U.S. patent application number 16/272599 was filed with the patent office on 2019-06-06 for compositions and methods for treating hcv.
The applicant listed for this patent is AbbVie Inc.. Invention is credited to Daniel E. Cohen, Christine A. Collins, Gennadiy Koev, Preethi Krishnan, Tami J. Pilot-Matias.
Application Number | 20190167684 16/272599 |
Document ID | / |
Family ID | 65242294 |
Filed Date | 2019-06-06 |
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United States Patent
Application |
20190167684 |
Kind Code |
A1 |
Collins; Christine A. ; et
al. |
June 6, 2019 |
COMPOSITIONS AND METHODS FOR TREATING HCV
Abstract
This disclosure is directed to pharmaceutical compositions that
comprise two or more therapeutic agents that, inter alia, are
useful for inhibiting hepatitis C virus (HCV) and methods for
inhibiting HCV by co-administering two or more anti-HCV therapeutic
agents.
Inventors: |
Collins; Christine A.;
(Skokie, IL) ; Cohen; Daniel E.; (Highland Park,
IL) ; Koev; Gennadiy; (Libertyville, IL) ;
Krishnan; Preethi; (Gurnee, IL) ; Pilot-Matias; Tami
J.; (Green Oaks, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AbbVie Inc. |
North Chicago |
IL |
US |
|
|
Family ID: |
65242294 |
Appl. No.: |
16/272599 |
Filed: |
February 11, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15398390 |
Jan 4, 2017 |
10201541 |
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16272599 |
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14247975 |
Apr 8, 2014 |
10201584 |
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15398390 |
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13474411 |
May 17, 2012 |
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14247975 |
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61486842 |
May 17, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/05 20130101;
A61K 31/513 20130101; A61K 31/4025 20130101; A61P 31/12 20180101;
A61K 31/427 20130101; A01N 37/18 20130101; A61K 45/06 20130101;
A61K 38/00 20130101; A61K 38/21 20130101; A61K 31/427 20130101;
A61K 2300/00 20130101; A61K 31/513 20130101; A61K 2300/00 20130101;
A61K 31/4025 20130101; A61K 2300/00 20130101; A61K 38/21 20130101;
A61K 2300/00 20130101 |
International
Class: |
A61K 31/513 20060101
A61K031/513; A01N 37/18 20060101 A01N037/18; A61K 45/06 20060101
A61K045/06; A61K 31/4025 20060101 A61K031/4025; A61K 38/05 20060101
A61K038/05; A61K 38/00 20060101 A61K038/00 |
Claims
1. A combination or pharmaceutical composition comprising an amount
of therapeutic agent A and an amount of therapeutic agent B,
wherein therapeutic agent A is compound A or a salt thereof:
##STR00005## and wherein therapeutic agent B is compound B or a
salt thereof: ##STR00006##
2. The combination or composition of claim 1, wherein the amount of
therapeutic agent A is about 25 mg.
3. The combination or composition of claim 1, wherein therapeutic
agent B is a sodium salt of compound B.
4. The combination or composition of claim 1, wherein the amount of
therapeutic agent B is from about 400 to about 800 mg.
5. The combination or composition of claim 1, wherein the amount of
therapeutic agent A and the amount of therapeutic agent B together
constitute a therapeutically effective amount.
6. The combination or composition of claim 1, wherein the
composition further comprises one or more additional therapeutic
agents.
7. The combination or composition of claim 6, wherein the
additional therapeutic agent is independently selected from the
group consisting of ribavirin, taribavirin, and HCV inhibitor.
8. The combination or composition of claim 6, wherein the one or
more additional therapeutic agents include an HCV protease
inhibitor.
9. The combination or composition of claim 8, wherein the one or
more additional therapeutic agents include a cytochrome P-450
inhibitor.
10. The combination or composition of claim 9, wherein the
cytochrome P-450 inhibitor is ritonavir.
11. The combination or composition of claim 6, wherein the amount
of therapeutic agent A, the amount of therapeutic agent B ant the
amount of the additional therapeutic agent together constitute a
therapeutically effective amount.
12. A pharmaceutical composition comprising an amount of
therapeutic agent A and an amount of therapeutic agent B, wherein
therapeutic agent A is compound A or a salt thereof: ##STR00007##
wherein therapeutic agent B is compound B or a salt thereof:
##STR00008## and wherein the ratio of therapeutic agent A to
therapeutic agent B is about 1:20 to about 1:2000.
13. The pharmaceutical composition of claim 12, wherein the amount
of therapeutic agent B is about 200 mg.
14. The pharmaceutical composition of claim 12, wherein the amount
of therapeutic agent A is from about 5 mg to about 200 mg.
15. The pharmaceutical composition of claim 12, wherein the
composition further comprises one or more additional therapeutic
agents
16. The pharmaceutical composition of claim 15, wherein the one or
more additional therapeutic agents include an HCV protease
inhibitor.
17. The pharmaceutical composition of claim 16, wherein the one or
more additional therapeutic agents include a cytochrome P-450
inhibitor.
18. The pharmaceutical composition of claim 12, further comprising
an HCV protease inhibitor and a cytochrome P-450 inhibitor.
19. The pharmaceutical composition of claim 18, wherein the
cytochrome P-450 inhibitor is ritonavir.
20. The pharmaceutical composition of claim 12, wherein therapeutic
agent B is a sodium salt of compound B.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATION
[0001] This patent application is a continuation of U.S. patent
application Ser. No. 13/474,411 filed May 17, 2012, which claims
priority to U.S. Provisional Patent Application No. 61/486,842,
filed May 17, 2011. The entire text of these applications is
incorporated by reference into this patent application.
TECHNICAL FIELD
[0002] This disclosure is directed to: (a) pharmaceutical
compositions that comprise two or more therapeutic agents that,
inter alia, are useful for inhibiting hepatitis C virus (HCV); (b)
methods for preparing such compositions; and (c) methods of use of
such compositions; as well as (d) methods for inhibiting HCV by
co-administering two or more anti-HCV therapeutic agents.
BACKGROUND
[0003] Hepatitis C is a blood-borne, infectious, viral disease that
is caused by an RNA virus belonging to the Hepacivirus genus in the
Flaviviridae family called HCV. The enveloped HCV virion contains a
positive stranded RNA genome encoding all known virus-specific
proteins in a single, uninterrupted, open reading frame. The open
reading frame comprises approximately 9500 nucleotides and encodes
a single large polyprotein of about 3000 amino acids. The
polyprotein comprises a core protein, envelope proteins E1 and E2,
a membrane bound protein p7, and the non-structural proteins NS2,
NS3, NS4A, NS4B, NS5A and NS5B.
[0004] At least six different HCV genotypes (with several subtypes
within each genotype) are known to date. In North America, HCV
genotype 1a predominates, followed by HCV genotypes 1b, 2a, 2b, and
3a. In the United States, HCV genotypes 1, 2, and 3 are the most
common, with about 80% of the hepatitis C patients having HCV
genotype 1. In Europe, HCV genotype 1b is predominant, followed by
HCV genotypes 2a, 2b, 2c, and 3a. HCV genotypes 4 and 5 are found
almost exclusively in Africa. As discussed below, the patient's HCV
genotype is clinically important in determining the patient's
potential response to therapy and the required duration of such
therapy.
[0005] An HCV infection can cause liver inflammation (hepatitis)
that is often asymptomatic, but ensuring chronic hepatitis can
result in cirrhosis of the liver (fibrotic scarring of the liver),
liver cancer (hepatocellular carcinoma), and/or liver failure. The
World Health Organization estimates that about 170 million persons
worldwide are chronically infected with HCV, and from about three
to about four million persons are newly infected globally each
year. According to the Centers for Disease Control and Prevention,
about four million people in the United States are infected with
HCV. Co-infection with the human immunodeficiency virus (HIV) is
common, and rates of HCV infection among HIV positive populations
are higher.
[0006] There is a small chance of clearing the virus spontaneously,
but the majority of patients with chronic hepatitis C will not
clear the virus without treatment. Indications for treatment
typically include proven HCV infection and persistent abnormal
liver function tests. These are two treatment regimens that are
primarily used to treat hepatitis C: monotherapy (using an
interferon agent--either a "conventional" or longer-acting
pegylated interferon) and combination therapy (using an interferon
agent and ribavirin). Interferon, which is injected into the
bloodstream, works by bolstering the immune response to HCV; and
ribavirin, which is taken orally, is believed to work by preventing
HCV replication. Taken alone, ribavirin does not effectively
suppress HCV levels, but an interferon/ribavirin combination is
more effective than interferon alone. Typically, hepatitis C is
treated with a combination of pegylated interferon alpha and
ribavirin for a period of 24 or 48 weeks, depending on the HCV
genotype.
[0007] The goal of treatment is sustained viral response--meaning
that HCV is not measurable in the blood after therapy is completed.
Following treatment with a combination of pegylated interferon
alpha and ribavirin, sustained cure rates (sustained viral
response) of about 75% occur in people with HCV genotypes 2 and 3
in 24 weeks of treatment, about 50% in those with HCV genotype 1
with 48 weeks of treatment, and about 65% in those with HCV
genotype 4 in 48 weeks of treatment.
[0008] Thus, there continues to be a need for new compositions and
methods of treatment to prevent the progression of liver damage
from hepatitis C. This disclosure provides compositions and methods
of treatment that generally address such a need.
SUMMARY
[0009] This disclosure is directed, in part, to the
co-administration of an amount of therapeutic agent A with an
amount of therapeutic agent B. Therapeutic agent A is compound A or
a salt thereof:
##STR00001##
Therapeutic agent B is compound B or a salt thereof:
##STR00002##
[0010] This disclosure is also directed, in part, to combinations
or pharmaceutical compositions comprising therapeutic agent A and
therapeutic agent B. The combinations or compositions may comprise
one or more additional therapeutic agents.
[0011] This disclosure is also directed, in part, to methods for
treating hepatitis C in a subject in need of such treatment. The
methods comprise administering to the subject an amount of
therapeutic agent A and an amount of therapeutic agent B. The
methods may optionally comprise administering to the subject an
amount of one or more additional therapeutic agents.
[0012] This disclosure is also directed, in part, to the use of
therapeutic agent A and therapeutic agent B, to prepare a
medicament. In embodiments, the medicament is useful for treating
hepatitis C.
[0013] This disclosure is also directed, in part, to methods of
using therapeutic agent A and therapeutic agent B, for example, to
inhibit replication of a ribonucleic acid (RNA) virus (including
HCV) or to treat a disease treatable by inhibiting HCV RNA
polymerase and/or the NS5A protein of HCV.
[0014] Further benefits of the disclosed embodiments will be
apparent to one skilled in the art from reading this
disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a three-dimensional surface plot illustrating the
statistically significant anti-HCV effect for the combination of
compound A and compound B in the HCV Genotype 1b (Con1) replicon.
FIG. 1 details the mean differences between the observed anti-HCV
effect and the calculated additivity of that effect in percent
inhibition at various concentrations of compound A and compound B
according to the Prichard and Shipman model. The concentrations for
each of compound A and compound B are expressed in a log.sub.2
scale.
[0016] FIG. 2 is a two-dimensional contour plot illustrating the
statistically significant synergistic, additive or antagonistic
anti-HCV effects at various concentrations of the combination of
compound A and compound B in the HCV Genotype 1b (Con1) replicon
using the Prichard and Shipman model as a reference.
[0017] FIG. 3 is a bar graph illustrating the percentage of
replicon colonies surviving exposure to various concentrations of
therapeutic agent A, therapeutic agent B and therapeutic agent C in
a replicon colony count assay.
DETAILED DESCRIPTION
[0018] This detailed description is intended only to acquaint
others skilled in the art with the disclosed embodiments, their
principles, and their practical applications so that others skilled
in the art may adapt and apply the disclosed embodiments in their
numerous forms, as they may be best suited to the requirements of a
particular use. This description and its specific examples are
intended for purposes of illustration only. This disclosure,
therefore, is not limited to the embodiments described in this
patent application, and may be variously modified.
[0019] The disclosure is directed, in part, to the
co-administration of an amount of therapeutic agent A with an
amount of therapeutic agent B. Therapeutic agent A is compound A or
a salt thereof.
##STR00003##
Compound A is also known as dimethyl
(2S2'S)-2,2'-(4,4'-((2S,5S)-1-(4-tert-butylphenyl)pyrrolidine-2,5-diyl)bi-
s(4,1-phenylene))bis(azanedily)bis(oxomethylene)bis(pyrrolidine-2,1-diyl))-
bis(3-methyl-1-oxobutane-2,1-diyl)dicarbamate. Compound A can be
prepared as described in, for example, U.S. Publication No.
2010/0317568, which is incorporated herein by reference.
[0020] Therapeutic agent B is compound B or a salt thereof.
##STR00004##
Compound B is also known as
N-(6-(3-tert-butyl-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl-2-methoxyph-
enyl)naphthalen-2-yl)methanesulfonamide. As described in, for
example, International Publication No. WO2009/039127, therapeutic
agent B includes various salts of compound B, such as sodium salts,
potassium salts, and choline salts. Therapeutic agent B also
includes crystalline forms of compound B and its salts such as
solvate, hydrate, and solvent-free crystalline forms of compound B
and its salts. Compositions comprising compound B can be prepared
as described in, for example, International Publication No.
WO2009/039127 which is incorporated herein by reference.
[0021] The total daily dose of the disclosed compounds of their
salts (administered in single or divided doses) may typically be
from about 0.001 mg/kg to about 200 mg/kg, or from about 0.001
mg/kg to about 30 mg/kg, or from about 0.01 mg/kg to about 10 mg/kg
(i.e., mg of the compound or salt per kg body weight).
[0022] Therapeutic agent A may be administered, for example and
without limitation, as a free acid or salt. Therapeutic agent A may
be administered in any suitable amount such as, for example, in
doses of from about 0.1 mg/kg to about 200 mg/kg body weight, or
from about 0.25 mg/kg to about 100 mg/kg, or from about 0.3 mg/kg
to about 3.0 mg/kg. As non-limiting examples, therapeutic agent A
may be administered in a total daily dose amount of from about 5 mg
to about 300 mg, or from about 25 mg to about 200 mg, or from about
25 mg to about 50 mg. In embodiments, the total daily dosage amount
for therapeutic agent A is about 25 mg. In embodiments, the total
daily dosage amount for therapeutic agent A is about 50 mg.
[0023] Therapeutic agent B may be administered as a free acid, salt
or particular crystalline form of compound B. In embodiments,
therapeutic agent B is administered as a sodium salt of compound B.
Therapeutic agent B may be administered in any suitable amount such
as, for example, in doses of from about 5 mg/kg to about 30 mg/kg.
As non-limiting examples, therapeutic agent B may be administered
in a total daily dose amount of from about 300 mg to about 1800 mg,
or from about 400 mg to about 1600 mg, or from about 600 mg to
about 1800 mg, or from about 800 mg to about 1600 mg. In
embodiments, the total daily dosage amount for therapeutic agent B
is about 300 mg. In embodiments, the total daily dosage amount for
therapeutic agent B is about 400 mg. In embodiments, the total
daily dosage amount for therapeutic agent B is about 600 mg. In
embodiments, the total daily dosage amount for therapeutic agent B
is about 800 mg. In embodiments, the total daily dosage amount for
therapeutic agent B is about 1200 mg. In embodiments, the total
daily dosage amount for therapeutic agent B is about 1600 mg.
[0024] Therapeutic agent A and therapeutic agent B may also be
co-administered with interferon. Interferon may include any
suitable form of interferon such as interferon alpha, interferon
alpha 2a, interferon alpha 2b such as LOCTERON.RTM., interferon
omega, interferon lambda, and albinterferon, such as ZALBIN.RTM.
and JOULFERON.RTM. or albinterferon as disclosed in International
Publication No. WO2007/021494A2. In embodiments, the interferon is
pegylated. Pegylated interferon may include pegylated interferon
alpha 2a, such as PEGASYS.RTM., or pegylated interferon alpha 2b,
such as PEGINTRON.RTM.; pegylated interferon omega, such as
Biomed-510, or pegylated interferon omega as disclosed in U.S.
Publication No. 2006/263433; and pegylated interferon lambda, such
as PEG-rIL-29, or pegylated interferon lambda as disclosed in
International Publication No. WO2007/041713A1.
[0025] Interferon may be administered in accordance with interferon
administration well known in the art. For example, interferon may
be administered in a total weekly dose amount of from about 0.1
mcg/kg to about 2.5 mcg/kg. In embodiments, alpha-2b pegylated
interferon is administered in a total weekly dose of about 0.5
mcg/kg to about 1.5 mcg/kg. Interferon may be administered in a
total weekly dose amount of 50 mcg to about 250 mcg. In
embodiments, alpha-2a pegylated interferon is administered in a
total weekly dose of from about 90 mcg to about 180 mcg.
[0026] LOCTERON.RTM. is an example of an interferon that can be
co-administered with the disclosed compositions, compounds and
their salts. LOCTERON.RTM. is a controlled-release formulation of
interferon alpha-2b interferon that allows the interferon to be
administered every two weeks rather than every week. LOCTERON.RTM.
may be administered in accordance with LOCTERON.RTM. administration
well known in the art. For example, the interferon may be
administered at least once every one to two weeks at a dose of from
about 250 mcg to about 750 mcg or from about 320 mcg to about 640
mcg as a single or as multiple subcutaneous injections at the same
or different doses in each injection. In embodiments, the
peginterferon is administered subcutaneously at a dose of 480 mcg
every two weeks.
[0027] ZALBIN.RTM. and JOULFERON.RTM. (formerly known as
Albuferon.RTM. and ABF-656) are other examples of an interferon
that can be co-administered with the disclosed compositions,
compounds and their salts. ZALBIN.RTM. and JOULFERON.RTM. are an
albumin interferon alpha-2b which is a recombinant fusion protein
composed of recombinant human albumin genetically fused at its
C-terminus to the N-terminus of recombinant human interferon
alfa-2b. ZALBIN.RTM. and JOULFERON.RTM. may be administered in
accordance with ZALBIN.RTM. and JOULFERON.RTM. administration well
known in the art. For example, the albinterferon may be
administered at least once every one to two weeks at a dose of from
about 1 to about 2000 mcg as a single or as multiple subcutaneous
injections at the same or different doses in each injection. In
embodiments, the albinterferon is administered subcutaneously at a
dose of from about 7 to about 900 mcg as single or double (14 days
apart) injections.
[0028] PEGASYS.RTM. is a further example of an interferon that can
be co-administered with the disclosed compositions, compounds and
their salts. PEGASYS.RTM. is a pegylated interferon alpha-2a which
is a covalent conjugate of recombinant alfa-2a interferon with a
single branched bis-monomethoxy polyethylene glycol (PEG) chain.
PEGASYS.RTM. may be administered in accordance with PEGASYS.RTM.
administration well known in the art. For example, the
peginterferon may be administered at least once every one to two
weeks at a dose of from about 100 mcg to about 400 mcg as a single
or as multiple subcutaneous injections at the same or different
doses in each injection. In embodiments, the peginterferon is
administered subcutaneously at a dose of about 180 mcg as a single
weekly injection.
[0029] PEGINTRON.RTM. is an additional example of an interferon
that can be co-administered with the disclosed compositions,
compounds and their salts. PEGINTRON.RTM. is a pegylated interferon
alpha-2b which is a covalent conjugate of recombinant alpha-2b
interferon with monomethoxy polyethylene glycol (PEG).
PEGINTRON.RTM. may be administered in accordance with
PEGINTRON.RTM. administration well known in the art. For example,
the peginterferon may be administered at least once every one to
two weeks at a dose of from about 1 mcg/kg to about 3 mcg/kg or
from about 40 mcg/m.sup.2 to about 80 mcg/m.sup.2. The
peginterferon may be administered at least once every one to two
weeks at a dose of from about 25 mcg to about 200 mcg or from about
50 mcg to about 150 mcg as a single or as multiple subcutaneous
injections at the same or different doses in each injection. In
embodiments, the peginterferon is administered subcutaneously at a
dose of about 1.5 mcg/kg as single weekly injection. In
embodiments, the peginterferon is administered subcutaneously at a
dose of about 60 mcg/m.sup.2as single weekly injection.
[0030] Therapeutic agent A and therapeutic agent B may also be
co-administered with ribavirin, or a pro-drug thereof, in the same
or separate pharmaceutical compositions. Ribavirin may include any
suitable form or formulation of ribavirin. Exemplary formulations
of ribavirin include COPEGUS.RTM., REBETOL.RTM. and
RIBASPHERE.RTM.. An exemplary pro-drug of ribavirin is taribavirin
having the chemical name of
1-.beta.-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine.
[0031] Ribavirin and taribavirin may be administered in accordance
with ribavirin and taribavirin administration well known in the
art. For example, ribavirin or taribavirin may be administered in a
total daily dose of from about 5 mg to about 1500 mg. In
embodiments, COPEGUS.RTM. or REBETOL.RTM. is administered in a
daily dosage amount of from about 500 mg to about 1500 mg in one
dose or in divided doses. In embodiments, COPEGUS.RTM. or
REBETOL.RTM. is administered in a daily dosage amount of about 800
mg. In embodiments, REBETOL.RTM. is administered in a daily dosage
amount of about 100 mg. In embodiments, COPEGUS.RTM. or
REBETOL.RTM. is administered in a daily dosage amount of about 1200
mg. In embodiments, REBETOL.RTM. is administered in a daily dosage
amount of about 1400 mg.
[0032] Ribavirin may be co-administered with the interferon,
together with therapeutic agent A and therapeutic agent B. In
embodiments, ribavirin is administered with pegylated interferon
alpha 2a, such as PEGASYS.RTM., together with therapeutic agent A
and therapeutic agent B. For example, in embodiments, a daily dose
of COPEGUS.RTM. of 800 mg to 1200 mg is administered in combination
with a weekly dose of PEGASYS.RTM. of 180 mcg, together with daily
administration of therapeutic agent A and therapeutic agent B. In
embodiments, ribavirin is administered with pegylated interferon
alpha 2b, such as PEGINTRON.RTM., together with therapeutic agent A
and therapeutic agent B. For example, in embodiments, a daily dose
of REBETOL.RTM. of 800 mg to 1400 mg is administered in combination
with a weekly dose of PEGINTRON.RTM. of 1.5 mcg/kg, together with
daily administration of therapeutic agent A and therapeutic agent
B.
[0033] Therapeutic agent A and therapeutic agent B may be
co-administered with interferon and ribavirin or a pro-drug
thereof.
[0034] Therapeutic agent A and therapeutic agent B may be
co-administered with an HIV inhibitor including an HIV protease
inhibitor, with or without a cytochrome P-450 inhibitor (e.g.,
ritonavir), in the same or separate pharmaceutical
compositions.
[0035] The cytochrome P-450 inhibitor may be administered in any
suitable amount such as, for example, in dose of from about 0.3
mg/kg to about 2 mg/kg or from about 0.6 mg/kg to about 1.5 mg/kg.
As non-limiting examples, the cytochrome P-450 inhibitor may be
administered in a total daily dose amount of from about 25 mg to
about 300 mg, or from about 50 mg to about 250 mg, or from about
100 mg to about 200 mg. In embodiments, the cytochrome P-450
inhibitor is administered in a total daily dose amount of about 25
mg. In embodiments, the cytochrome P-450 inhibitor is administered
in a total daily dose amount of about 50 mg. In embodiments, the
cytochrome P-450 inhibitor is administered in a total daily dose
amount of about 75 mg. In embodiments, the cytochrome P-450
inhibitor is administered in a total daily dose amount of about 100
mg. In embodiments, the cytochrome P-450 inhibitor is administered
in a total daily dose amount of about 125 mg.
[0036] Therapeutic agent A and therapeutic agent B may be
co-administered with an HCV protease inhibitor in the same or
separate pharmaceutical compositions. HCV protease inhibitors may
include, for example, ACH-1625 (Achillion), ACH-2684 (Achillion),
AVL-181 (Avila Therapeutics), AVL-192 (Avila Therapeutics), BI
201335 (Boehringer Ingelheim), BMS-791325 (Bristol-Myers Squibb),
GS 9256 (Gilead), IDX320 (Idenix), danoprevir or ITMN-191 or R7227
(RO5190591) (Intermune/Roche), TMC435 (Medivir/Tibotec/JnJ),
Boceprevir or SCH503034 (Merck), Vaniprevir or MK-7009 (Merck),
PHX1766 (Phenomix), Telaprevir or VX-950 (Vertex), VX-985 (Vertex)
and VX-500 (Vertex).
[0037] In embodiments, therapeutic agent A and therapeutic agent B
are co-administered with interferon and an HCV protease inhibitor.
In embodiments, therapeutic agent A and therapeutic agent B are
co-administered with ribavirin and an HCV protease inhibitor. In
embodiments, therapeutic agent A and therapeutic agent B are
co-administered with interferon, ribavirin and an HCV protease
inhibitor. In embodiments, therapeutic agent A and therapeutic
agent B are co-administered with an HCV protease inhibitor (with or
without a cytochrome P-450 inhibitor such as ritonavir). In
embodiments, therapeutic agent A and therapeutic agent B are
co-administered with ribavirin and an HCV protease inhibitor (with
or without a cytochrome P-450 inhibitor such as ritonavir). In
embodiments, therapeutic agent A and therapeutic agent B are
co-administered with interferon, ribavirin, and an HCV protease
inhibitor (with or without a cytochrome P-450 inhibitor such as
ritonavir).
[0038] Factors affecting the dosage regimen include the route of
administration; the type, age, weight, sex, diet, and condition of
the patient; the severity of the pathological condition;
pharmacological considerations, such as the activity, efficacy,
pharmacokinetic, and toxicology profiles of the particular compound
or salt used; whether a drug delivery system is utilized; and the
specific drug combination. Thus, the dosage regimen actually
employed can vary widely and, therefore, can deviate from the
disclosed dosage regimen set forth above.
[0039] In embodiments, the combination or pharmaceutical
composition comprises an amount of therapeutic agent A and an
amount of therapeutic agent B. The amount of therapeutic agent A
and therapeutic agent B may be any suitable amount that provides
the desired total periodic dosing amount such as the total daily
dosing amount. For example, the amount of therapeutic agent A in
the combination or pharmaceutical composition may be any suitable
amount such as from about 5 mg to about 200 mg or from about 10 to
about 100 mg or from about 25 mg to about 50 mg. In embodiments,
the total daily dosage amount for therapeutic agent A is about 25
mg. In embodiments, the total daily dosage amount for therapeutic
agent A is about 50 mg.
[0040] The amount of therapeutic agent B in the combination or
pharmaceutical composition may be from about 100 mg to about 1800
mg or from about 300 to about 1600 mg or from about 400 mg to about
1200 mg. In embodiments, the amount of therapeutic agent B in a
combination or pharmaceutical composition is about 100 mg. In
embodiments, the amount of therapeutic agent B in a combination or
pharmaceutical composition is about 200 mg. In embodiments, the
amount of therapeutic agent B in a combination or pharmaceutical
composition is about 300 mg. In embodiments, the amount of
therapeutic agent B in a combination or pharmaceutical composition
is about 400 mg. In embodiments, the amount of therapeutic agent B
in a combination or pharmaceutical composition is about 600 mg. In
embodiments, the amount of therapeutic agent B in a combination or
pharmaceutical composition is about 800 mg. In embodiments, the
amount of therapeutic agent B in a combination or pharmaceutical
composition is about 1000 mg. In embodiments, the amount of
therapeutic agent B in a combination or pharmaceutical composition
is about 1200 mg. In embodiments, the amount of therapeutic agent B
in a combination or pharmaceutical composition is about 1600
mg.
[0041] The combinations or pharmaceutical compositions may also
comprise other therapeutic agents and combinations thereof, used to
treat hepatitis C, such as any suitable amount of ribavirin and
pro-drugs thereof, HCV inhibitors such as, for example, HCV
helicase inhibitors, HCV polymerase inhibitors, HCV protease
inhibitors, HCV NS5A inhibitors, CD81 inhibitors, cyclophilin
inhibitors, or internal ribosome entry site (IRES) inhibitors; and
HIV inhibitors.
[0042] In embodiments, the combination or pharmaceutical
composition comprises an amount of therapeutic agent A and an
amount of therapeutic agent B. In embodiments, the combination or
pharmaceutical composition comprises an amount of therapeutic agent
A, an amount of therapeutic agent B and an amount of HCV protease
inhibitor. In embodiments, the combination or pharmaceutical
composition comprises an amount of therapeutic agent A, an amount
of therapeutic agent B and ribavirin. In embodiments, the
combination or pharmaceutical composition comprises an amount of
therapeutic agent A, an amount of therapeutic agent B, and an
amount of HCV protease inhibitor (with or without a cytochrome
P-450 inhibitor such as ritonavir). In embodiments, the combination
or pharmaceutical composition comprises an amount of therapeutic
agent A, an amount of therapeutic agent B, an amount of HCV
protease inhibitor (with our without a cytochrome P-450 inhibitor
such as ritonavir), and ribavirin. In embodiments, interferon is
co-administered with the above-mentioned combination or
pharmaceutical composition.
[0043] Dosage unit compositions may contain such amounts or
submultiples thereof to make up the total daily dose. The
administration of the therapeutic agent may be repeated a plurality
of times. Multiple doses per day may be used to achieve the total
daily dose, if desired. For example, a combination or
pharmaceutical composition comprising a dose of about 25 mg or 50
mg of therapeutic agent A may be administered at least twice per
day to achieve a total daily dosage amount of about 50 mg or 100 mg
of therapeutic agent A, respectively. A dose of about 400 mg or 800
mg of therapeutic agent B may be administered at least twice per
day to achieve a total daily dosage amount of about 800 mg or 1600
mg of therapeutic agent B, respectively.
[0044] The disclosed compositions may comprise one or more
conventional pharmaceutically acceptable carriers, adjuvants,
and/or vehicles (together referred to as "excipients"). The
disclosed compositions may be prepared in a form for oral
administration such as in a solid dosage form. Such solid dosage
forms include, for example, capsules, tablets, pills, powders, and
granules. In such solid dosage forms, the compounds or salts may be
combined with one or more excipients. If administered per os, the
compounds or salts may be mixed with, for example, lactose,
sucrose, starch powder, cellulose esters of alkanoic acids,
cellulose alkyl esters, talc, stearic acid, magnesium stearate,
magnesium oxide, sodium and calcium salts of phosphoric and
sulfuric acids, gelatin, acacia gum, sodium alginate,
polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted
or encapsulated for convenient administration. Such capsules or
tablets may contain a controlled-release formulation, as may be
provided in, for example, a dispersion of the compound or its salt
in hydroxypropylmethyl cellulose. In the case of capsules, tablets,
and pills, the dosage forms also may comprise buffering agents,
such as sodium citrate, or magnesium or calcium carbonate or
bicarbonate. In addition, tablets and pills may be prepared with
enteric coatings or other sustained/delayed/controlled release
excipients known in the art. In embodiments, therapeutic agent A
may be formulated as described in U.S. Provisional Application No.
61/353,553, filed Jun. 10, 2010, which is incorporated herein by
reference.
[0045] One or more of interferon, an HCV protease inhibitor (with
or without a cytochrome P-450 inhibitor, such as ritonavir), and
ribavirin may be co-administered with therapeutic agent A and
therapeutic agent B.
[0046] The disclosed combination(s)/composition(s) may be
administered at any suitable frequency such as at least three times
daily (e.g., every 8 hours in a 24-hour period), at least two times
daily (e.g., every 12 hours in a 24-hour period), at least once
daily (e.g., once in a 24-hour period), or at least once weekly
(e.g., once in a 7-day period).
[0047] This disclosure is also directed, in part, to methods of
using the disclosed combination(s)/compositions(s). The disclosed
combination(s)/composition(s) may be used in a method for
inhibiting replication of an RNA virus. In embodiments, the method
comprises exposing the virus to a disclosed
combination(s)/composition(s) and, optionally one or more
additional therapeutic agents. The disclosed
combination(s)/composition(s) may be administered with one or more
of an HCV protease inhibitor (with or without a cytochrome-P-450
inhibitor such as ritonavir), interferon and ribavirin in the same
or separate pharmaceutical compositions to inhibit replication of
an RNA virus. In embodiments, replication of the RNA virus is
inhibited in vitro. In embodiments, replication of the RNA virus is
inhibited in vivo. In embodiments, the RNA virus whose replication
is being inhibited is a single-stranded, positive sense RNA virus.
In embodiments, the RNA virus whose replication is being inhibited
is a virus from the Flaviviridae family. In embodiments, the RNA
virus whose replication is being inhibited is HCV.
[0048] The disclosed combination(s)/composition(s) may be used in a
method for inhibiting HCV RNA polymerase. In embodiments, the
method comprises exposing the polymerase to a disclosed
combination(s)/composition(s) and, optionally one or more
additional therapeutic agents. The disclosed
combination(s)/composition(s) may be administered with one or more
of an HCV protease inhibitor (with or without a cytochrome P-450
inhibitor such as ritonavir), interferon and ribavirin in the same
or separate pharmaceutical compositions to inhibit HCV RNA
polymerase. In embodiments, HCV RNA polymerase activity is
inhibited in vitro. In embodiments, HCV RNA polymerase activity is
inhibited in vivo.
[0049] The disclosed combination(s)/composition(s) may be used in a
method for inhibiting the HCV non-structural protein 5A (NS5A
protein). In embodiments, the method comprises exposing the
polymerase to a disclosed combination(s)/composition(s) and,
optionally one or more additional therapeutic agents. The disclosed
combination(s)/composition(s) may be administered with one or more
of an HCV protease inhibitor (with or without a cytochrome P-450
inhibitor such as ritonavir), interferon and ribavirin in the same
or separate pharmaceutical compositions to inhibit the HCV NS5A
protein. In embodiments, the HCV NS5A protein is inhibited in
vitro. In embodiments, the HCV NS5A protein is inhibited in
vivo.
[0050] The term "inhibiting" means reducing the level of RNA virus
replication/HCV polymerase activity either in vitro or in vivo. for
example, if a disclosed combination(s)/composition(s) reduces the
level of RNA virus replication by at least about 10% compared to
the level of RNA virus replication before the virus was exposed to
the combination(s)/composition(s), then the
combination(s)/composition(s) inhibits RNA virus replication. In
some embodiments, the disclosed combination(s)/composition(s) can
inhibit RNA virus replication by at least about 20%, at least about
30%, at least about 40%, at least about 50%, at least abut 60%, at
least about 70%, at least about 80%, at least about 90%, or at
least about 95%.
[0051] The disclosed combination(s)/compositions(s) may be used in
a method for reducing HCV viral load. In embodiments, the method
comprises exposing the polymerase to a disclosed
combination(s)/composition(s) and, optionally one or more
additional therapeutic agents. The disclosed
combination(s)/composition(s) may be administered with one or more
of an HCV protease inhibitor (with or without a cytochrome P-450
inhibitor such as ritonavir), interferon and ribavirin in the same
or separate pharmaceutical compositions to reduce HCV viral load.
In embodiments, HCV viral load is reduced in vitro. In embodiments,
HCV viral load is reduced in vivo. For example, if a disclosed
combination(s)/composition(s) reduces the HCV viral load by at
least about 10% compared to the HCV viral load before the virus was
exposed to the combination(s)/composition(s), then the
combination(s)/composition(s) reduces the HCV viral load. In some
embodiments, the disclosed combination(s)/composition(s) can reduce
viral load by at least about 20%, at least about 30%, at least
about 40%, at least about 50%, at least about 60%, at least about
70%, at least about 80%, at least about 90%, or at least about
95%.
[0052] The disclosed combination(s)/composition(s) may be used in a
method for treating a disease that can be treated by inhibiting HCV
RNA polymerase and/or the HCV NS5A protein. Thus, this disclosure
is also directed, in part, to a method for treating hepatitis C in
an animal in need of such treatment. These methods comprise
administering to the animal a disclosed
combination(s)/composition(s) and, optionally one or more
additional therapeutic agents. The disclosed
combination(s)/composition(s) may be administered with one or more
of an HCV protease inhibitor (with or without a cytochrome P-450
inhibitor such as ritonavir), interferon and ribavirin in the same
or separate pharmaceutical compositions to treat hepatitis C. In
some embodiments, a therapeutically effective amount of the
disclosed combination(s)/composition(s) is administered to the
animal.
[0053] "Treating" means ameliorating, suppressing, eradicating,
preventing, reducing the risk of, and/or delaying the onset of the
disease being treated. The term "treating" encompasses
administration of the disclosed combination(s)/composition(s) to an
HCV-negative patient that is a candidate for an organ
transplant.
[0054] The methods of treatment are particularly suitable for use
with humans, but may be used with other animals, particularly
mammals. A "therapeutically-effective amount" or "effective amount"
is an amount that will achieve the goal of treating the targeted
condition.
[0055] In embodiments, therapeutic agent A is administered in
combination with therapeutic agent B to reduce side effects
associated with the administration of an interferon and ribavirin,
either alone or in combination.
[0056] This disclosure is also directed, in part, to use of the
disclosed combination(s)/composition(s), and, optionally one or
more additional therapeutic agents in preparation of a medicament
for use in one or more of the disclosed methods. The disclosed
combination(s)/composition(s) may be combined with one or more of
an HCV protease inhibitor (with or without a cytochrome P-450
inhibitor such as ritonavir), interferon and ribavirin in the same
or separate medicaments for use in one or more of the disclosed
methods.
[0057] In embodiments, therapeutic agent A and therapeutic agent B
are used in the preparation of a medicament. In embodiments,
therapeutic agent A, therapeutic agent B and an HCV protease
inhibitor (with or without a cytochrome P-450 inhibitor such as
ritonavir) are used in the preparation of a medicament. In
embodiments, therapeutic agent A, therapeutic agent B and ribavirin
are used in the preparation of a medicament. In embodiments,
therapeutic agent A, therapeutic agent B, an HCV protease inhibitor
(with or without a cytochrome P-450 inhibitor such as ritonavir),
and ribavirin are used in the preparation of a medicament.
[0058] In embodiments, the disclosed medicaments are for inhibiting
replication of an RNA virus.
[0059] In embodiments, the disclosed medicaments are for inhibiting
HCV RNA polymerase activity.
[0060] In embodiments, the disclosed medicaments are for inhibiting
the HCV NS4A protein.
[0061] In embodiments, the disclosed medicaments are for decreasing
HCV viral load in a subject.
[0062] In embodiments, the disclosed medicaments are for treating
hepatitis C.
[0063] In embodiments, the disclosed medicaments are for reducing
side effects associated with the administration of an interferon
and ribavirin, either alone or in combination.
[0064] The disclosed medicaments may be for co-administration with
one or more additional therapeutic agents. For example, the
medicaments may be for co-administration with one or more of an HCV
protease inhibitor (with or without a cytochrome P-450 inhibitor
such as ritonavir), interferon and ribavirin.
[0065] In embodiments, the disclosed medicaments may be for
co-administration with interferon. In embodiments, the disclosed
medicaments are for co-administration with one or more of an HCV
protease inhibitor (with or without a cytochrome P-450 inhibitor
such as ritonavir), interferon and ribavirin for inhibiting
replication of an RNA virus.
[0066] In embodiments, the disclosed medicaments are for
co-administration with one or more of an HCV protease inhibitor
(with or without a cytochrome P-450 inhibitor such as ritonavir),
interferon and ribavirin for inhibiting HCV RNA polymerase
activity.
[0067] In embodiments, the disclosed medicaments are for
co-administration with one or more of an HCV protease inhibitor
(with or without a cytochrome P-450 inhibitor such as ritonavir),
interferon and ribavirin for inhibiting HCV NS5A protein.
[0068] In embodiments, the disclosed medicaments are for
co-administration with one or more of an HCV protease inhibitor
(with or without a cytochrome P-450 inhibitor such as ritonavir),
interferon and ribavirin for decreasing HCV viral load in a
subject.
[0069] In embodiments, the disclosed medicaments are for
co-administration with one or more of an HCV protease inhibitor
(with or without a cytochrome P-450 inhibitor such as ritonavir),
interferon and ribavirin for treating hepatitis C.
EXAMPLES
[0070] The following examples are for illustration purposes and do
not limit the scope of this disclosure in any way.
Materials.
[0071] The replicon cell line was derived from the human hepatoma
cell line Huh7. It was derived from HCV genotype 1b (Con1), and is
a bicistronic subgenomic replicon, essentially similar to those
described in Science 285(5424):110-3 (1999). The first cistron of
the construct contains a firefly luciferase reporter and a neomycin
phosphotransferase selectable marker.
Replicon Cell Culture.
[0072] Replicon cells were seeded at a density of 5000 cells per
well of a 96-well plate in 100 .mu.l Dulbecco's Modified Eagle
Media (DMEM) containing 5% FBS. Replicon cells were maintained in
DMEM containing 100 IU/ml penicillin, 100 mg/ml streptomycin
(Invitrogen), 200 mg/ml G418 (Invitrogen) and 10% fetal bovine
serum (FBS) at 37.degree. C. and 5% CO.sub.2.
Combination Studies.
[0073] The replicon cell culture was used to determine the dose or
concentration of therapeutic agent A that produces a synergistic,
additive or antagonistic inhibitory effects on HCV replication when
combined with therapeutic agent B.
[0074] The compounds were diluted in dimethyl sulfoxide (DMSO) to
generate a 200X stock in a series of 6 two-fold dilutions. The
dilution series was then further diluted 100-fold in the medium
containing 5% FBS.
[0075] The dilutions of each compound were combined in a
checkerboard fashion in the cell culture plates. Three experiments
with three plates in each experiment were performed. In particular,
six concentrations of compound A alone and six concentrations of
the sodium salt of compound B alone were assayed in each plate. In
addition, 36 combinations of various concentrations of the two
compounds were assayed for each plate. The concentrations of
compound A and compound B were chosen to ensure that the EC.sub.50s
of the compounds were substantially in the middle of the serial
dilution range. For compound A, concentrations ranged from 0.0002
nM (1.95.times.10.sup.-4 nM) to 0.0063 nM (6.25.times.10.sup.-3
nM), and for compound B, concentrations ranged from 0.10 nM (0.0977
nM) to 3.13 nM. The cells were incubated in a tissue culture
incubator at 37.degree. C. and 5% CO.sub.2 for three days.
[0076] The inhibitor effects of compounds on HCV replication were
analyzed by determining the fraction of inhibition of the
luciferase signal which was determined by measuring activity of a
luciferase reporter gene using a Luciferase Assay System kit
(Promega) according to the manufacturer's instructions. Passive
Lysis buffer (30 .mu.l, Promega) was added to each well, and the
plates were incubated for 15 minutes with rocking to lyse the
cells. Luciferin solution (100 .mu.l, Promega) was added to each
well and the luciferase activity was measured using a Victor II
luminometer (Perkin-Elmer). To determine the EC.sub.50, the
luciferase inhibition data were analyzed using GraphPad Prism 4
software.
Combination Analysis.
[0077] Synergy or antagonism from combining therapeutic agent A
with therapeutic agent B was quantified for direct comparison of
inhibitor effects on HCV replication. The percent inhibition
results were analyzed for synergy, additivity and antagonism
according to the Bliss independence, Lowe additivity, and
Pritchard-Direct models (Pharmacol. Rev. 47(2):331-85 (1995);
Antiviral Research 14:181-206 (1990)).
[0078] An E.sub.max model in the following form is used to fit the
data from each single drug for each plate in each experiment using
the NLIN procedure of SAS (SAS 9.1, SAS Institute Inc. 2004),
f o = 1 - 1 ( 1 + ( C I ) h ) g , ##EQU00001##
where f.sub.a is the fraction of inhibition, C is the
concentration, l is the location of the concentration-response
curve's inflection point (point of greatest slope), g is the degree
of asymmetry, and h is the shape parameter of the curve. Using the
estimated g, l, and h for each single drug for each plate in an
experiment, the fraction of inhibition for any concentration
combination of the two drugs is predicted by one of two reference
models: Loewe additivity and Bliss independence (Pharmacol. Rev.
47(2):331-85 (1995)). A difference between the actual observed
fraction of inhibition and the predicted value is calculated for
each concentration combination for each plate in each experiment to
determine whether the observed combined effect is greater than that
predicted by Loewe additivity or Bliss independence. For each
concentration combination, the replicates (across all plates and
experiments) were used to calculate a mean difference between
observed and predicted fraction of inhibition, its standard error
and its two-sided 95% confidence interval (CI).
[0079] The Prichard-Shipman method, similar to the E.sub.max
methods, is used to calculate the difference between the actual
observed fraction of inhibition and the predicted value for each
concentration combination for each plate in each experiment to
determine whether the observed combined effect is greater than the
theoretical additive effect determined directly from the individual
dose-response curves in the assays described above (Antiviral
Research 14:181-206 (1990)). The calculated theoretical additivity
is then compared to the experimental dose-response surface, and
subsequently subtracted to reveal any areas of aberrant
interaction. The following equation is used to calculate the
theoretical additive effects:
Z=X+Y(1-X)=X+Y-XY,
where Z is the total inhibition produced by the combination of
drugs X and Y, with X and Y representing the inhibition produced by
drugs X and Y alone, respectively.
[0080] A difference between the actual observed fraction of
inhibition and the predicted value is calculated for each
concentration combination for each plate in each experiment to
determine whether the observed combined effect is greater than the
theoretical additive effect, Z, calculated from equation (1). The
mean difference between the observed and predicted fraction of
inhibition, its standard error and its two-sided 95% CI is then
calculated for each concentration combination across all plates and
experiments.
[0081] Synergy or antagonism for a concentration combination is
determined by calculating at each concentration combination the 95%
confidence interval (CI) of the mean difference between observed
and predicted fraction of inhibition. If the lower bound of 95% CI
is larger than zero, then the drug combination is considered to
have a synergistic effect; it the upper bound of 95% CI is less
than zero, then the drug combination is considered to have an
antagonistic effect; otherwise, the effect of the combination is
considered to be purely additive, and no significant antagonism or
synergy exists at this concentration combination. Small differences
of statistical significance caused by very small variance were
excluded if the relative mean difference (i.e., the absolute mean
difference divided by its corresponding observed mean inhibition)
of the synergistic or antagonistic effect is less than about one
percent.
Results.
[0082] The results of the replicon assay analysis using the
Prichard-Shipman Model are illustrated in Table 1 and FIGS. 1 and
2.
[0083] Table 1 below lists various combinations of concentrations
of compound A and compound B. For each combination of
concentrations, Table 1 includes the mean difference in the
observed and predicted fraction of inhibition, the standard
deviation or error of the mean difference, and the upper and lower
limits of the 95% confidence interval of the mean difference
between observed and predicted fractions of inhibition.
TABLE-US-00001 TABLE 1 Mean difference in Standard Lower 95% Upper
95% Compound A, Compound B, fraction of inhibition: error of mean
confidence confidence nM nM Observed-Predicted difference limit
limit 0.000195 0.390625 -0.16428 0.054657 -0.29032 -0.03824
0.000391 0.097656 0.17174 0.035757 0.08929 0.25420 0.000781
0.097656 0.16218 0.063815 0.01502 0.30933 0.000781 0.195313 0.13851
0.054433 0.01298 0.26403 0.000781 0.390625 0.09246 0.021657 0.04252
0.14240 0.000781 0.781250 0.08495 0.016567 0.04674 0.12315 0.001563
0.195313 0.07811 0.032064 0.00417 0.15205 0.001563 0.390625 0.05619
0.016132 0.01900 0.09339 0.001563 0.781250 0.05043 0.012437 0.02175
0.07911
[0084] According to Table 1, all but one of the concentration
combinations of compound A and compound B listed in the table have
statistically significant synergistic effects.
[0085] FIG. 1 illustrates deviations from expected interactions
between compound A and compound B are purely additive at
concentrations associated with a horizontal plane at 0%.
Synergistic interactions between compound A and compound B appear
as a peak above the horizontal plane with a height corresponding to
the percent above calculated additivity. Antagonistic interactions
between compound A and compound B appear as a pit or trough below
the horizontal plane with a negative value signifying the percent
below the calculated additivity. It is apparent from FIG. 1 that
synergistic interactions between compound A and compound B exist at
many of the concentration combinations of compounds A and B.
[0086] The contour plot of FIG. 2 displays the region of
concentration combinations with a statistically significant
synergistic, antagonistic, or additive effect. Synergistic
interactions appear as dark grey, additive interactions appear
white, and antagonistic interactions appear as light grey. As
illustrated in FIG. 2, an additive or synergistic effect exists at
most of the concentrations for compound A and compound B. In
particular, there is a concentration region showing synergy at the
lower dose concentrations of compounds A and B.
[0087] The results presented in Table 1 and FIGS. 1 and 2
demonstrate that the combination of therapeutic agent A and
therapeutic agent B achieves additivity or synergy at most
concentration combinations of therapeutic agent A and therapeutic
agent B. Taken together, these in vitro replicon results suggest
that therapeutic agent A should produce a significant antiviral
effect in patients when administered in combination with
therapeutic agent B in patients infected with HCV.
Colony Counting Assay
[0088] Replicon colonies were exposed to therapeutic agent A,
therapeutic agent B, an HCV protease inhibitor (a macrocyclic
compound comprising a 9-membered fused bicycle, herein referred to
as "therapeutic agent C"), and various combinations of these
agents, to quantify the frequency of resistance of these replicon
colonies to these agents.
[0089] The stable subgenomic bicistronic replicon cell line derived
from HCV genotype 1a (H77; Genbank accession number AF011751) was
generated by introducing the constructs into cell lines derived
from the human hepatoma cell line Huh-7. The replicon also contains
a firefly luciferase reporter and a neomycin phosphotransferase
(Neo) selectable marker. The first cistron and the second cistron
of the bicistronic replicon construct are separated by the FMDV 2a
protease, and the second cistron comprises the HCV NS3-NS5B coding
region with addition of adaptive mutations E1202G, K1691R, K2040R
and S22041.
[0090] The HCV replicon cell line was maintained in Dulbecco's
modified Eagles medium (DMEM; Invitrogen) containing 10% (v/v)
fetal bovine serum, 100 IU/ml penicillin, 100 .mu.g/ml
streptomycin, and 200 .mu.g/ml G418 (all from Invitrogen). 1a-H77
replicon cells (10.sup.5-10.sup.6) were plated in 150 mm cell
culture plates and grown in the presence of G418 (400 .mu.g/ml) and
therapeutic agent A, the potassium salt of compound B and/or
therapeutic agent C at concentrations that were either 10-fold or
100-fold above the EC.sub.50 value for the HCV genotype 1a replicon
cell line. After three weeks of treatment, the majority of replicon
cells were cleared of replicon RNA and, therefore, were unable to
survive in the G418-containing medium. The cells containing
resistant replicon variants survived and formed colonies. These
colonies were stained with 1% crystal violet in 10% Protocol
SafeFix II reagent (Fisher Scientific) and counted.
[0091] As shown in FIG. 3, the combination of therapeutic agent A
and therapeutic agent B, and the combination of therapeutic agent A
and therapeutic agent C, at concentrations either 10-fold or
100-fold above their respective EC.sub.50 values, resulted in
significantly fewer colonies than therapeutic agent A, therapeutic
agent B or therapeutic agent C alone at concentrations 10-fold or
100-fold above their respective EC.sub.50 values.
[0092] All references (patent and non-patent) cited above are
incorporated by reference into this patent application. The
discussion of those references is intended merely to summarize
assertions made by their authors. No admission is made that any
reference (or a portion of a reference) is relevant prior art (or
prior art at all). Applicants reserve the right to challenge the
accuracy and pertinence of the cited references.
* * * * *