32) after IV
infusion. High concentration (C.sub.max) was associated with bile
in the duct, which was 14 times greater after IV (C.sub.max 508
.mu.g equiv/g) than oral (C.sub.max=36 .mu.g equiv/g)
administration, suggesting a possible source for the high small
intestinal tissue concentrations after IV administration. Tissues
with lowest concentrations of radioactivity were brain, spinal
cord, skeletal muscle, white adipose tissue and bone. Association
of radioactivity in the brain and spinal cord was higher after IV
administration (.about.20% of plasma C.sub.max concentration and
.about.5% of plasma C.sub.max concentration after oral
administration). No evidence of specific association with melanin
containing tissues (eye, uvea) was detected.
[0621] The predicted exposure in humans following a 100 .mu.Ci oral
dose was estimated using four different methods (including single
tissue and whole body exposure estimates), all of which resulted in
a human exposure that was 4% or less of the FDA and ICRP allowed
limits.
[0622] There were no observed SOP and/or protocol deviations that
occurred on the study that had a detrimental effect on the quality
or integrity of the study.
IV BCV Single Ascending Dose Study in Healthy Subjects
[0623] Brincidofovir was administered to healthy subjects at 10 mg
and 25 mg. IV brincidofovir at 10 mg was found to provide similar
exposure as orally administered brincidofovir at 100 mg. It was
found that both IV brincidofovir doses (10 mg and 25 mg) were
generally safe and well-tolerated. No drug related adverse events,
no gastrointestinal adverse events, and no graded lab abnormalities
(e.g., no hematologic toxicity and no kidney toxicity) were
observed.
[0624] A single dose of oral BCV at 100 mg and 200 mg resulted in
about 5% instance of diarrhea, and a 350 mg dose resulted in about
20% instance of diarrhea.
[0625] FIG. 6 shows a plot of the plasma brincidofovir
concentration (AUC.sub.inf (ng*h/mL)) at different 100 mg oral
administration, 10 mg IV administration, and 25 mg IV
administration. As shown in FIG. 6, IV administration of 10 mg
brincidofovir provided substantially the same plasma concentration
as oral administration of 100 mg brincidofovir, while IV
administration of 25 mg brincidofovir provided higher plasma
concentration than both the IV 10 mg dose and the oral 100 mg
dose.
Example 10--Brincidofovir: A 28-Day Intravenous Infusion Toxicity
Study in Rats with a 14-Day Recovery Period
Summary
[0626] Sprague-Dawley CD.RTM. rats (15/sex/group) were administered
0 (2.times. Sodium Phosphate Buffer Solution), 1, 4 or 15
mg/kg/dose brincidofovir twice per week via 2-hour intravenous
infusion on a total of 9 occasions over 29 days. The dose rate was
10 mL/kg/hr for all dose groups. At the end of the treatment phase,
up to 10 animals/sex/group were euthanized and necropsied. The
remaining animals (up to 5 animals/sex/group) were held for a
14-day recovery phase in order to determine progression or
reversibility of any brincidofovir-related effects. Satellite
animals (up to 3/sex/group) were similarly dosed and serial blood
samples were collected from each animal on Days 1 and 29 for
toxicokinetic analysis of brincidofovir and the brincidofovir
metabolite (Cidofovir, CDV). Parameters evaluated during the study
were: viability, clinical observations, ophthalmology, body
weights, food consumption, clinical pathology (termination of
dosing and end of recovery), organ weights, macroscopic
observations and microscopic pathology.
[0627] Brincidofovir exposure increased with increasing dose on Day
1 and Day 29 with a trend towards a greater than proportional
increase in AUC.sub.last versus dose. Without wishing to be bound
by theory, in general, there was no accumulation of brincidofovir
over the course of the 29 day study. Male to female ratios for
brincidofovir indicated there were no sex differences in exposures
(based on C.sub.max and AUC.sub.last).
[0628] On Days 1 and 29, following intravenous administration of
brincidofovir, CDV C.sub.max and AUC.sub.last values increased with
increasing brincidofovir dose (from 1 to 15 mg/kg). In general,
there was little to no accumulation of CDV over the course of the
study and male to female ratio generally indicated there were no
sex differences in CDV exposures.
[0629] There were no brincidofovir-related mortalities. There were
3 unscheduled deaths on the study, 2 deaths in toxicity animals
were determined not to be brincidofovir-related and 1 death in a TK
animal for which the cause could not be determined.
[0630] There were no brincidofovir-related effects on
ophthalmology, body weights, hematology, coagulation, clinical
chemistry, or urinalysis.
[0631] Administration of brincidofovir resulted in intermittent
(generally following infusion) clinical signs in some but not all
animals (rapid breathing and hunched posture with and without
piloerection, partially closed eyes, decreased activity irregular
breathing, and/or flattened posture) in both sexes at .gtoreq.4
mg/kg/dose and minimally decreased food consumption at .gtoreq.1
mg/kg/dose in males and at 15 mg/kg/dose in females.
[0632] At the end of dosing, brincidofovir-related findings were
present in the male reproductive tract (testes, epididymides and
seminal vesicles) and the intestinal tract (duodenum, jejunum,
and/or ileum) at .gtoreq.4 mg/kg/dose and skin (sebaceous glands)
of males and females at 15 mg/kg/dose. In the male reproductive
tract, testicular changes were characterized by decreased
testicular weights and slight to marked germ cell depletion at
.gtoreq.4 mg/kg, with minimal spermatid retention and Sertoli cell
vacuolation at 15 mg/kg/dose. In addition, males at 15 mg/kg/dose
had minimal luminal cell debris in the epididymides and minimally
increased epithelial apoptosis in the seminal vesicles. In the
intestinal tract, single cell necrosis was observed in the small
intestine (crypts) at .gtoreq.4 mg/kg/dose and in the large
intestine (glands) at 15 mg/kg/dose in both sexes. Sebaceous gland
atrophy in the skin was present in males and females at 15
mg/kg.
TABLE-US-00082 TABLE 79 Comparison of toxicity of orally and
intravenously administered brincidofovir ORAL BRINCIDOFOVIR (Single
Dose) IV BRINCIDOFOVIR (Repeat Dose) Exposure Exposure DOSE (ng/mL
or DOSE (ng/mL or Preliminary (mg/kg) ng hr/mL) Findings (mg/kg) ng
hr/mL) Findings 30 C.sub.max-426 Single cell necrosis of 1
C.sub.max-298 No GI findings AUC-5564 the epithelial cells lining
AUC-410 mucosal crypts or glands at the base of small intestinal
villi 100 C.sub.max-1086 Diarrhea, dehydration, 4 C.sub.max-1200
Minimal to slight AUC-13552 .dwnarw. Body weight AUC- 2759 small
intestine Gross - distended necrosis crypt; stomach minimal crypt
Enteritis with epithelial hyperplasia loss and inflammation 38%
mortality 300 C.sub.max-1186 Clinical: diarrhea, 15 C.sub.max-4500
Slight to minimal AUC-20506 dehydration, .dwnarw. Body AUC-9893
small intestine crypt weight necrosis; minimal Gross - distended
crypt hyperplasia stomach, jejunum, Minimal epithelial cecum, colon
gland necrosis large Extensive loss of intestine mucosal epithelial
cells with inflammatory infiltrate into lamina propria 100%
mortality 1000 C.sub.max-1375 Clinical: Diarrhea, Note: Infusion of
BCV resulted in AUC-26449 dehydration, .dwnarw. body intermittent
clinical signs of weight rapid breathing, hunched posture- Gross -
distended with or without piloerection in stomach, jejunum, some
animals during infusion; and cecum, colon fully resolved shortly
following GI toxicity similar to infusion. 300 mg/kg 100%
mortality
[0633] Following a 14-day recovery period, all findings completely
recovered with the exception of testicular weight decreases and
germ cell depletion in males at .gtoreq.4 mg/kg/dose; however,
without wishing to be bound by theory, germ cell depletion would
not be expected to completely recover in 2 weeks. In addition, a
few animals at 15 mg/kg/dose (3/5 males; females) had bone marrow
decreased cellularity and soft and/or small testes and epididymides
(males). Without wishing to be bound by theory, germ cell depletion
in the testes and decreased sperm in the head region of the
epididymides were reflective of maturation depletion secondary to
the loss of germ cell populations observed at the end of the dosing
period.
[0634] Under the conditions of the study, based on the testicular
germ cell depletion in males at .gtoreq.4 mg/kg/dose and moderate
intestinal single cell crypt necrosis in 1 female at 15 mg/kg/dose,
the no-observed-adverse-effect level (NOAEL) for brincidofovir was
considered to be 1 mg/kg/dose in males and 4 mg/kg/dose for in
females.
Introduction
[0635] The following abbreviations are used in this study: [0636] M
Male(s) [0637] F Female(s) [0638] .about.approximately [0639] mg/kg
milligrams of test item per kilogram of body weight [0640] mL/kg
milliliters of dose formulation per kilogram of body weight [0641]
mL/kg/hr milliliters of dose formulation per kilogram of body
weight per hour [0642] mg/mL milligrams of test item per milliliter
of dose formulation [0643] IV Intravenous [0644] MTD Maximum
tolerated dose [0645] DRF Dose range finding
[0646] The purpose of this study was to assess the toxicity and
toxicokinetics of brincidofovir when administered via intravenous
(2-hour) infusion to rats 2 times per week for a total of 9 doses
over the course of 29 days, and to evaluate the progression or
reversibility of brincidofovir effects during a drug-free
post-treatment recovery period of 14 days.
[0647] The study design incorporates elements of general regulatory
guidelines for toxicity studies.
[0648] This study was conducted in compliance with the principles
of Good Laboratory Practice as set forth in: United States Food and
Drug Administration's Good Laboratory Practice Regulations (Part 58
of 21 CFR); and OECD Principles of Good Laboratory Practice (as
revised in 1997), ENV/MC/CHEM(98)17
[0649] These principles are compatible with Good Laboratory
Practice regulations specified by regulatory authorities throughout
the European Community, the United States (EPA and FDA), and Japan
(MHLW, MAFF and METI), and other countries that are signatories to
the OECD Mutual Acceptance of Data Agreement.
[0650] This study complied with all appropriate parts of the Animal
Welfare Act Regulations: 9 CFR Parts 1 and 2 Final Rules, Federal
Register, Volume 54, No. 168, Aug. 31, 1989, pp. 36112-36163
effective Oct. 30, 1989 and 9 CFR Part 3 Animal Welfare Standards;
Final Rule, Federal Register, Volume 56, No. 32, Feb. 15, 1991, pp.
6426-6505 effective Mar. 18, 1991.
[0651] Pre-clinical studies in two or more species (one rodent, one
non-rodent) are recommended by regulatory agencies such as the FDA
to support administration in humans. The rat is an animal model
commonly utilized in toxicity studies. In addition, a historical
data base is available for comparative evaluation.
[0652] The test item was administered intravenously up to 2 times
per week.
[0653] The number of animals in this study was considered to be the
minimum necessary for statistical, regulatory and scientific
reasons. The purpose of this study was to monitor for toxicity and
progression or reversibility of effects of the test item.
Historical control data indicate that clinical laboratory data,
organ weight data and microscopic examination of tissues vary among
individual animals. The number of rats utilized in the conduct of
subchronic studies (10 animals/sex/group) is recommended by
regulatory guidance documents and was considered the minimum number
that would account for the expected variability among animals. To
determine progression or reversibility of potential effects of the
test item, additional post-treatment (recovery) animals (5
animals/sex/group) were included in the study. Three test
item-treated groups receiving low, intermediate and high multiples
of the proposed human dose and a negative control group were
considered the minimum number of groups necessary to provide a
range of effects and allowed for extrapolation of results to
humans.
[0654] The number of animals selected for toxicokinetic evaluations
(3/sex/treated group) was considered the minimum number necessary
to provide meaningful data, given the inherent variability in
absorption, distribution, metabolism and excretion processes. A
control group with 3 animals/sex was necessary to evaluate the
absence of the test item.
[0655] The doses for this study (0, 1, 4 and 15 mg/kg/dose) were 2,
4, 10 or 15 mg/kg/dose over the course of 2 hours in the maximum
tolerated dose (MTD) phase, and a second set of animals were then
administered 3 doses of 1 or 15 mg/kg/dose over the course of 2
hours in the dose range finding (DRF) phase over the course of 9
days. The high dose (15 mg/kg) administered on this 2-phase study,
was determined to be the maximum feasible dose (MFD) based on
hemolysis observed at higher concentrations.
[0656] Because the infusion catheter receded or was not usable on
the scheduled day of dosing and insufficient time to reposition or
repair the line, the following animals were dosed via intravenous
injection into the tail vein on Day 29; Animal Nos. 1511 and 1519
were found on Day 28 with receded catheters, and Animal No. 4581
was found on Day 29 with an ulceration near the exteriorization
site. These animals were restrained into individual plastic cones
for no more than 2.25 hours (15 minutes for dose site preparation
activities and 2 hours for dose administration) during this
procedure.
TABLE-US-00083 TABLE 80 Experimental Outline Number of animals Dose
Toxicity Recovery Satellite Treat- (mg/ study phase study.sup.b
Group ment kg).sup.a Male Female Male Female Male Female 1 Control
0 15 15 5 5 3 3 2 BCV 1 15 15 5 5 3 3 3 BCV 4 15 15 5 5 3 3 4 BCV
15 15 15 5 5 3 3 .sup.aDoses represent active ingredient.
.sup.bToxicokinetic samples were collected on Day 1 and Day 29.
[0657] The first day of dosing was defined as Day 1 of the study.
Brincidofovir was provided as a white powder and was stored at room
temperature. The purity was determined to be 99.8%.
[0658] The diluent was a 5% dextrose solution for injection (USP)
and was supplied as a clear liquid (96.8% pure). It was stored at
room temperature.
[0659] 2.times. Sodium Phosphate buffer solution was used as a
control. It was prepared from monobasic sodium phosphate
(anhydrous), supplied as a white crystalline powder and stored at
room temperature (100% pure). The phosphate buffer (control)
solution was dissolved in sterile water and pH was adjusted with
sodium hydroxide.
Preparation of Dose Formulations
[0660] A vehicle solution of 2.times. sodium phosphate buffer
solution (400 mM, pH 8.0.+-.0.04) was prepared by mixing the
appropriate amounts of monobasic sodium phosphate solution (400 mM)
and dibasic sodium phosphate solution (400 mM). The pH of the
solution was adjusted with monobasic sodium phosphate solution (400
mM), when necessary. The solution was filtered through a 0.22 .mu.m
Millex.RTM.-GP (PES) filter within a laminar flow hood into a
sterile vessel.
[0661] The vehicle (control item) was stored refrigerated at
2-8.degree. C. Fresh vehicle solution was formulated twice; once
prior to Dose 1 and again prior to Dose 4, and each formulation was
used within one month of preparation.
[0662] A brincidofovir stock of 15 mg/mL was prepared by mixing the
appropriate amount of brincidofovir with 2.times. sodium phosphate
buffer solution, sterile water for injection, USP and 1N sodium
hydroxide (NaOH). The solution was filtered through a 0.22 .mu.m
Millex.RTM.-GP filter under a laminar flow hood into a sterile
vessel. Brincidofovir stock was prepared once prior to Dose 1 and
again prior to Dose 4, and stored refrigerated at 2-8.degree.
C.
[0663] Dosing formulations were prepared by diluting the
appropriate amounts of the brincidofovir stock (15 mg/mL) or stock
vehicle (2.times. sodium phosphate buffer solution) with the
appropriate amounts of 5% dextrose for injection, USP, into a
sterile vial under a laminar flow hood and inverting 10 times to
mix. Fresh dosing formulations were prepared weekly and were stored
refrigerated 2-8.degree. C. when not in use.
TABLE-US-00084 TABLE 81 Dosing Dose Concentration Volume dose Group
Treatment (mg/kg) (mg/mL) (mL/kg/hr) 1 Control 0 0 10 2 BCV 1 0.05
10 3 BCV 4 0.2 10 4 BCV 15 0.75 10
[0664] Detailed records of brincidofovir usage were maintained. The
amount of brincidofovir necessary to prepare the formulations and
the amount actually used were determined at each preparation.
Stability
[0665] Stability analyses demonstrated that brincidofovir
formulations at concentrations bracketing those used in this study
(0.05 mg/mL and 2.0 mg/mL) are stable for 24 hours when stored at
room temperature, and for 8 days when stored refrigerated at 2 to
8.degree. C. The brincidofovir stock solution of 15.0 mg/mL was
stable for 24 hours when stored at room temperature (nominally
20.degree. C.) and for 8, 14 and 28 days when stored at 2 to
8.degree. C.
Confirmation Analysis
[0666] Two samples (5 mL each) were taken on the day of preparation
from the middle region of each dosing formulation (including
control) prepared for each week. One sample was analyzed, in
duplicate, for dose confirmation analysis and one sample was
retained refrigerated (2 to 8.degree. C.) (Due to initial results
outside the study acceptance plan criteria (.+-.10% of nominal),
the Group 2 retained samples were re-analyzed for Weeks 1 and
2).
Storage Conditions
[0667] All dose formulation samples were stored refrigerated (2 to
8.degree. C.) until analysis or final disposition.
Brincidofovir Administration
[0668] Brincidofovir was administered as an intravenous infusion
over 2 hours. Animals were treated at constant doses in 20 mL/kg/2
hours.
[0669] Infusion Catheter Implantation Procedure
[0670] Catheters for infusion were implanted approximately 1-3
weeks prior to dose administration. All animals, including the
spares, were surgically implanted with a catheter.
[0671] The surgical site was prepared as per Testing Facility's SOP
for aseptic, recovery surgical procedures. Animals received
analgesics pre-emptively (flunixin meglumine (USP) 2 mg/kg,
subcutaneously).
[0672] All animals were anesthetized (isoflurane) and shaved in the
inguinal and dorsal regions. A small incision was made and the
femoral vein isolated. A small incision in the vessel was performed
and a sterile catheter was placed into the vessel. The catheter was
inserted via the femoral vein and passed retrograde into the
inferior vena cava, just caudal to the kidneys. The catheter was
secured with sutures and passed subcutaneously so as to exit at the
nape of the neck. A small pocket was made at the exteriorization
site in which a loop of catheter remained. The inguinal region was
closed by sutures.
[0673] The animals were placed in jackets and the implanted
catheters were attached to pins with capped septum connectors. The
catheters were locked with taurolidine citrate locking solution.
Each animal received enrofloxacin 5.0 mg/kg intramuscularly on the
day of surgery.
[0674] Surgical repairs (1 repair per animal limit) to restore
catheter patency were performed as necessary, during the pretest
period and at the discretion of the Study Director during the
on-test period. Analgesics were administered preemptively (flunixin
meglumine 2 mg/kg (USP), subcutaneously). Animals undergoing a
surgical repair received an appropriate dose of antibiotics (as per
the Standard Operating Procedures of the Testing Facility) on the
day of surgery.
[0675] Catheters were tied off after the completion of dose
administration just prior to recovery (a knot was placed in the
catheter and the catheter was receded under the skin) and jackets
were removed.
Infusion Catheter Maintenance Procedure
[0676] Implanted femoral vein catheters were assessed for patency
as per Testing Facility's SOP prior to placing animals on
study.
Volume Dose (Rate)
[0677] 20 mL/kg/2 hours (10 mL/kg/hour).
Individual Dose Concentration
[0678] The individual animal concentrations were calculated from
the most recently recorded scheduled bodyweight.
Frequency and Duration
[0679] The test/control item was administered via 2 hours
intravenous infusion twice weekly for a total of 9 individual doses
over 29 days. Test/control item administration continued through
the day prior to terminal necropsy. Recovery animals were held for
a 14 day observation period after the last dose.
Dose Site
[0680] Surgically implanted cannula in the femoral vein/vena cava
or via peripheral tail vein.
Dosing Procedure
[0681] One to two days prior to dose initiation, the lock solution
was withdrawn from each animal's implanted catheter (if possible)
and the catheter was flushed with saline before connecting to a
tether and infusion dosing set. After connecting the animals'
catheters, the animals were infused with sterile saline (0.9% NaCl,
USP) at a rate of 0.5 mL/hr by a calibrated Medfusion syringe pump
until dose administration on Day 1.
[0682] A saline flush was infused to deliver the test item in the
infusion lines and to ensure the delivery of a complete dose (an
additional -0.5 to 1 mL volume, at the same rate as the test item,
was administered to flush the catheter line).
[0683] Animals were maintained on saline in between dose
administrations. Saline syringes were changed at least every 4
days.
[0684] Animals with an unusable/non-functioning catheter were
surgically repaired when possible. If repair was not possible,
these animals were dosed via a peripheral tail vein in a
restrainer, at the discretion of the Study Director. Animals were
placed into individual plastic cones for no more than 2.25 hours
(15 minutes for dose site preparation activities and 2 hours for
dose administration via tail vein injection).
[0685] On Day 29, Animal Nos. 1511, 1519 and 4581 were administered
Dose 9 via peripheral tail vein.
Dosing Accuracy
[0686] The accountability (confirmation of actual dose
administered) for each day of infusion was calculated based on the
weight of the each syringe predose and postdose, and reviewed for
adjustments daily; the data is retained in the study files. Pumps
were checked for accuracy prior to dose initiation with saline
using the same length of dose at rate as the study, and post dose
accountability was used to monitor pump performance throughout the
study.
Animals
Strain/Species
[0687] Albino Rats (Outbred) VAF/Plus.RTM. CD.RTM. (Sprague-Dawley
derived) [Crl:CD.RTM. (SD)BR]
Number of Animals
[0688] 170 Total received (85 males and 85 females); 144 placed on
test (72 males and 72 females).
Pretest Period
[0689] The pretest period was approximately 4 weeks. All animals
were examined during the pretest period to confirm suitability for
study. Pretest procedures were not performed until animals had been
allowed to stabilize for 5 days.
Age of the Main Study Animals at Start of Treatment
[0690] Approximately 10 weeks
Weight Range of the Main Study Animals at the Start of
Treatment
[0691] Males: 290.6 g to 422.4 g. Females: 203.8 g to 294.7 g
Assignment
[0692] More animals than required for the study were purchased and
stabilized. Animals considered unsuitable for the study on the
basis of pretest physical examinations and catheter patency were
eliminated prior to random selection for group assignment. Animals
considered suitable for study were distributed into 4 groups of 18
animals per sex by a computerized random sort program so that body
weight means for each group were comparable. Due to pretest
ophthalmology findings, several toxicity animals were swapped with
satellite animals but remained within the same group. Individual
weights of animals placed on test were within .+-.20% of the mean
weight for each sex.
Identification of Animals
[0693] Each rat was implanted with a BMDS IMI-1000 Implantable
Radio Frequency Transponder (microchip) programmed with a unique
number. This number was cross referenced with an animal number
assigned by the Testing Facility; this number plus the study number
comprised a unique identification number for each animal. In
addition, each cage was provided with a cage card that was
color-coded for dose level identification and contained study
number and facility-assigned animal number information.
Veterinary Care
[0694] Animals were monitored by the technical staff for any
conditions requiring possible veterinary care and treated as
necessary. Miscellaneous, non-test item-related veterinarian
evaluations for individual animals were reviewed by the study
director and are documented in the study file.
[0695] Serial observations performed on the animals from the
satellite groups were confined to clinical observations, body
weights, ophthalmology and food consumption; these data are not
reported.
[0696] Blood samples were obtained for the determination of plasma
concentrations of brincidofovir and the brincidofovir metabolite,
cidofovir.
[0697] On Days 1 and 29, blood samples were obtained for
toxicokinetic determinations at the timepoints shown in the table
below:
TABLE-US-00085 TABLE 82 Collection times and number of animals
Number of Toxicokinetic Intervals Timepoints Animals Day 1 2 and 24
hours post initiation of 3 animals/sex/group dose administration
(Group 1) Predose, 1, 2, 8, 24, and 48 hours 3 animals/sex/group
post initiation of dose administration (Groups 2, 3, 4) Day 29 2
and 24 hours post initiation of 3 animals/sex/group dose
administration (Group 1) Predose, 1, 2, 8, 24, and 48 hours 3
animals/sex/group post initiation of dose administration (Groups 2,
3, 4)
Collection Procedures
[0698] Approximately 0.40 mL of whole blood was obtained via the
tail vein from each animal at each time point. Animals were not
fasted prior to blood collection. Blood was collected into tubes
containing K.sub.2EDTA anticoagulant and placed on wet ice in an
upright position. Plasma was separated by centrifugation (for 10
minutes at approximately 2000 g, at approximately 2-8.degree. C.).
Approximately 0.150 mL of plasma was transferred into a single
cryotube appropriately labeled as to study number, animal number,
time point, date of sampling and sample type. Remaining plasma was
transferred into a second cryotube and retained as a backup sample.
Plasma was obtained and frozen at approximately -80.+-.10.degree.
C. within approximately 2 hours after collection of each blood
sample until analysis. Animals were euthanized (CO.sub.2
inhalation) after the final blood collection.
Storage and Disposition of Samples
[0699] All sample tubes were stored frozen (-80.+-.10.degree. C.)
and shipped (frozen, on dry ice) to Pyxant Labs, Colorado Springs,
Colorado for analysis.
Analysis/Reporting
[0700] Bioanalytical samples were analyzed using a validated liquid
chromatographic-triple quadruple mass spectrometric (LC-MS/MS)
assay at Pyxant Labs.
[0701] Upon receipt of the plasma concentration data,
noncompartmental toxicokinetic analysis was performed by Nuventra,
Inc. Durham, N.C.
Daily Observations
[0702] Animals were observed in their cages twice daily for
mortality and signs of severe toxic or pharmacologic effects.
Animals in extremely poor health or in a possible moribund
condition were identified for further monitoring and possible
euthanasia.
Dosing Observations
[0703] Observations were made immediately and 2 hours after the
completion of dosing for any signs of poor health or toxic or
pharmacologic effects (e.g., abnormalities in general condition,
appearance, activity, behavior, respiration, etc.).
Signs
[0704] Animals were removed from their cages and examined once
pretest and weekly during the study period. Examinations included
observations of general condition, skin and fur, eyes, nose, oral
cavity, abdomen and external genitalia as well as evaluations of
respiration.
Ophthalmoscopic Examination
[0705] All animals were examined pretest. All surviving toxicity
animals were examined at end of dosing and end of recovery. Only
animals which were within normal limits at the pretest examination
were placed on test.
[0706] Lids, lacrimal apparatus and conjunctiva were examined
visually. The cornea, anterior chamber, lens, iris, vitreous humor,
retina and optic disc were examined by indirect ophthalmoscopy.
[0707] The pupils of each animal were dilated prior to examination
using tropicamide ophthalmic solution.
Body Weight
[0708] Animals were removed from their cages and weighed twice
pretest, on the day prior to each dose during the treatment period
and weekly during the recovery period. Terminal, fasted body
weights were obtained just prior to the scheduled necropsy
intervals. Animals euthanized for welfare reasons were weighed
prior to necropsy.
Food Consumption
[0709] Food consumption was measured (weighed) weekly, beginning
one week prior to treatment.
Clinical Pathology
[0710] Blood obtained via the orbital sinus (retrobulbar venous
plexus) under light isoflurane anesthesia was used to analyze
hematology, coagulation and clinical chemistry parameters for up to
10 toxicity animals/sex/group at the termination of dosing and up
to 5 recovery animals/sex/group at the end of recovery. Animals
were fasted overnight prior to each blood collection interval.
[0711] Urine obtained via a 16-hour overnight collection period was
analyzed for up to 10 toxicity animals/sex/group at the termination
of dosing and up to 5 recovery animals/sex/group at the end of
recovery. Animals were fasted but not water-deprived during the
collection period.
Hematology
[0712] Blood samples (approximately 0.25 mL) were collected into
tubes containing K.sub.2EDTA anticoagulant and analyzed for the
following using a Siemens ADVIA 120 Hematology Analyzer: Hemoglobin
concentration (HGB); Hematocrit (HCT); Erythrocyte count (RBC);
Platelet count (PLT); Mean corpuscular volume (MCV); Mean
corpuscular hemoglobin (MCH); Mean corpuscular hemoglobin
concentration (MCHC); Red cell distribution width (RDW); Total
leukocyte count (WBC); Reticulocyte count (RETIC); Differential
leukocyte count (Manual differential leukocyte counts were
performed for verification and absolute values were calculated if
necessary).; Neutrophils (ANEU); Lymphocytes (ALYM); Eosinophils
(AEOS); Basophils (ABASO); Monocytes (AMONO); and Large unstained
cells (ALUC).
[0713] A peripheral blood smear was prepared for each animal at
each blood collection interval and was available for confirmation
of automated results and/or other evaluations deemed necessary.
Coagulation
[0714] Blood samples (approximately 1.0 mL) were collected into
tubes containing sodium citrate anticoagulant and analyzed for the
following using a Diagnostica Stago Products STA Compact.RTM.
mechanical clot detection system: Prothrombin time (PT): and
Activated partial thromboplastin time (APTT).
Clinical Chemistry
[0715] Blood samples (approximately 1.0 mL) were collected into
tubes with no anticoagulant, allowed to clot, centrifuged to obtain
serum and analyzed for the following using a Siemens ADVIA 1800
Chemistry Analyzer:
[0716] Aspartate aminotransferase (AST) (Kinetic--Modified
Bergmeyer); Alanine aminotransferase (ALT) (Kinetic--Modified
Bergmeyer); Alkaline phosphatase (ALKP) (Kinetic Tietz AMP Buffer);
Blood urea nitrogen (BUN) (Enzymatic--Roch-Ramek with Urease);
Creatinine (CREAT) (Jaffe Picric Acid in Alkaline Medium); Glucose
(GLU) (Glucose Hexokinase II Method); Cholesterol (CHOL) (Enzymatic
esterase/oxidase--Trinder Endpoint); Triglycerides (TRIG) (Fossati
Three Step Enzymatic--Trinder Endpoint); Total protein (TP) (Biuret
Technique); Albumin (ALB) (Bromocresol Green Method); Total
bilirubin (TBILI) (Oxidation with Vandate); Sodium (NA.sup.+) (Ion
Selective Electrode) Potassium (K.sup.+) (Ion Selective Electrode);
Chloride (Cl.sup.-) (Ion Selective Electrode); Calcium (Ca.sup.++)
(Michaylova & Ilkova, Arsenazo III); and Inorganic phosphorus
(PHOS) (Phosphomolybdate--UV Method)
Urinalysis
[0717] Urine was collected into ice-chilled containers overnight
(approximately 16 hours) from toxicity study animals housed in
metabolism cages.
[0718] Urine samples were analyzed for the following using
Multistix reagent strips, interpreted using a Siemens Clinitek
Advantus: pH; Protein (PRO); Glucose (GLU); Ketones (KET);
Bilirubin (BIL); and Occult blood (BLD).
[0719] The samples were also analyzed for the following using
manual methods:
[0720] Appearance (APP); Specific gravity (Sp.G.) (Clinical
Refractometer, Atago Uricon-N); Volume (VOL).
Postmortem
Method of Euthanasia
[0721] Animals sacrificed for welfare reasons or at the scheduled
sacrifice intervals were euthanized by exsanguination following
isoflurane inhalation. Toxicokinetic animals were euthanized via
CO.sub.2 inhalation.
Scheduled Necropsy
[0722] Necropsy was performed on up to 10 toxicity study
animals/sex/group after animals had been treated for 29 days and on
up to 5 toxicity study animals/sex/group after a 14 day
treatment-free recovery period. Animals were fasted overnight prior
to necropsy. Necropsy schedules were established to ensure that
examination of animals of both sexes from each group were performed
at similar times of the day throughout the necropsy periods.
[0723] Toxicokinetic animals were euthanized following the final
blood collection on Day 29 and discarded without examination.
Macroscopic Examinations
[0724] Complete macroscopic examinations were performed on all
animals. The macroscopic examination included examination of the
external surface and all orifices; the external surfaces of the
brain and spinal cord; the organs and tissues of the cranial,
thoracic, abdominal and pelvic cavities and neck; and the remainder
of the carcass for the presence of macroscopic morphologic
abnormalities.
[0725] A macroscopic examination was performed for TK Animal No.
4107, which was sacrificed for welfare reasons, tissues were
preserved but not examined microscopically.
Organ Weights
[0726] Organs indicated below were weighed for all surviving
toxicity and recovery study animals at the scheduled sacrifice
intervals. Prior to weighing, the organs were carefully dissected
and properly trimmed to remove adipose and other contiguous tissues
in a uniform manner. Organs were weighed as soon as possible after
dissection in order to avoid drying. Paired organs were weighed
together.
Tissues Preserved and Examined Histopathologically
[0727] The tissues listed below were obtained from all animals and
preserved. In addition, slides of the indicated tissues were
prepared and examined microscopically. Any abnormalities not noted
during macroscopic examinations which were seen during histology
processing were recorded.
TABLE-US-00086 TABLE 83 Tissues weighed, preserved and examined
microscopically EXAMINED PRE- MICRO- ORGAN NAME WEIGHED SERVED
SCOPICALLY animal identity X adrenal glands X X X aorta (thoracic)
X X bone marrow smear X (femur) bone and marrow X X (femur with
joint) bone and marrow X X.sup.a (sternum) brain (medulla, X X X
pons, cerebrum and cerebellum) epididymides X X X esophagus X X
eyes X X Harderian gland X X heart X X X infusion site X X kidneys
X X X lacrimal glands X X large intestine X X.sup.b (cecum, colon,
rectum) liver X X X lungs (with X X mainstem bronchi) lymph nodes X
X (axillary, mesenteric) mammary gland X X.sup.c (inguinal) nerve
(sciatic) X X optic nerve X ovaries X X X pancreas X X pituitary
gland X.sup.d X X prostate gland X.sup.e X X salivary glands X X
(mandibular) seminal vesicles X.sup.e X X skeletal muscle X X
{rectus femoris) skin (dorsal - X X base of tail) small intestine X
X.sup.c (duodenum, ileum, jejunum, Peyer's patches\GALT) spinal
cord X X (cervical, thoracic, lumbar) spleen X X X stomach X X
testes X X X thymus X X X thyroid/ X.sup.d X X parathyroid glands
tongue X trachea X X urinary bladder X X uterus (body/homs) X X X
with cervix vagina X X Zymbal's gland X tissues with X X
macroscopic findings including tissue masses .sup.aQualitative
examination (no differential count). .sup.bRectum was not examined
microscopically. .sup.cMammary gland for males and GALT were
evaluated only if present in routine sections. .sup.dWeighed
post-fixation. .sup.eProstate and seminal vesicles were weighed
together.
Preservatives
[0728] All tissues were preserved in 10% neutral buffered formalin
(NBF). The eyes and testes were placed in Modified Davidson's
solution initially and then retained in 10% NBF. Lungs were infused
with 10% NBF prior to their immersion into a larger volume of the
same fixative. Smear preparations of the marrow from the femur were
air dried and fixed in absolute methanol.
Processing
[0729] After fixation, the tissues and organs from all animals were
routinely processed, embedded in paraffin, cut at a microtome
setting of 4-7 microns, mounted on glass slides, stained with
hematoxylin and eosin and examined by light microscopy. The bones
were decalcified in Decalcifier II.TM..
[0730] The data collection system used for collecting the in-life
and post-life data divides the study into phases. The start of each
phase begins with Day 1/Week 1. The phases presented in this report
are:
[0731] Pre-Treatment: Begins the day the animals arrive. Phase
days/weeks are labeled P1, P2, P3, etc. During this phase, animals
are not being dosed and they have not been assigned their permanent
identification numbers.
[0732] Randomization: Begins the day the animals are sorted into
groups and assigned their permanent identification numbers. Phase
days/weeks are labeled Ra1, Ra2, Ra3, etc. Animals are not being
dosed during this phase.
[0733] Treatment: Begins for each animal when it receives its first
administration of test/control item as specified in the study
plan.
[0734] Recovery: Begins the day after each designated animal stops
receiving its specified dose and continues until study completion.
Phase days/weeks are labeled R1, R2, R3, etc.
[0735] The death codes used have the following meanings:
[0736] "FD": Found dead; "S1": Scheduled euthanasia 1; "WE":
euthanized for welfare reasons; "Z1": Recovery euthanasia 1.
Clinical Observations
[0737] Clinical observations are presented for each animal that
showed signs, providing detail of the type of sign, day or week of
occurrence and information on the duration of the sign applicable.
"AM": Morning Observation; "PM": Afternoon Observation; "Unsched":
Unscheduled Observation; "Post Dose": Immediately After Completion
of Dosing; "2 Hr PD": 2 Hours After Completion of Dosing.
Ophthalmic Examination
[0738] Observations are presented for each animal that showed
findings. Observations were bilateral unless otherwise
indicated.
Food Consumption
[0739] Food consumption (g/day) was calculated as: grams of food
consumed/#days
Food consumption (g/animal/day) was calculated as: grams of food
consumed/animal days.sup.a
.sup.aSum of whole days each animal is alive in the cage for each
consumption period. If an animal dies the day of death is not
counted as an animal day. For example: 4 animals in a cage for 5
days equals 20 animal days; if one animal died on the 3.sup.rd day
then the animal days equals 1
Hematology
[0740] The following abbreviations were used: "ACMO": Automated
count invalidated by manual observations; "CS": Clotted specimen;
"NSAD": No sample animal died.
[0741] Sample comments (SCM1) were described using the following
abbreviations: "CLSE": Severe platelet clumping noted; "CLSL":
Slight platelet clumping noted; "NCLP": No clumping.
Coagulation
[0742] The following abbreviations were used: "CS": Clotted
specimen; "NS": No specimen; "NSAD": No sample animal dead; "NVIR":
Not valid due to irreproducible result.
[0743] Sample comments (SCM1) were described using the following
abbreviations: "SLH": Slightly hemolyzed.
Blood Chemistry
[0744] BUN values below the analytical limit (<10) are excluded
from calculations. TBILI values below the analytical limit
(<0.2) are excluded from calculations.
[0745] The following abbreviations were used: "NS": No specimen;
"NSAD": No sample animal dead; "1+H": Minimal hemolysis.
Globulin (GLOB) was calculated as: total protein-albumin
Albumin/globulin ratio (A/G) was calculated as:
albumin/globulin
Urinalysis
[0746] Group means are presented for volume, pH, and specific
gravity only.
[0747] The following other abbreviations were used: "NS": No
specimen.
[0748] The appearance and color were described using the following
abbreviations: "CLOG": Cloudy; "CLR": Clear; "SLCL": Slightly
cloudy; "STR": Straw; "YELL": Yellow.
TABLE-US-00087 TABLE 84 Qualitative results of Multistix diagnostic
reagents are reported according to the following convention: NEG
Negative Protein TR Trace levels detected 30 30 mg/dL 100 100 mg/dL
.gtoreq.300 .gtoreq.300 mg/dL Glucose 100 100 mg/dL 250 250 mg/dL
500 500 mg/dL .gtoreq.1000 .gtoreq.1000 mg/dL Ketones TR Trace
levels detected 15 15 mg/dL 40 40 mg/dL .gtoreq.80 .gtoreq.80 mg/dL
Bilirubin SM Small amount detected MOD Moderate amount detected LG
Large amount detected Occult Blood TR Trace levels detected SM
Small amount detected MOD Moderate amount detected LG Large amount
detected
Organ Weights
[0749] Organ weights were presented as absolute, relative to
terminal body weight using the weight recorded on the day of
necropsy and relative to brain weight.
Pathology
[0750] Unless otherwise specified in a histopathology note, the
organ/tissue examined was the required (routine) section.
[0751] The following abbreviations were used: "B-": Benign; "Cath":
Catheter; "GALT": Gut-associated lymphoid tissue; "Infuse":
Infusion; "LS": Longitudinal; "M-": Malignant; "TS":
Transverse.
[0752] Histopathology findings were graded according to the
following scale:
TABLE-US-00088 Minimal The change is barely discernible and/or very
few/very small foci or areas are affected. Slight The change is
more noticeable but only evident as few/small foci or areas
affected. Moderate The change is obviously present, and of
appreciable size and/or number. Marked The change is abundant in
many areas of the section and/or is of prominent size. Severe The
change affects a large proportion of the tissue and/or is of a
large size.
[0753] All statistical analyses were carried out separately for
males and females using the individual animal as the basic
experimental unit.
[0754] The following data types were analyzed at each timepoint
separately: body weight; body weight change from interval to
interval; food consumption; hematology; coagulation; clinical
chemistry; urinalysis; and organ weights.
[0755] The parameters to analyze were identified as continuous,
discrete or binary. Test-item treated groups were then compared to
the control using the following procedures.
Study Plan Deviations
[0756] The following study plan deviations occurred during the
study, but without wishing to be bound by theory, were not
considered to have compromised the validity or integrity of the
study.
[0757] On Day 1, the processed 8 hour post-dose toxicokinetic
samples for Groups 2 to 4 were left on dry ice overnight. Samples
were found frozen the following morning. However, the required
temperature (-80.degree. C. (.+-.10.degree. C.)) was not monitored
overnight. Samples were immediately transferred to freezer SL301 at
nominal -80.degree. C. (.+-.10.degree. C.).
[0758] On Pretest Day 13, Animal No. 1015 did not have surgery
performed due to significantly low body weight.
[0759] On Day 13, viability checks were not performed on the spare
animals. However, animals were within normal limits the day prior
to and the day after Day 13.
[0760] Surgical repairs for Animal Nos. 1592 (Day 21) and 4078 (Day
10) were not acknowledged by the Study Director until 7 and 2 days
after repair, respectively.
[0761] On Day 29, low blood volumes were collected for
toxicokinetic samples from Animal Nos. 2036 (8 hour post dose
timepoint) and 3050 (2 hour post dose timepoint) because the
animals were "difficult to bleed" and "slow to bleed",
respectively.
[0762] The pituitary gland tissue for Animal No. 4074 was collected
at the recovery necropsy, but was later found to be missing at the
post-fix trimming; a weight was not obtained.
[0763] On Day 29, the immediate post-dose observation was not
performed on Animal No. 4580.
[0764] On Day 1, the 24 hour toxicokinetic samples for Animal Nos.
1016 and 2036 were processed 1 minute over the "within the 2 hours"
specified in the study plan.
[0765] At the terminal necropsy, the collection site for clinical
pathology samples was not documented for Animal No. 1004.
[0766] On Day 23, toxicokinetic Animal No. 4107 was transferred to
necropsy due to welfare reasons, however, the animal was no
documented on the appropriate records but examination was
performed.
[0767] Spare Animal No. 1015 was euthanized rather than transferred
to the in-house colony as per study plan.
[0768] There is no documentation present on the surgery records
documenting the catheterized animals were individually housed
post-surgery.
[0769] On Day 19, the dose infusion time was interrupted for 14
minutes for female TK Animal No. 1528 due to changing of vehicle
syringes.
[0770] Between Days 4 and 12, the saline syringe change was not
documented on the Saline and Injection Cap Change Record for the
toxicokinetic animals.
[0771] On Day 29, toxicokinetic samples were collected from only 2
males, not 3 males/group/time-point in Group 4, due to the
euthanasia of the male Animal No. 4107 for welfare reasons on Day
23.
[0772] On Days 29, the following sample was collected more than
.+-.3 minutes (the time permitted per SOP) after the indicated time
point:
TABLE-US-00089 Animal Scheduled Actual Deviation Number
Day/Interval Time Time in Minutes 2029 29-1 hr 10:59 11:04 2
[0773] Various tissues were noted missing at the time of trimming
or microscopic examination for various animals and necropsy
intervals.
[0774] Due to the timing of the validation study, it was
anticipated that stability might not have been established before
the first day of dosing, therefore the formulation preparation was
changed to daily preparations rather than weekly as per original
study plan. The Day 1 dose formulation was prepared on the day
before dosing thereby deviating from the study plan in place at
that time. Once stability was established, formulation preparation
was changed back to weekly preparations.
[0775] Some dose intervals had inconsistencies in the dose
accountability, resulting in deviations from the study plan in that
animals received more or less than the intended dose (Table 84A).
These may or may not have resulted in out of range dose
accountability criteria which are .+-.10.0%.
TABLE-US-00090 TABLE 84A Samples with inconsistencies in dose
accountability. Infusion Theoretical Actual Percent Animal Rate
Volume Volume Difference Number Dose No. (ml/hr) (ml) (ml) (%) Code
*3566 Dose 1 2.51 5.05 5.00 -0.40 A 4061 Dose 8 4.08 8.16 9.76
19.61 B 3046 Dose 5 4.04 8.08 10.25 26.86 B 3551 Dose 5 2.47 4.94
4.99 1.01 A 2037 Dose 6 3.55 7.10 8.21 15.63 B 2030 Dose 6 4.04
8.08 7.21 -10.77 B 1511 Dose 9 2.67 5.34 1.07 -79.96 C 1519 Dose 9
2.79 5.58 6.22 11.47 B 4063 Dose 9 4.00 8.00 10.96 37.00 B *TK
animal A = Catheter found leaking for an indeterminable amount of
time B = No apparent reason C = Tail vein catheter difficulties;
dosing stopped due to welfare reasons
[0776] On one occasion for some animals dosing was interrupted or
pump settings needed to be adjusted affecting the accountability
documentation. However, variations were less than 10% deviation
from desired dose. Additionally, Animal No. 1017 (TK) did not
receive the last 2 minutes of saline catheter flush.
[0777] On the day of terminal necropsy, Animal Nos. 1010, 3047,
3048, 3049, 3057, 2538, 2539 and 2594 were given feed for welfare
reasons after being fasted close to 26 hours.
Analytical Chemistry
[0778] Analysis of preliminary mixes confirmed that the test item
was stable in the vehicle for at least 24 hours at room temperature
(nominally 20.degree. C.) and for at least 8 days at 2 to 8.degree.
C. (under storage conditions used in this study). Analyses
conducted during the treatment period confirmed that with the
exception of the Day 1, 1 mg/mL (Group 2) formulation, dose
solutions of appropriate concentration were administered. The Day
1, 1 mg/mL dose solution was 86.7% of nominal. Without wishing to
be bound by theory, it was concluded that due to interactions at
the meniscus, the volume collected for dose formulation analysis
may have lowered the analysis results. The volume collected for
analysis was increased for subsequent weekly preparations and
confirmed to be of appropriate concentration.
TABLE-US-00091 TABLE 85 Mean analytical concentrations Day 1
NOMINAL ANALYTICAL CONCENTRATION CONCENTRATION GROUP (ppm) OR
(mg/mL) (% of Nominal) 2 1 86.7 2 Back-up 1 76.9 2 Repeat 1 75.5
Preparation 3 4 95.7 4 15 100.7
TABLE-US-00092 TABLE 86 Mean analytical concentrations Week 2
NOMINAL ANALYTICAL CONCENTRATION CONCENTRATION GROUP (ppm) OR
(mg/mL) (% of Nominal) 2 1 90.1 2 Back-up 1 86.8 3 4 95.4 4 15
102.9
TABLE-US-00093 TABLE 87 Mean analytical concentrations Week 3
NOMINAL ANALYTICAL CONCENTRATION CONCENTRATION GROUP (ppm) OR
(mg/mL) (% of Nominal) 2 1 93.2 3 4 94.8 4 15 100.2
TABLE-US-00094 TABLE 88 Mean analytical concentrations Week 4
NOMINAL ANALYTICAL CONCENTRATION CONCENTRATION GROUP (ppm) OR
(mg/mL) (% of Nominal) 2 1 92.8 3 4 96.9 4 15 101.0
TABLE-US-00095 TABLE 89 Mean analytical concentrations Week 5
NOMINAL ANALYTICAL CONCENTRATION CONCENTRATION GROUP (ppm) OR
(mg/mL) (% of Nominal) 2 1 94.9 3 4 97.7 4 15 101.1
Brincidofovir
[0779] In the brincidofovir-treated animals on Day 1 peak plasma
concentrations of brincidofovir occurred at 1 to 2 hours post start
of infusion with mean concentrations ranging from 215 ng/mL to 3670
ng/mL for doses ranging from 1 to 15 mg/kg. Plasma concentrations
of brincidofovir remained quantifiable at the 8-hour time point for
the 4 and 15 mg/kg dose groups, but only through the 2-hour time
point for the 1 mg/kg dose group. Plasma concentrations of
brincidofovir increased as the dose increased from 1 to 15 mg/kg on
Day 1.
TABLE-US-00096 TABLE 90 Summary of combined male and female plasma
brincidofovir toxicokinetic parameters by treatment on Day 1 in
rats (TK parameter population) AUC.sub.last/Dose C.sub.max
C.sub.max/Dose T.sub.max C.sub.last T.sub.last AUC.sub.last
((hr*ng/mL)/ Treatment Statistic (ng/mL) (kg*ng/mL/mg) (hr) (ng/mL)
(hr) (hr*ng/mL) (mg/kg)) 1 mg/kg N 6 6 6 6 6 6 6 Mean 219 219 NC
209 NC 319 319 SD 39.8 39.8 NC 39.5 NC 58.4 58.4 CV % 18.2 18.2 NC
18.9 NC 18.3 18.3 Min 171 171 1.00 157 2.00 249 249 Median 210 210
1.00 206 2.00 305 305 Max 271 271 2.00 271 2.00 398 398 4 mg/kg N 6
6 6 6 6 6 6 Mean 1010 252 NC 2.20 NC 2215 554 SD 159 39.8 NC 0.819
NC 717 179 CV % 15.8 15.8 NC 37.2 NC 32.3 32.3 Min 825 206 1.00
1.26 8.00 1340 335 Median 1000 250 2.00 2.31 8.00 2317 579 Max 1220
305 2.00 3.38 8.00 3056 764 15 mg/kg N 6 6 6 6 6 6 6 Mean 3800 254
NC 9.68 NC 8815 588 SD 565 37.7 NC 3.56 NC 1260 83.8 CV % 14.9 14.9
NC 36.7 NC 14.3 14.3 Min 2950 197 1.00 5.06 8.00 7368 491 Median
3760 251 1.00 10.3 8.00 8777 585 Max 4460 297 2.00 13.6 8.00 10565
704 NC = Not calculated; For the 1 mg/kg dose group, AUC.sub.last
values were calculated using only 2 quantifiable data points.
[0780] In the Day 29 brincidofovir-treated animals, peak plasma
concentrations of brincidofovir occurred at 1 to 2 hours post start
of infusion with mean concentrations ranging 297 ng/mL to 4500
ng/mL for doses ranging from 1 to 15 mg/kg. Plasma concentrations
of brincidofovir remained quantifiable at the 8 hour time point for
the 4 and 15 mg/kg dose groups and for 1 of 6 animals in the 1
mg/kg dose group. For the 1 mg/kg dose group, plasma concentrations
were quantifiable at the 2-hour time point for all animals. For the
majority of the animals, there were no quantifiable concentrations
at the 24 or 48-hour time points at any dose administered;
exclusive of one animal in the 4 mg/kg dose group that showed
measurable concentrations out to 48 hours. Plasma concentrations of
brincidofovir increased as the dose increased from 1 to 15 mg/kg on
Day 29.
TABLE-US-00097 TABLE 91 Summary of combined male and female plasma
brincidofovir toxicokinetic parameters by treatment on Day 29 in
rats (TK parameter population) AUC.sub.last/Dose C.sub.max
C.sub.max/Dose T.sub.max C.sub.last T.sub.last AUC.sub.last
((hr*ng/mL)/ Treatment Statistic (ng/mL) (kg*ng/mL/mg) (hr) (ng/mL)
(hr) (hr*ng/mL) (mg/kg)) 1 mg/kg N 6 6 6 6 6 6 6 Mean 298 298 NC
126 NC 410 410 SD 130 130 NC 88.2 NC 190 190 CV % 43.7 43.7 NC 69.9
NC 46.4 46.4 Min 52.6 52.6 1.00 1.12 2.00 73.9 73.9 Median 324 324
1.00 144 2.00 434 434 Max 421 421 2.00 229 8.00 653 653 4 mg/kg N 6
6 6 6 6 6 6 Mean 1200 301 NC 2.06 NC 2759 690 SD 297 74.3 NC 0.519
NC 851 213 CV % 24.7 24.7 NC 25.2 NC 30.8 30.8 Min 935 234 1.00
1.38 8.00 2119 530 Median 1160 290 1.00 2.14 8.00 2549 637 Max 1700
425 2.00 2.67 48.00 4415 1104 15 N 5 5 5 5 5 5 5 mg/kg Mean 4500
300 NC 10.9 NC 9893 660 SD 742 49.5 NC 4.14 NC 1840 122 CV % 16.5
16.5 NC 37.9 NC 18.6 18.6 Min 3550 237 1.00 7.75 8.00 8001 533
Median 4460 297 1.00 8.90 8.00 9528 635 Max 5600 373 1.00 18.0 8.00
12928 862 NC = Not calculated; For the 1 mg/kg dose group,
AUC.sub.last values were calculated using only 2 quantifiable data
points.
Brincidofovir Metabolite Cidofovir
[0781] In the brincidofovir-treated animals on Day 1, individual
plasma concentrations of the metabolite CDV were generally not
quantifiable with the first sample collected after initiation of
dosing (1-hour post-start of the infusion) in the 1 mg/kg (0 of 6
rats) and 4 mg/kg (1 of 6 rats) dose groups. However, individual
plasma concentrations of CDV were quantifiable at the 1-hour sample
collection time point in 5 of 6 rats in the 15 mg/kg dose
group.
[0782] On Day 29, individual plasma concentrations of the
metabolite CDV were generally not quantifiable with the first
sample collected after initiation of dosing (1-hour post-start of
the infusion) in the 1 mg/kg (1 of 6 rats) dose group. However,
individual plasma concentrations of CDV were quantifiable at the
1-hour sample collection time point in 4 of 6 rats in the 4 mg/kg
and 5 of 5 rats in the 15 mg/kg dose group.
[0783] Plasma concentrations of CDV generally remained quantifiable
at the 24-hour time point for the 4 mg/kg dosed animals, and at the
48-hour time point for the 15 mg/kg dosed animals, but only through
the 8-hour time point for the 1 mg/kg dose group. Plasma
concentrations of CDV generally increased as the brincidofovir dose
increased from 1 to 15 mg/kg.
TABLE-US-00098 TABLE 92 Summary of combined male and female plasma
CDV toxicokinetic parameters by treatment on Day 1 in rats (TK
parameter population) AUC.sub.last Treat- Sta- C.sub.max T.sub.max
C.sub.last T.sub.last T.sub.lag (hr*ng/ ment tistic (ng/mL) (hr)
(ng/mL) (hr) (hr) mL) 1 N 6 5 5 5 5 6 mg/kg Mean 5.64 NC 5.66 NC NC
27.3 SD 2.96 NC 0.486 NC NC 20.2 CV % 52.4 NC 8.6 NC NC 74.1 Min 0
2.00 5.01 2.00 1.00 0 Median 6.06 2.00 5.87 8.00 1.00 37.8 Max 8.60
8.00 6.18 8.00 1.00 45.2 4 N 6 6 6 6 6 6 mg/kg Mean 18.0 NC 7.69 NC
NC 280 SD 5.85 NC 1.12 NC NC 68.7 CV % 32.5 NC 14.5 NC NC 24.5 Min
11.2 2.00 6.52 24.00 0 204 Median 16.7 8.00 7.54 24.00 1.00 258 Max
27.8 8.00 9.62 24.00 1.00 394 15 N 6 6 6 6 6 6 mg/kg Mean 32.1 NC
7.29 NC NC 825 SD 7.86 NC 1.32 NC NC 122 CV % 24.5 NC 18.0 NC NC
14.8 Min 23.5 8.00 5.53 48.00 0 699 Median 33.0 8.00 7.42 48.00 0
817 Max 39.6 8.00 9.18 48.00 1.00 973 NC = Not calculated; For the
1 mg/kg dose group, AUC.sub.last values were calculated using only
2 quantifiable data points.
TABLE-US-00099 TABLE 93 Summary of combined male and female plasma
CDV toxicokinetic parameters by treatment on Day 29 in rats (TK
parameter population) AUC.sub.last Treat- Sta- C.sub.max T.sub.max
C.sub.last T.sub.last T.sub.lag (hr*ng/ ment tistic (ng/mL) (hr)
(ng/mL) (hr) (hr) mL) 1 N 6 5 5 5 5 6 mg/kg Mean 7.81 NC 8.31 NC NC
38.6 SD 4.18 NC 1.90 NC NC 28.9 CV % 53.5 NC 22.9 NC NC 75.0 Min 0
2.00 5.24 2.00 0 0 Median 8.41 2.00 8.61 8.00 1.00 43.0 Max 12.5
8.00 10.4 8.00 1.00 74.8 4 N 6 6 6 6 6 6 mg/kg Mean 20.4 NC 8.76 NC
NC 399 SD 9.41 NC 1.69 NC NC 248 CV % 46.2 NC 19.3 NC NC 62.3 Min
10.6 8.00 6.47 24.00 0 195 Median 20.0 8.00 8.92 24.00 0 347 Max
37.3 8.00 10.7 48.00 1.00 883 15 N 5 5 5 5 5 5 mg/kg Mean 41.3 NC
8.05 NC NC 1068 SD 16.0 NC 1.04 NC NC 327 CV % 38.8 NC 13.0 NC NC
30.6 Min 23.6 2.00 6.99 48.00 0 732 Median 44.0 8.00 7.81 48.00 0
1117 Max 60.9 8.00 9.40 48.00 0 1473 NC = Not calculated; For the 1
mg/kg dose group, AUC.sub.last values were calculated using only 2
quantifiable data points. N = 5 in for the 15 mg/kg dose group
because animal 4107 was euthanized prior to the collection of TK
samples on Day 29.
Mortality
Dosing Phase
[0784] There were three unscheduled deaths on the study; none of
which were considered to be brincidofovir-related. The causes of
death in two animals (Tox Animal Nos. 2595 and 1005) were
determined not to be brincidofovir-related. The cause of death in
the third animal (TK Animal No. 4107) could not be determined.
[0785] Animal No. 2595 (toxicity female at 1 mg/kg/dose) was
euthanized for welfare reasons on Day 23 because of a swollen
cranium which was likely related to accidental trauma. Dark areas
on the cranium observed macroscopically correlated microscopically
with multifocal hemorrhage. On Day 23, this rat was abnormally cold
to touch and displayed decreased activity and abnormal (labored and
irregular) breathing.
[0786] Animal No. 4107 (toxicokinetics male at 15 mg/kg/dose) was
euthanized for welfare reasons on Day 23. On Day 23, this rat was
abnormally cold to touch and exhibited decreased activity,
partially closed eyelids and dull eyes, and irregular breathing.
Enlarged liver and spleen, and correct catheter tip placement were
noted at gross necropsy. The cause of death could not be determined
from macroscopic data and microscopic evaluations were not
conducted to further determine cause of death.
[0787] Animal No. 1005 (toxicity male control) was found dead on
Day 27. On Day 27, this animal had decreased activity and a red
aqueous nasal discharge. Macroscopic and microscopic findings were
consistent with bacterial infection that originated at the catheter
site and disseminated to multiple other organs.
Recovery Phase
[0788] There was no brincidofovir-related mortality.
Clinical Observations
Dosing Phase
[0789] Administration of brincidofovir at 4 and 15 mg/kg/dose
resulted in intermittent clinical signs in some animals (both
sexes). The intermittent clinical signs consisted of rapid
breathing and hunched posture with and without piloerection,
partially closed eyes, decreased activity, irregular breathing,
and/or flattened posture. Manual differential leukocyte counts were
performed for verification and absolute values were calculated if
necessary, these signs were generally seen following infusion
between Days 22 and 29 and were dose-related in incidence.
Recovery Phase
[0790] All clinical signs observed during the treatment phase
completely recovered by the first day of the recovery period. There
were no brincidofovir-related clinical signs during the recovery
phase.
Opthalmology
[0791] There were no brincidofovir-related effects on ophthalmology
at the end of dosing recovery phases.
Body Weights
[0792] There were no brincidofovir-related effects on body weight
or body weight change.
Food Consumption
Dosing Phase
[0793] There were minimal, statistically significant
brincidofovir-related decreases in food consumption in males
administered .gtoreq.1 mg/kg/dose on Weeks 1, 2, 3 and/or 4 (to
-16% concurrent controls on Week 4), and in females administered 15
mg/kg/dose on Weeks 3 and 4 (to -9.8% concurrent controls on Week
3). The decreases in food consumption had no effect on body weight
or body weight gain.
Recovery Phase
[0794] The brincidofovir-related decreases in food consumption
observed during the treatment phase were fully reversible as there
were no brincidofovir-related effects on food consumption in any
animals during the recovery period.
Clinical Pathology
Hematology: Dosing Phase
[0795] There were no test item-related hematology changes.
[0796] Without wishing to be bound by theory, all hematologic
abnormalities were considered to be related to the infusion
procedure and exhibited no distinct dose-related trends. These
included minimal to slight decreases in red cell mass (hemoglobin,
hematocrit and red blood cells) accompanied by increases in
reticulocytes, increases in platelet counts, and increases in
neutrophil and/or monocyte counts. These findings were considered
to reflect an appropriate hematopoietic and immune response to
catheter vein infection, inflammation, and increased blood loss or
destruction.
Hematology: Recovery Phase
[0797] There were no test item-related hematology findings after a
14-day recovery.
Coagulation: Dosing Phase
[0798] There were no test item-related effects on coagulation
times.
[0799] Without wishing to be bound by theory, all differences from
controls, statistically significant or otherwise, were not
considered to be test item-related due to their direction, small
magnitude, infrequent occurrence, lack of relation to dose, lack of
concordance between related analytes, and/or because values were
comparable to the study control range.
Coagulation: Recovery Phase
[0800] After a 14-day recovery, all values were comparable to
controls.
Clinical Chemistry
Dosing Phase
[0801] There were no test item-related clinical chemistry
changes.
[0802] Without wishing to be bound by theory, all abnormalities
were considered to be related to the infusion procedure and
exhibited no distinct dose-related trends. These included minimal
to moderate increases in aspartate aminotransferase (AST), alanine
aminotransferase (ALT), alkaline phosphatase (ALKP), blood urea
nitrogen (BUN), creatinine, and globulins and decreases in albumin
and albumin to globulin ratio. These findings correlated with
microscopic evidence of inflammation, necrosis, abscesses, and
infarction in the liver, inflammation, abscesses, urothelial
hyperplasia, tubular basophilia and eosinophilic tubular globules
in the kidneys, and general inflammation in a variety of other
tissues.
Recovery Phase
[0803] There were no test item-related clinical chemistry findings
after a 14-day recovery.
Urinalysis
Dosing Phase
[0804] There were no test item-related effects on urinalysis.
[0805] All differences from controls, statistically significant or
otherwise, were not considered to be test item-related due to their
direction, small magnitude, infrequent occurrence, lack of relation
to dose, lack of concordance between related analytes, and/or
because values were comparable to the study control range.
Recovery Phase
[0806] After a 14-day recovery, all values were comparable to
controls.
Organ Weights
[0807] Brincidofovir-related, minimal to mild, dose-dependent lower
testes weights were present in males administered .gtoreq.4
mg/kg/dose compared to controls (Table 94). Lower testes weights
correlated microscopically with germ cell depletion. The 15
mg/kg/dose males had minimally lower epididymal weights. Without
wishing to be bound by theory, however, since there were no
correlative microscopic findings, the lower weights were considered
incidental and unrelated to the microscopic changes in the
testes.
TABLE-US-00100 TABLE 94 Brincidofovir-related organ weight changes
(% difference relative to controls) in rats dosed twice weekly for
a 29 day period Group/sex 2M 3M 4M 2F 3F 4F Dose (mg/kg/dose) 1 4
15 1 4 15 Testes Absolute weight (%) -- -9.sup.a -24.sup.a NA NA NA
vs. body weight (%) -- -3 -16.sup.a NA NA NA vs. brain weight (%)
-- -9.sup.a -22.sup.a NA NA NA Epididymides Absolute weight (%) --
-- -14.sup.a NA NA NA vs. body weight (%) -- -- -6 NA NA NA vs.
brain weight (%) -- -- -13.sup.a NA NA NA .sup.aStatistically
significant difference between mean values for
brincidofovir-treated and control groups. -- = not CMX00-related;
NA = Not applicable
[0808] All other organ weight differences at the end of dosing,
statistically significant or otherwise, had no microscopic
correlations and were considered to be incidental due to biologic
variability.
[0809] At the end of the recovery period, testes weights in males
administered .gtoreq.4 mg/kg/dose (Table 95) were still lower than
controls and differences were of greater magnitude than at the end
of dosing. Without wishing to be bound by theory, these changes
were, without wishing to be bound by theory, reflective of
maturation depletion secondary to the loss of germ cell stages
observed microscopically at the end of the dosing period.
TABLE-US-00101 TABLE 95 Brincidofovir-related organ weight changes
(% difference relative to controls) in rats dosed twice weekly for
a 29 day period and following a 14-day recovery period Group/sex 2M
3M 4M 2F 3F 4F Dose (mg/kg/dose) 1 4 15 1 4 15 Testes Absolute
weight (%) -- -21.sup.a -42.sup.a NA NA NA vs. body weight (%) --
-26.sup.a -40.sup.a NA NA NA vs. brain weight (%) -- -18.sup.a
-41.sup.a NA NA NA .sup.aStatistically significant difference
between mean values for brincidofovir-treated and control. -- = not
CMX00-related; NA = Not applicable
[0810] All other organ weight differences had no microscopic
correlations and were considered to be incidental and due to
biologic variability.
Pathology: Macroscopic
[0811] There were three unscheduled deaths during the dosing phase
of the study. Two animals, a control male and a female administered
1 mg/kg/dose, died or was euthanized during the dosing phase from
causes unrelated to brincidofovir. A third animal, a toxicokinetics
male administered 15 mg/kg/dose, was euthanized during the dosing
phase; the cause of death could not be determined. There was no
brincidofovir-related mortality during the recovery phase.
[0812] A control male (Animal No. 1005) was found dead on Day 27.
The cause of death was sepsis. Macroscopic and microscopic findings
were consistent with bacterial infection that originated at the
catheter site and disseminated to multiple other organs, resulting
in masses in many organs that correlated microscopically with
inflammation, abscessation, thrombosis, infarction and necrosis.
Inflammation was also present in a swollen extremity (left
femorotibial joint). Increased cellularity in the bone marrow was
considered to be an adaptive reactive response to infection and
decreased cellularity and/or necrosis in the thymus, spleen and
lymph nodes, adrenal cortical hypertrophy were considered to be
related to physiologic stress.
[0813] One female administered 1 mg/kg/dose (Animal No. 2595) was
euthanized on Day 23 because of a swollen cranium which was likely
related to accidental trauma. Dark areas on the cranium were
observed macroscopically and correlated microscopically with
multifocal hemorrhage.
[0814] One toxicokinetics male administered 15 mg/kg/dose (Animal
No. 4107) was euthanized on Day 23. Enlarged liver and spleen, and
correct catheter tip placement were noted at gross necropsy. The
cause of death could not be determined from macroscopic data and
microscopic evaluations were not conducted to further determine the
cause of death.
Terminal Sacrifice
Brincidofovir-Related Findings
[0815] No brincidofovir-related macroscopic findings were observed
at the end of the dosing period.
Procedure-Related Findings
[0816] Most macroscopic findings were related to inflammation in
the infusion vein and were present across all groups including the
controls. These findings included scabs or thickening of the
catheter exteriorization site and abnormal contents, masses,
thickening, and/or edema associated with the infusion vein,
abnormal color and masses in the kidneys, irregular surface or
masses in the liver and dark or pale areas and masses in the lung.
Microscopically, these findings correlated with inflammation and
necrosis. Enlargement or abnormal color of lymph nodes and spleen
correlated microscopically with reactive lymphoid processes
(increased follicle cellularity in lymph nodes and spleen),
drainage of blood from sites of inflammation
(erythrocytosis/erythrophagocytosis in lymph nodes) or
extramedullary hematopoiesis (spleen). All these changes were
considered to be systemic reactions associated with inflammation
related to the infusion site/procedure.
[0817] Without wishing to be bound by theory, all other macroscopic
findings occurred sporadically or in control groups and were
considered incidental and due to biological variability. Abnormal
color (blue) and soft left testis and small left epididymis in one
15 mg/kg/dose male (Animal No. 4063) were considered incidental and
spontaneous background findings unrelated to the
brincidofovir-related change of testicular germ cell depletion.
Sporadic unilateral testicular seminiferous tubular atrophy is not
uncommon in rodents.
Recovery Sacrifice
Test Item-Related Findings
[0818] At the end of the recovery period, brincidofovir-related
macroscopic findings were limited to the testes and epididymides in
the 15 mg/kg/dose males.
[0819] Two of 5 males had bilaterally soft and/or small testes and
epididymides. Without wishing to be bound by theory, these findings
correlated with germ cell depletion in the testes and decreased
sperm in the head region of the epididymides. These findings were
reflective of maturation depletion secondary to the loss of germ
cell populations observed microscopically at the end of the dosing
period. One male (Animal No. 4071) had a unilaterally (left) soft
and/or small testis and epididymis which correlated with tubular
atrophy in the testis and decreased sperm throughout the
epididymis; these unilateral findings in this male were considered
to be incidental spontaneous background changes.
Procedure-Related Findings
[0820] Major macroscopic findings related to the infusion procedure
included abnormal contents, masses, thickening, and/or edema of the
infusion vein, enlarged spleens and an enlarged renal lymph node.
These findings correlated microscopically with inflammation and
abscess formation of the infusion vein and increased follicular
cellularity and plasmacytosis in the lymphoid organs. An enlarged
adrenal gland correlated with cortical hypertrophy (related to
physiologic stress).
Pathology: Microscopic
[0821] Brincidofovir-related microscopic findings were present in
the testes, epididymides, and/or seminal vesicles in males at
.gtoreq.4 mg/kg/dose, in the intestinal tract in males and females
at .gtoreq.4 mg/kg/dose, and in the sebaceous glands in males and
females at 15 mg/kg/dose.
Terminal Sacrifice
Brincidofovir-Related Findings
Male Reproductive Tract (Testes, Epididymides, Seminal
Vesicles)
[0822] Brincidofovir-related germ cell depletion was present in the
testes in males at .gtoreq.4 mg/kg/dose (Table 96). These changes
were bilateral and minimal to slight at 4 mg/kg/dose and slight to
moderate at 15 mg/kg/dose with the severity grading generally
reflecting the number of cell types affected by the depletion. At 4
mg/kg/dose, germ cell depletion was characterized by a variable
loss of spermatogonia through preleptotene spermatocytes. At 15
mg/kg/dose, the germ cell depletion was characterized by a variable
loss of spermatogonia through pachytene spermatocytes, often with
some sparing of pachytene spermatocytes in late stage tubules and
occasional active degeneration of germ cells in late stage
tubules.
[0823] Brincidofovir-related findings at 15 mg/kg/dose also
included minimal luminal cell debris in the epididymides and
minimally increased epithelial apoptosis in the seminal vesicles
compared to controls. The latter change was occasionally
accompanied by an increase in mitotic figures. Minimally increased
epithelial apoptosis was also present in 4 male administered 4
mg/kg/dose.
[0824] Unilateral tubular atrophy observed in one 15 mg/kg/dose
male (Animal No. 4063) was considered an incidental background
finding that occurs sporadically in laboratory rodents.
TABLE-US-00102 TABLE 96 Brincidofovir-related findings in the male
reproductive tract in rats dosed with Brincidofovir twice weekly
for a 29 day period Group/sex 1M 2M 3M 4M 1F 2F 3F 4F Dose
(mg/kg/dose) 0 1 4 15 0 1 4 15 Number of tissues examined 10 10 10
10 NA NA NA NA Testes Depletion, Germ Cell Minimal -- -- 6 -- NA NA
NA NA Slight -- -- 3 5 NA NA NA NA Moderate -- -- -- 5 NA NA NA NA
Epididymides Cell Debris, Luminal Minimal -- -- -- 9 NA NA NA NA
Seminal Vesicles Apoptosis, Epithelial Minimal -- -- 4 8 NA NA NA
NA Mitotic Figures, Epithelial Minimal -- -- -- 4 NA NA NA NA Total
Animals Represented 0 0 10 10 NA NA NA NA -- = Finding not present.
NA = not applicable
Intestinal Tract
[0825] Single cell necrosis of the crypt epithelium (minimal to
moderate) was present in the small intestine (duodenum, jejunum,
and/or ileum) in males and females at .gtoreq.4 mg/kg/dose and was
dose-dependent with respect to severity and/or incidence (Table
97). This change was occasionally accompanied by minimal crypt
epithelial hyperplasia. Minimal single cell necrosis was also
present in the glandular epithelium of the large intestine (cecum
and/or colon) in males and females at 15 mg/kg/dose.
TABLE-US-00103 TABLE 97 Brincidofovir-related findings in the
intestinal tract in rats dosed with brincidofovir twice weekly for
a 29 day period Group/sex 1M 2M 3M 4M 1F 2F 3F 4F Dose (mg/kg/dose)
0 1 4 15 0 1 4 15 Number of tissues examined 10.sup.c 10 10 10 10
11.sup.d 10 10 Small intestine.sup.a Necrosis, Epithelium, Crypts
Minimal -- -- 8 2 -- -- 9 3 Slight -- -- 1 8 -- -- -- 5 Moderate --
-- -- -- -- -- -- 1 Hyperplasia, Epithelium, Crypts Minimal -- -- 5
2 -- -- -- 5 Large intestine.sup.b Necrosis, Epithelium, glands
Minimal -- -- -- 9 -- -- -- 4 Total Animals Represented 0 0 9 10 0
0 9 9 .sup.a, bIncidence of severity grades represent highest grade
recorded for duodenum, jejunum and ileum.sup.a and for cecum and
colon..sup.b .sup.cDuodenum was autolytic in one animal and not
readable. .sup.dIncidence includes one unscheduled death (Animal
No. 2595). -- = Finding not present.
Skin (Sebaceous Gland)
[0826] Sebaceous gland atrophy (slight to marked) was present in
males and females at 15 mg/kg/dose (Table 98). Marked severity
indicated absence of sebaceous glands microscopically with no
change in remaining adnexa (hair follicles).
TABLE-US-00104 TABLE 98 Brincidofovir-related findings in the skin
in rats dosed with brincidofovir twice weekly for a 29 day period
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F Dose (mg/kg/dose) 0 1 4 15 0 1 4
15 Number of tissues examined 10 10 10 10 10 11.sup.a 10 10
Atrophy, Sebaceous Glands Slight -- -- -- 3 -- -- -- 5 Moderate --
-- -- 3 -- -- -- 3 Marked -- -- -- 4 -- -- -- 2 Total Animals
Represented 0 0 0 10 0 0 0 10 .sup.aIncidence includes one
unscheduled death (Animal No. 2595). -- = Finding not present.
Procedure-Related Findings
[0827] Microscopic findings related to the infusion procedure were
observed in the vehicle control group and in animals administered
brincidofovir. Without wishing to be bound by theory, many findings
were those commonly associated with catheterization, including
pericatheteral thrombi, suture granulomas, and intimal
proliferation in the catheter vein (Weber, 2011) and perivascular
eosinophilic inflammatory cell infiltrates in the lung (Morton,
1997). Some animals had chronic-active inflammation, abscess
formation and thrombosis with bacterial colonies in the catheter
vein and epidermal necrosis and/or inflammatory tracts and
abscesses in the skin at the catheter exteriorization site.
Chronic-active inflammation, abscess formation, infarction and/or
necrosis were occasionally present in other organs, primarily the
kidney and liver. Without wishing to be bound by theory, these
changes were likely the result of bacterial infection of the
catheter vein. Findings indicative of an immune response to
infection included overall increased cellularity or increased
granulocytes in the bone marrow, increased follicular cellularity
and plasmacytosis in lymph nodes and increased follicular
cellularity, increased macrophages and/or extramedullary
hematopoiesis in the spleen. Other findings in the lymphoid organs,
such as decreased cellularity and necrosis, as well as cortical
hypertrophy in the adrenal glands and vaginal mucification were
considered related to stress.
[0828] Additional procedure-related microscopic findings were also
observed in the stomach (serosal inflammation), heart (valvular
endothelial hypertrophy), and lung (granulomas, emboli, macrophage
aggregates, alveolar and vascular inflammation, and/or vascular
intimal proliferation). Instances of minimal focal/multifocal
myocardial inflammation were also observed and, without wishing to
be bound by theory, could have been secondary to the infusion
procedures or could have represented common background findings in
laboratory rodents.
Miscellaneous Findings
Kidney
[0829] Eosinophilic globules were present in the cortical tubular
epithelium of the kidney in a male (Animal No. 3048) and a female
(Animal No. 3560) dosed at 4 mg/kg/dose. There were no associated
inflammatory or degenerative changes. These globules exhibited
negative staining with Prussian blue for iron and Martius Scarlet
Blue as a general stain for erythrocytes.
[0830] All other microscopic findings occurred sporadically or at
similar incidence and severity in control and brincidofovir groups
and without wishing to be bound by theory were considered
incidental and due to biological variability. These included a
poorly differentiated carcinoma of the salivary gland in one 4
mg/kg/dose male (Animal No. 3044) and a renal tubular adenoma in a
15 mg/kg/dose male (Animal No. 4068). Without wishing to be bound
by theory, these tumors have been reported to occur spontaneously
in rats and were considered incidental (Nishikawa 2010, Hardisty
2013) and unrelated to brincidofovir.
[0831] Following the 14-day recovery period, microscopic findings
remained present in the testes and/or epididymides in males at
.gtoreq.4 mg/kg/dose, and a new finding noted in the bone marrow in
males and females at 15 mg/kg/dose. There was complete recovery of
all microscopic findings in the intestinal tract and sebaceous
glands observed at the end of dosing. Infusion procedure-related
findings were also present at the end of the recovery period.
Male Reproductive Tract (Testes, Epididymides)
[0832] At the end of the recovery period, there was continued
depletion of the cell populations identified in terminal necropsy
animals along with maturation depletion in the testes in males at
.gtoreq.4 mg/kg/dose (Table 99). At 15 mg/kg/dose, this change was
characterized by a depletion of most germ cell populations
generally sparing only elongating spermatids often with multifocal
tubular atrophy. At 4 mg/kg/dose, there was variable loss of
spermatogonia, spermatocytes, and round spermatids in early stage
tubules through about stage XIII tubules.
[0833] In the males at 15 mg/kg/dose, maturation depletion was
accompanied by reduced sperm and luminal cell debris in the head of
the epididymides. One 15 mg/kg/dose male (Animal No. 4071) had
unilateral tubular atrophy in the testes accompanied by reduced
sperm with cribiform change (a finding related to reduced sperm) in
the epididymis. Without wishing to be bound by theory, these
unilateral findings in this male were considered spontaneous
background changes and unrelated to brincidofovir.
TABLE-US-00105 TABLE 99 Test item-related findings in the male
reproductive tract in rats dosed with brincidofovir twice weekly
for a 29 day period and following a 14-day recovery period
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F Dose (mg/kg/dose) 0 1 4 15 0 1 4
15 Number of tissues examined 5 5 5 5 NA NA NA NA Testes Depletion,
Germ Cell Minimal -- -- 1 -- NA NA NA NA Slight -- -- 4 -- NA NA NA
NA Moderate -- -- -- 1 NA NA NA NA Marked -- -- -- 4 NA NA NA NA
Epididymides Sperm, Reduced, Luminal Marked -- -- -- 4 NA NA NA NA
Severe -- -- -- 1 NA NA NA NA Cell Debris, Luminal Minimal -- -- --
5 NA NA NA NA Total Animals Represented 0 0 5 5 NA NA NA NA -- =
Finding not present. NA = not applicable
Bone Marrow
[0834] At the 15 mg/kg/dose, a few recovery males and females had
minimal to slight decreased cellularity (Table 100) in the bone
marrow, a finding which was not observed at the end of the dosing
period.
TABLE-US-00106 TABLE 100 Test item-related findings in the bone
marrow in rats dosed with brincidofovir twice weekly for a 29 day
period and following a 14-day recovery period Group/sex 1M 2M 3M 4M
1F 2F 3F 4F Dose (mg/kg/dose) 0 1 4 15 0 1 4 15 Number of tissues
examined 5 5 5 5 5 4 5 5 Cellularity, Decreased.sup.a Minimal -- --
-- -- -- -- -- 2 Slight -- -- -- 3 -- -- -- -- Total Animals
Represented 0 0 0 3 0 0 0 2 .sup.aIncidence of severity grades
represent highest grade recorded for sternal or femoral bone
marrow. -- = Finding not present.
Procedure-Related Findings
[0835] At the end of the recovery period, a range of
procedural-related microscopic observations were present similar to
those observed at the end of the dosing period and described above
These were related to infection of the infusion catheter and
without wishing to be bound by theory were not considered to be
related to brincidofovir.
[0836] All other microscopic findings occurred sporadically or at
similar incidence and severity in control and brincidofovir groups
and were considered incidental and due to biological
variability.
[0837] Brincidofovir was administered twice weekly for 29 days via
2-hour intravenous infusion to Sprague-Dawley CD.RTM. rats
(15/sex/group) at 0 (2.times. Sodium Phosphate Buffer Solution), 1,
4 or 15 mg/kg/dose. Up to 5 animals/sex/group were held for a
14-day post-dosing recovery.
[0838] Administration of brincidofovir resulted in intermittent
(generally following infusion) clinical signs in some animals
(primarily abnormal breathing and hunched posture) in both sexes at
.gtoreq.4 mg/kg/dose and minimally decreased food consumption in
males and females at .gtoreq.1 mg/kg/dose and 15 mg/kg/dose,
respectively.
[0839] Brincidofovir-related findings were present in the male
reproductive tract (testicular germ cell depletion, luminal cell
debris in the epididymides and increased epithelial apoptosis in
the seminal vesicles) and intestinal tract (single cell necrosis)
at .gtoreq.4 mg/kg/dose and sebaceous glands (atrophy) at 15
mg/kg/dose. All brincidofovir-related findings completely recovered
with the exception of testicular changes in males at .gtoreq.4
mg/kg/dose, which would not be expected to fully recover in a 2
week period. In addition, a few animals administered 15 mg/kg/dose
had decreased bone marrow cellularity and unilaterally or
bilaterally soft and/or small testes and epididymides at the end of
recovery.
[0840] Under the conditions of the study, based on the testicular
germ cell depletion in males at .gtoreq.4 mg/kg/dose and moderate
intestinal single cell necrosis in 1 female at 15 mg/kg/dose, the
no-observed-adverse-effect level (NOAEL) for brincidofovir was
considered to be 1 mg/kg/dose in males and 4 mg/kg/dose for in
females.
Example 11--IV BCV Ascending Dose Study in Healthy Human
Subjects
Summary
[0841] Brincidofovir was administered to healthy subjects at 10 mg,
25 mg and 50 mg. IV brincidofovir at 10 mg was found to provide
similar exposure as orally administered brincidofovir at 100 mg. It
was found that both IV brincidofovir doses (10 mg and 25 mg) were
generally safe and well-tolerated. No drug related adverse events,
no gastrointestinal adverse events, and no graded lab abnormalities
(e.g., no hematologic toxicity and no kidney toxicity) were
observed.
[0842] This study also evaluated the peripheral blood mononuclear
cell (PBMC) PK of CDV-PP following IV and oral suspension BCV
administration. BCV is converted intracellularly to active
cidofovir diphosphate (CDV-PP).
[0843] As background, a single dose of oral BCV at 100 mg and 200
mg resulted in about 5% instance of diarrhea, and a 350 mg dose
resulted in about 20% instance of diarrhea.
Materials and Methods
[0844] In this double-blind study, subjects were randomized 3:1 to
receive IV BCV or placebo in sequential single ascending dose
cohorts (Tables 101 and 102, below). Plasma PK samples were
collected over 7 days and assayed by HPLC-MS. Plasma BCV PK
parameters were determined by non-compartmental analysis and dose
proportionality was assessed. Safety assessments were collected
over 14 days.
TABLE-US-00107 TABLE 101 Study Design IV Infusion Cohort N Actual
Doses Duration 1 6 active and 2 placebo BCV 10 mg or placebo 2
hours 2 6 active and 2 placebo BCV 25 mg or placebo 2 hours 3 9
active and 3 placebo BCV 50 mg or placebo 2 hours 4 9 active and 3
placebo BCV 50 mg or placebo 4 hours
TABLE-US-00108 TABLE 102 Subject Demographics Cohort Male Sex Age
(years) 1 8/8 (100%) 26 (18-34) 2 8/8 (100%) 23 (20-37) 3 12/12
(100%) 21 (18-26) 4 12/12 (100%) 26 (18-46) Male sex presented as
n/N (%) Age presented as mean (minimum-maximum)
PBMC PK of CDV-PP
[0845] Two single dose BCV studies in healthy subjects contributed
data for this analysis. The first study was a 2-period crossover
bioequivalence study that enrolled 24 subjects who received two
formulations of BCV oral suspension 100 mg fasted, one in each
period. Data from 12 subjects who had PBMCs collected in Period 1
were included in this analysis; these subjects were 100% female,
31-59 years, and 58-88 kg. The second study, outlined above,
enrolled 40 subjects who received IV BCV or placebo. Data from 18
subjects who received BCV 50 mg as either a 2-h or 4-h IV infusion
were included in this analysis; these subjects were 100% male,
18-46 years, 64-106 kg. Serial PBMC samples were collected over 14
days and assayed by HPLC-MS-MS. PBMC CDV-PP PK parameters were
determined by non-compartmental analysis.
[0846] Forty healthy male subjects (18-46 y, 83% White) were
enrolled and completed the study. Plasma BCV C.sub.max and
AUC.sub..infin. increased in proportion to dose (Table 103, below).
Adverse events and alanine aminotransferase (ALT) elevations were
dose- and infusion duration-related. Gastrointestinal adverse
events were mild. All adverse events and ALT elevations were
transient and no serious adverse events occurred. No significant
differences between placebo and IV BCV in other chemistries or
hematologic parameters were observed.
[0847] FIG. 6 shows a plot of the plasma brincidofovir
concentration (AUC.sub.inf (ng*h/mL)) at different 100 mg oral
administration, 10 mg IV administration, and 25 mg IV
administration. As shown in FIG. 6, IV administration of 10 mg
brincidofovir provided substantially the same plasma concentration
as oral administration of 100 mg brincidofovir, while IV
administration of 25 mg brincidofovir provided higher plasma
concentration than both the IV 10 mg dose and the oral 100 mg
dose.
[0848] A dose-related, reversible ALT increase was observed with IV
BCV. In prior studies, oral BCV had reversible dose-related ALT
increases in humans and preclinical studies. In the IV BCV SAD
study, at therapeutic IV exposures (cohorts 1 and 2), no grade 2 or
higher ALT elevations were observed. At supratherapeutic exposures
(cohorts 3 and 4), transient grade 2-3 ALT elevations were noted in
cohort 4 only. No Grade 2 or higher bilirubin was observed in any
cohort.
TABLE-US-00109 TABLE 103 Preliminary Summary of Plasma BCV PK Data
following IV infusion Cohort 1 Cohort 2 Cohort 3 Cohort 4 BCV 10 mg
BCV 25 mg BCV 50 mg BCV 50 mg Pooled 2 h Infusion 2 h Infusion 2 h
Infusion 4 h Infusion Placebo (n = 6) (n = 6) (n = 9) (n = 9) (n =
10) PK C.sub.max (ng/mL) 613 (25%) 1412 (27%) 2952 (19%) 1586 (14%)
NA [439-799] [973-1900] [2200-3940] [1360-2180] AUC.sub..infin. (ng
h/mL) 1312 (26%) 2889 (37%) 5948 (19%) 6570 (15%) NA [875-1710]
[1742-4790] [4446-7274] [5347-9048] t.sub.1/2 (h) 3.42 (27%) 5.89
(23%) 6.54 (49%) 5.10 (13%) NA [2.70-5.76] [4.06-8.00] [3.98-18.09]
[4.23-6.33] CL (L/h) 7.62 (26%) 8.65 (37%) 8.41 (19%) 7.61 (15%) NA
[5.85-11.4] [5.22-14.4] [6.87-11.3] [5.53-9.35] V.sub.SS (L) 16.4
(24%) 19.8 (25%) 19.5 (21%) 10.8 (19%) NA [12.0-22.2] [15.0-30.3]
[13.9-24.9] [7.67-14.5] Drug-Related AEs Diarrhea 0 0 1 (11%) 3
(33%) 0 Nausea 0 0 0 2 (22%) 0 Decreased appetite 0 0 0 1 (11%)
Headache 0 0 2 (22%) 2 (22%) 0 Pain, phlebitis at infusion site 0 0
1 (11%) 0 0 Elevated liver transaminases.sup.1 0 0 0 1 (11%) 0
C.sub.max and AUC.sub..infin. presented as geometric mean (% CVb)
[minimum-maximum] T.sub.max was 2 hours for Cohorts 1-3 (2-hour
infusion) and 2.5 to 4 hours (4-hour infusion) .sup.1ALT >2x ULN
in 2 BCV 50 mg 4 h infusion and 1 placebo subjects; 1 ALT elevation
considered an AE 2. In Cohort 3, 5 drug-related AEs in 4 subjects.
3. In Cohort 4, 9 drug-related AEs in 5 subjects.
TABLE-US-00110 TABLE 104 IV BCV PK and Safety: Grade 3 or 4
Drug-Related Adverse Events Cohort 1 Cohort 2 Cohort 3 Cohort 4 BCV
10 mg BCV 25 mg BCV 50 mg BCV 50 mg Pooled 2 h Infusion 2 h
Infusion 2 h Infusion 4 h Infusion Placebo (n = 6) (n = 6) (n = 9)
(n = 9) (n = 10) Grade 3 or 4 Drug-Related AEs Diarrhea 0 0 0 0 0
Nausea 0 0 0 0 0 Decreased appetite 0 0 0 0 Headache 0 0 0 0 0
Pain, phlebitis at 0 0 0 0 0 infusion site Elevated liver 0 0 0 1
(11%) 0 transaminases.sup.1
[0849] FIG. 9 shows a mean plasma BCV concentration as a function
of time for subjects in cohorts 1-4. FIG. 9 also shows mean plasma
BCV concentration as a function of time for subjects administered
BCV orally.
[0850] Table 105 shows a summary of plasma BCV PK data following IV
and oral dosing (Single dose administration).
TABLE-US-00111 TABLE 105 Summary of Plasma BCV PK Data Following IV
and Oral Dosing 10 mg 25 mg 50 mg 50 mg 100 mg 200 mg 200 mg 350 mg
Plasma 2 h IV 2 h IV 2 h IV 4 h IV Oral Oral Oral Oral BCV PK
infusion infusion infusion infusion Tablet Tablet Tablet Tablet
Parameter N = 6 N = 6 N = 9 N = 9 N = 52 N = 15 N = 63 N = 70
C.sub.max 613 1412 2952 1586 258 622 802 1482 (ng/mL) (25%) (27%)
(19%) (14%) (42%) (33%) (38%) (34%) [439- [973- [2200- [1360-
[97.0- [384- [344- [732- 799] 1900] 3940] 2180] 586] 1030] 1980]
3600] AUC.sub.inf 1312 2889 5948 6570 1380 3307 3744 6938 (ng h/mL)
(26%) (37%) (19%) (15%) (41%) (35%) (35%) (31%) [875- [1742- [4446-
[5347- [555- [1741- [1605- [3293- 1710] 4790] 7274] 9048] 3070]
5190] 8227] 14132] t1/2 (h) 3.42 5.89 6.54 5.10 7.62 12.0 -- --
(27%) (23%) (49%) (13%) (47%) (27%) [2.70- [4.06- [3.98- [4.23-
[3.05- [3.93- 5.76] 8.00] 18.09] 6.33] 24.1] 17.7] Data presented
as geometric mean (CVb %) [minimum, maximum] AUC.sub.last presented
in place of AUC.sub.inf for Study CMX001-108
[0851] Table 106 shows a summary of preliminary plasma CDV PK data
cohorts 1-4.
TABLE-US-00112 TABLE 106 Preliminary Summary of Plasma CDV PK Data
following IV infusion 10 mg Single 25 mg Single 50 mg Single 50 mg
Single Plasma Dose 2 h Dose 2 h Dose 2 h Dose 4 h CDV PK IV
infusion IV infusion IV infusion IV infusion Parameter N = 6 N = 6
N = 9 N = 9 C.sub.max ND 5.94 (22%) 12.7 (19%) 12.1 (14%) (ng/mL)
[4.62-7.83] [9.44-16.6] [9.83-15.6] AUC.sub.last ND 189 (81%) 652
(38%) 654 (30%) (ng h/mL).sup.5 [77.6-419] [341-1367] [477-1151]
T.sub.max (h) ND 9.0 10.0 10.0 [8.0-10.0] [8.00-12.0] [8.00, 10.00]
Data presented as geometric mean (CVb %) [minimum-maximum] ND: not
done because majority of CDV concentrations were below the lower
limit of quantification AUC.sub.inf and t1/2 not reported due to
high AUC percent extrapolated
[0852] FIG. 10 shows median plasma CDV concentration as a function
of time following IV and Oral BCV Doses.
PBMC PK of CDV-PP
[0853] Single doses of BCV 50 mg administered as 2- or 4-h IV
infusions delivered intracellular CDV-PP exposures comparable to
BCV 100 mg oral suspension (Table 107). PBMC CDV-PP exposure was
inversely related to infusion duration (Table 107).
[0854] FIG. 8 shows the mean (+/-SE) ALT levels observed in the IV
BCV SAD trial.
TABLE-US-00113 TABLE 107 Summary of PBMC CDV-PP PK following BCV
Oral and IV Dosing BCV 100 mg BCV 50 mg IV PBMC CDV-PP PK Oral
suspension 2 h Infusion 4 h Infusion Parameter (n = 12) (n = 9) (n
= 9) C.sub.max (pg/10.sup.6 cells) 7.68 (41%) 14.3 (63%) 8.41 (57%)
AUC.sub.last (pg h/ 1107 (50%) 1409 (36%) 963 (36%) 10.sup.6
cells)
[0855] Single doses of BCV 10-50 mg administered as a 2 h IV
infusion were well tolerated and not associated with significant
clinical or laboratory abnormalities. BCV IV 10 mg and BCV IV 50 mg
achieved geometric mean plasma BCV AUC.sub..infin. similar to and
4.5-fold, respectively, values achieved with BCV oral 100 mg
tablets (C.sub.max=251 ng/mL and AUC.sub..infin.=1394 ngh/mL).
These data support evaluation of repeat dose administration in
healthy subjects and virally-infected patients.
[0856] Additionally, IV BCV provided comparable PBMC CDV-PP
exposures with lower doses when compared with oral BCV. Without
wishing to be bound by theory, in some embodiments this can allow
for improvements in BCV tolerability and efficacy.
EQUIVALENTS
[0857] While the present disclosure has been described in
conjunction with the specific embodiments set forth above, many
alternatives, modifications and other variations thereof will be
apparent to those of ordinary skill in the art. All such
alternatives, modifications and variations are intended to fall
within the spirit and scope of the present disclosure.
* * * * *