U.S. patent application number 13/564286 was filed with the patent office on 2012-11-22 for combination therapy for the treatment of hcv infection.
This patent application is currently assigned to VERTEX PHARMACEUTICALS INCORPORATED. Invention is credited to Ann Kwong, Chao Lin, Nagraj Mani, Yi Zhou.
Application Number | 20120295843 13/564286 |
Document ID | / |
Family ID | 39711894 |
Filed Date | 2012-11-22 |
United States Patent
Application |
20120295843 |
Kind Code |
A1 |
Kwong; Ann ; et al. |
November 22, 2012 |
COMBINATION THERAPY FOR THE TREATMENT OF HCV INFECTION
Abstract
The present invention relates to therapeutic combinations
comprising a protease inhibitor and a polymerase inhibitor for the
treatment of HCV. The present invention also relates to therapeutic
combinations comprising VX-950 and a polymerase inhibitor. Also
within the scope of the invention are methods using the therapeutic
combinations of the present invention for treating HCV infection or
alleviating one or more symptoms thereof in a patient. The present
invention also provides kits comprising the combinations of the
present invention.
Inventors: |
Kwong; Ann; (Cambridge,
MA) ; Mani; Nagraj; (Natick, MA) ; Zhou;
Yi; (Lexington, MA) ; Lin; Chao; (Winchester,
MA) |
Assignee: |
VERTEX PHARMACEUTICALS
INCORPORATED
Cambridge
MA
|
Family ID: |
39711894 |
Appl. No.: |
13/564286 |
Filed: |
August 1, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12598802 |
Mar 24, 2010 |
8258116 |
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PCT/US08/05758 |
May 5, 2008 |
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13564286 |
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60927581 |
May 4, 2007 |
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60931425 |
May 23, 2007 |
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Current U.S.
Class: |
514/4.3 ;
514/21.8 |
Current CPC
Class: |
A61K 31/7068 20130101;
A61P 1/16 20180101; A61K 45/06 20130101; A61P 31/14 20180101; A61K
31/7068 20130101; A61K 31/497 20130101; A61P 31/12 20180101; A61P
43/00 20180101; A61K 31/497 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
514/4.3 ;
514/21.8 |
International
Class: |
A61K 38/08 20060101
A61K038/08; A61P 31/14 20060101 A61P031/14 |
Claims
1. A therapeutic combination comprising VX-950 and a polymerase
inhibitor selected from the group consisting of R7128 and
IDX184.
2. The therapeutic combination of claim 1, wherein the polymerase
inhibitor is R7128.
3. The therapeutic combination of claim 1, wherein the polymerase
inhibitor is IDX184.
4. A kit comprising: (i) a therapeutic combination of claim 1; and
(ii) instructions for utilizing said combination.
5. A method of treating HCV infection, or alleviating one or more
symptoms thereof in a patient, comprising administering VX-950 in
combination with a polymerase inhibitor selected from the group
consisting of R7128 and IDX184.
6. The method of claim 5, wherein the polymerase inhibitor is
R7128.
7. The method of claim 5, wherein the polymerase inhibitor is
IDX184.
8. The method of claim 5, wherein the HCV infection is genotype
1.
9. The method of claim 5, wherein said patient is a treatment naive
patient.
10. The method of claim 5, wherein said patient is non-responsive
to interferon monotherapy.
11. The method of claim 5, wherein said patient is non-responsive
to a combination therapy using ribavirin and an interferon.
12. The method of claim 5, wherein the patient is infected by an
HCV variant.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. provisional
applications Ser. Nos. 60/927,581, filed May 4, 2007, and
60/931,425, filed May 23, 2007, the contents of which are
incorporated herein by reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] Infection by hepatitis C virus ("HCV") is a compelling human
medical problem. HCV is recognized as the causative agent for most
cases of non-A, non-B hepatitis, with an estimated human
sero-prevalence of 3% globally [A. Alberti et al., "Natural History
of Hepatitis C," J. Hepatology, 31., (Suppl. 1), pp. 17-24 (1999)].
Nearly four million individuals may be infected in the United
States alone [M. J. Alter et al., "The Epidemiology of Viral
Hepatitis in the United States, Gastroenterol. Clin. North Am., 23,
pp. 437-455 (1994); M. J. Alter "Hepatitis C Virus Infection in the
United States," J. Hepatology, 31, (Suppl. 1), pp. 88-91
(1999)].
[0003] Upon first exposure to HCV only about 20% of infected
individuals develop acute clinical hepatitis while others appear to
resolve the infection spontaneously. In almost 70% of instances,
however, the virus establishes a chronic infection that persists
for decades [S. Iwarson, "The Natural Course of Chronic Hepatitis,"
FEMS Microbiology Reviews, 14, pp. 201-204 (1994); D. Lavanchy,
"Global Surveillance and Control of Hepatitis C," J. Viral
Hepatitis, 6, pp. 35-47 (1999)]. This usually results in recurrent
and progressively worsening liver inflammation, which often leads
to more severe disease states such as cirrhosis and hepatocellular
carcinoma [M. C. Kew, "Hepatitis C and Hepatocellular Carcinoma",
FEMS Microbiology Reviews, 14, pp. 211-220 (1994); I. Saito et.
al., "Hepatitis C Virus Infection is Associated with the
Development of Hepatocellular Carcinoma," Proc. Natl. Acad. Sci.
USA, 87, pp. 6547-6549 (1990)]. Unfortunately, there are no broadly
effective treatments for the debilitating progression of chronic
HCV.
[0004] The HCV genome encodes a polyprotein of 3010-3033 amino
acids [Q. L. Choo, et. al., "Genetic Organization and Diversity of
the Hepatitis C Virus," Proc. Natl. Acad. Sci. USA, 88, pp.
2451-2455 (1991); N. Kato et al., "Molecular Cloning of the Human
Hepatitis C Virus Genome From Japanese Patients with Non-A, Non-B
Hepatitis," Proc. Natl. Acad. Sci. USA, 87, pp. 9524-9528 (1990);
A. Takamizawa et. al., "Structure and Organization of the Hepatitis
C Virus Genome Isolated From Human Carriers," J. Virol., 65, pp.
1105-1113 (1991)]. The HCV nonstructural (NS) proteins are presumed
to provide the essential catalytic machinery for viral replication.
The NS proteins are derived by proteolytic cleavage of the
polyprotein [R. Bartenschlager et. al., "Nonstructural Protein 3 of
the Hepatitis C Virus Encodes a Serine-Type Proteinase Required for
Cleavage at the NS3/4 and NS4/5 Junctions," J. Virol., 67, pp.
3835-3844 (1993); A. Grakoui et. al., "Characterization of the
Hepatitis C Virus-Encoded Serine Proteinase: Determination of
Proteinase-Dependent Polyprotein Cleavage Sites," J. Virol., 67,
pp. 2832-2843 (1993); A. Grakoui et. al., "Expression and
Identification of Hepatitis C Virus Polyprotein Cleavage Products,"
J. Virol., 67, pp. 1385-1395 (1993); L. Tomei et. al., "NS3 is a
serine protease required for processing of hepatitis C virus
polyprotein", J. Virol., 67, pp. 4017-4026 (1993)].
[0005] The HCV NS protein 3 (NS3) contains a serine protease
activity that helps process the majority of the viral enzymes, and
is thus considered essential for viral replication and infectivity.
It is known that mutations in the yellow fever virus NS3 protease
decrease viral infectivity [Chambers, T. J. et. al., "Evidence that
the N-terminal Domain of Nonstructural Protein NS3 From Yellow
Fever Virus is a Serine Protease Responsible for Site-Specific
Cleavages in the Viral Polyprotein," Proc. Natl. Acad. Sci. USA,
87, pp. 8898-8902 (1990)]. The first 181 amino acids of NS3
(residues 1027-1207 of the viral polyprotein) have been shown to
contain the serine protease domain of NS3 that processes all four
downstream sites of the HCV polyprotein [C. Lin et al., "Hepatitis
C Virus NS3 Serine Proteinase: Trans-Cleavage Requirements and
Processing Kinetics", J. Virol., 68, pp. 8147-8157 (1994)].
[0006] The HCV NS3 serine protease and its associated cofactor,
NS4A, help process all of the viral enzymes, and are thus
considered essential for viral replication. This processing appears
to be analogous to that carried out by the human immunodeficiency
virus aspartyl protease, which is also involved in viral enzyme
processing. HIV protease inhibitors, which inhibit viral protein
processing, are potent antiviral agents in man indicating that
interrupting this stage of the viral life cycle results in
therapeutically active agents. Consequently HCV NS3 serine protease
is also an attractive target for drug discovery.
[0007] Until recently, the only established therapy for HCV disease
was interferon treatment. However, interferons have significant
side effects [M. A. Wlaker et al., "Hepatitis C Virus: An Overview
of Current Approaches and Progress," DDT, 4, pp. 518-29 (1999); D.
Moradpour et al., "Current and Evolving Therapies for Hepatitis C,"
Eur. J. Gastroenterol. Hepatol., 11, pp. 1199-1202 (1999); H. L. A.
Janssen et al. "Suicide Associated with Alfa-Interferon Therapy for
Chronic Viral Hepatitis," J. Hepatol., 21, pp. 241-243 (1994); P.
F. Renault et al., "Side Effects of Alpha Interferon," Seminars in
Liver Disease, 9, pp. 273-277 (1989)] and induce long term
remission in only a fraction (.about.25%) of cases [O. Weiland,
"Interferon Therapy in Chronic Hepatitis C Virus Infection", FEMS
Microbiol. Rev., 14, pp. 279-288 (1994)]. Recent introductions of
the pegylated forms of interferon (PEG-INTRON.RTM. and
PEGASYS.RTM.) and the combination therapy of ribavirin and
pegylated interferon (REBETROL.RTM.) have resulted in only modest
improvements in remission rates and only partial reduction of side
effects. Moreover, the prospects for effective anti-HCV vaccines
remain uncertain.
[0008] Thus, there is a need for more effective anti-HCV therapies.
Such inhibitors would have therapeutic potential as protease
inhibitors, particularly as serine protease inhibitors, and more
particularly as HCV NS3 protease inhibitors. Specifically, such
compounds may be useful as antiviral agents, particularly as
anti-HCV agents.
SUMMARY OF THE INVENTION
[0009] This invention relates to therapeutic combinations
comprising VX-950 and a polymerase inhibitor.
[0010] The invention also relates to methods of treating HCV
infection or alleviating one or more symptoms thereof in a patient,
comprising administering to said patient a therapeutic combination
of the present invention.
[0011] It is yet another object of the present invention to provide
a pharmaceutical regimen for treating HCV infection in a
patient.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0012] For purposes of this invention, the chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 75.sup.th Ed.
Additionally, general principles of organic chemistry are described
in "Organic Chemistry", Thomas Sorrell, University Science Books,
Sausalito: 1999, and "March's Advanced Organic Chemistry", 5.sup.th
Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New
York: 2001, the entire contents of which are hereby incorporated by
reference.
[0013] As described herein, compounds of the invention may
optionally be substituted with one or more substituents, or as
exemplified by particular classes, subclasses, and species of the
invention.
[0014] As used herein, "patient" refers to a mammal, including a
human.
[0015] The term "polymerase inhibitor," as used herein, refers to
compounds that inhibit the activity of HCV RNA dependent RNA
polymerase (RdRp). Polymerase inhibitors of the following invention
include, but are not limited to, the compounds of formula I, II and
III. The polymerase inhibitor can be a nucleoside or non-nucleoside
inhibitor. A nucleoside polymerase inhibitor binds to the substrate
of the enzyme; whereas, the polymerase non-nucleoside binds to
different site while still inhibiting the enzyme, e.g., an
allosteric inhibitor.
[0016] The term "protease inhibitor," as used herein refers to
means an agent (compound or biological) that is effective to
inhibit the function of HCV NS3 protease in a mammal. Protease
inhibitors of the following invention include, but are not limited
to, VX-950.
[0017] As used herein, "VX-950" refers to an HCV inhibitor shown
below and described in PCT Publication Number WO 02/18369, which is
incorporated herein by reference in its entirety.
##STR00001##
[0018] The term "therapeutic combination" as used herein means a
combination of one or more active drug substances, i.e., compounds
having a therapeutic utility. Typically, each such compound in the
therapeutic combinations of the present invention will be present
in a pharmaceutical composition comprising that compound and a
pharmaceutically acceptable carrier. The compounds in a therapeutic
combination of the present invention may be administered
simultaneously or separately, as part of a regimen.
[0019] Unless otherwise stated, structures depicted herein are also
meant to include all isomeric (e.g., enantiomeric, diastereomeric,
and geometric (or conformational)) forms of the structure; for
example, the R and S configurations for each asymmetric center, (Z)
and (E) double bond isomers, and (Z) and (E) conformational
isomers. Therefore, single stereochemical isomers as well as
enantiomeric, diastereomeric, and geometric (or conformational)
mixtures of the present compounds are within the scope of the
invention. Unless otherwise stated, all tautomeric forms of the
compounds of the invention are within the scope of the invention.
Additionally, unless otherwise stated, structures depicted herein
are also meant to include compounds that differ only in the
presence of one or more isotopically enriched atoms. For example,
compounds having the present structures except for the replacement
of hydrogen by deuterium or tritium, or the replacement of a carbon
by a .sup.13C- or .sup.14C-enriched carbon are within the scope of
this invention. Such compounds are useful, for example, as
analytical tools or probes in biological assays.
Therapeutic Combinations
[0020] The present invention relates to therapeutic combinations
comprising a protease inhibitor and a polymerase inhibitor for the
treatment of HCV.
Protease Inhibitors
[0021] In one aspect of the invention, the protease inhibitor is
VX-950.
[0022] VX-950, an HCV inhibitor with its structure shown below is
such a compound in need. VX-950 is described in PCT Publication
Number WO 02/18369, which is incorporated herein by reference in
its entirety.
##STR00002##
[0023] VX-950, a potent and specific NS3-4A protease inhibitor
demonstrated substantial antiviral activity in a phase 1b trial of
subjects infected with HCV genotype 1 (Study VX04-950-101). The
degree to which a subject responds to treatment and the rate at
which viral rebound is observed could in part be due to genotypic
differences in sensitivity to the protease inhibitor. The rapid
replication rate of HCV, along with the poor fidelity of its
polymerase, gives rise to an accumulation of mutations throughout
its genome [P. Simmonds, "Genetic diversity and evolution of
hepatitis C virus--15 years on," J. Gen. Virol., 85, pp. 3173-88
(2004)]. The degree to which sequence variability in the protease
region affects the catalytic efficiency of the enzyme or the
binding of an inhibitor is not known. Additionally, the generation
of numerous viral genomes with remarkable sequence variation
presents potential problems of emerging drug resistant virus in
subjects treated with antiviral therapy. Indeed, drug resistance
against antiviral drugs, such as HIV protease inhibitors, is well
documented [Johnson et al., Top. HIV Med., 12, pp. 119-24 (2004)].
Drug resistant mutations have already been shown to develop in
vitro in the presence of HCV protease inhibitors [Lin et al., "In
vitro studies of cross-resistance mutations against two hepatitis C
virus serine protease inhibitors, VX-950 and BILN 2061," J. Biol.
Chem., 280, pp. 36784-36791 (2005), which is incorporated herein by
reference in its entirety; Lin et al., "In vitro resistance studies
of hepatitis C virus serine protease inhibitors, VX-950 and BILN
2061: Structural analysis indicates different resistance
mechanisms," J. Biol. Chem., 279, pp. 17508-17514 (2004), which is
incorporated herein by reference in its entirety; Lu et al.,
Antimicrob. Agents Chemother., 48, pp. 2260-6 (2004); Trozzi et
al., "In vitro selection and characterization of hepatitis C virus
serine protease variants resistant to an active-site peptide
inhibitor," J. Virol. 77, pp. 3669-79 (2003)]. Mutations resistant
to the protease inhibitor BILN 2061 have been found at positions
R155Q, A156T, and D168V/A/Y in the NS3 gene, but no mutations have
yet been observed in the NS4 region or in the protease cleavage
sites. A VX-950 resistance mutation has also been found in vitro at
position A156S. Cross-resistant mutations against both VX-950 and
BILN 2061 have also been shown to develop in vitro at position 156
(A156V/T) (Lin et al., 2005, supra).
[0024] In other embodiments, the protease inhibitor is described in
international patent application publication number WO2007/025307,
the entire contents of which is incorporated herein.
[0025] In still other embodiments, the protease inhibitor is
described in one or more of the following publications:
WO1997/43310, US20020016294, WO2001/81325, WO2002/08198,
WO2001/77113, WO2002/08187, WO2002/08256, WO2002/08244,
WO2003/006490, WO2001/74768, WO1999/50230, WO1998/17679,
WO2002/48157, US20020177725, WO2002/060926, US20030008828,
WO2002/48116, WO2001/64678, WO2001/07407, WO1998/46630,
WO2000/59929, WO1999/07733, WO2000/09588, US20020016442,
WO2000/09543, WO1999/07734, U.S. Pat. No. 6,018,020, U.S. Pat. No.
6,265,380, U.S. Pat. No. 6,608,027, US20020032175, US20050080017,
WO1998/22496, U.S. Pat. No. 5,866,684, WO2002/079234, WO2000/31129,
WO1999/38888, WO1999/64442, WO2004/072243, and WO2002/18369, the
contents of all of which are incorporated herein by reference in
their entireties.
Polymerase Inhibitors
[0026] The polymerase inhibitors can be a nucleoside or a
non-nucleoside inhibitor.
[0027] In an embodiment of the first aspect, the polymerase
inhibitor is the compound of formula (I)
##STR00003##
or a pharmaceutically acceptable salt thereof.
[0028] In another embodiment of the first aspect, the polymerase
inhibitor is the compound of formula (II)
##STR00004##
or a pharmaceutically acceptable salt thereof.
[0029] In another embodiment of the first aspect, the polymerase
inhibitor is the compound of formula (III)
##STR00005##
or a pharmaceutically acceptable salt thereof.
[0030] In another aspect, the invention provides a therapeutic
combination of VX-950 and the polymerase inhibitor of compound
(I)
##STR00006##
or a pharmaceutically acceptable salt thereof.
[0031] In another aspect, the invention provides a therapeutic
combination of VX-950 and the polymerase inhibitor of compound
(II)
##STR00007##
or a pharmaceutically acceptable salt thereof.
[0032] In another aspect, the invention provides a therapeutic
combination of VX-950 and the polymerase inhibitor of compound
(III)
##STR00008##
or a pharmaceutically acceptable salt thereof.
[0033] In other embodiments, the polymerase inhibitor is selected
from the inhibitors known in the art. For instance, the polymerase
inhibitor is described in one or more of the following
international patent application publications: WO2008/011337;
WO2006/093801; WO2005/019191; WO2004/041818; WO2007/150001;
WO2006/066079; WO2006/137706; WO2006/011719; WO2004/108719;
WO2004/108068; WO2004/033450; WO2003/084953; WO2008/019477;
WO2007/019674; WO2006/007693; WO2005/080388; WO2004/065367;
WO2004/064925; WO2003/010141; WO2003/010140; WO2003/007945;
WO2002/004425; WO2008/021928; WO2008/021927; WO2007/143521;
WO2006/020082; WO2004/014852; WO2004/014313; WO2003/026587;
WO2003/002518; WO2002/079187; WO2008/011521; WO2008/008912;
WO2006/138744; WO2007/039144; WO2006/045613; WO2006/045615;
WO2005/103045; WO2005/092863; WO2005/079799; WO2004/076415;
WO2004/037818; WO2004/009543; WO2003/037894; WO2003/037893;
WO2007/082554; WO2007/028789; WO2006/119975; WO2006/046030;
WO2006/046039; WO2006/029912; WO2006/027628; WO2006/008556;
WO2005/034941; WO2004/087714; WO2003/062211; WO2002/006246;
WO2006/052013; WO2005/080399; WO2005/049622; WO2005/014543;
WO2003/000254; WO2007/023381; WO2006/018725; WO2004/074270;
WO2004/073599; WO2003/082848; WO2004/002977; WO2004/002944;
WO2004/002940; WO2006/117306; WO2006/050035; WO2002/100851;
WO2007/147794; WO2007/088148; WO2007/071434; WO2006/100106;
WO2006/119646; WO2005/112640; WO2004/052885; WO2004/052879;
WO2004/041201; WO2007/092558; WO2005/016932; WO2004/091724;
WO2003/099824; WO2003/099275; WO2008/009078; WO2007/034127;
WO2007/041632; WO2007/005779; WO2007/002639; WO2006/065590;
WO2006/019831; WO2004/080453; WO2003/101993; WO2003/082265;
WO2001/077091; and WO1994/12192. The entire content of each of the
foregoing publications is incorporated herein in its entirety.
[0034] In other examples, the polymerase inhibitor is described in
one or more of the following U.S. patent application publications:
US2004167123; US2004162285; US20040097492; US20040087577;
US20070275947; US20070275930; US20070270406; US20070270405;
US2005075376; US20070032488; US20030050320; US2005154056;
US2006040927; US2006094706; US2006258682; US2006223834;
US2006217390; US2006019976; US2005107364; US2006183751;
US2006063821; and US2005176701. The entire content of each of the
foregoing publications is incorporated herein in its entirety.
[0035] In still further examples, the polymerase inhibitor is
described in U.S. Pat. No. 7,112,600 or European Patent No. EP
1321463. The entire content of each of the foregoing publications
is incorporated herein.
[0036] In still further embodiments, the polymerase inhibitor is
selected from A-837093, being developed by Abbott Laboratories;
GS-9190, being developed by Gilead Sciences; PF-868 and PF-554,
being developed by Pfizer; R1479, R1626 (prodrug of R1479), R7128,
and R1728, being developed by Roche Pharmaceuticals; PST 7081,
being developed by Pharmasset and Roche Pharmaceuticals; IDX102,
IDX184, NM107, NM283 (prodrug of NM107) called Valopicitabine,
being developed by Idenix; and VCH-916 and VCH-759, being developed
by ViroChem; and HCV-796, being developed by Wyeth. Other
polymerase inhibitors are in development by Genelabs; Boehringer
Ingelheim; Anadys; Celera; Schering; and Medavir.
[0037] According to another aspect, the present invention provides
kits for use in treating HCV infection in a patient. The kits of
the present invention comprise any one of the therapeutic
combinations of the present invention. The kits further comprise
instructions for utilizing the therapeutic combinations. The kits
may be tailored to the needs of classes or types of patients or
other clinically relevant factors such as age, body weight,
concomitant diseases/conditions, severity and stage of HCV
infection, responsiveness or non-responsiveness to prior
treatments, propensity for side effects, etc. For example, the
therapeutic combination in a kit may be tailored for dosages
suitable for patients having a body weight of, e.g., 75 kg. Or, the
therapeutic combination in a kit may be tailored for dosages
suitable for patients having a body weight of, e.g., less than or
equal to 75 kg. Or, the therapeutic combination in a kit may be
tailored for pediatric use, wherein the dosage for children is
varied depending on factors such as age, body weight, severity of
disease, etc.
[0038] According to another aspect, the present invention provides
a kit comprising: (i) a plurality of VX-950 compositions; (ii) a
plurality of polymerase inhibitor compositions; and (iii)
instructions for utilizing the above compositions.
[0039] In another aspect, the invention provides methods of using
the therapeutic combinations of the present invention for treating
HCV infection or alleviating one or more symptoms thereof in a
patient.
[0040] In one embodiment, the HCV infection is genotype.
[0041] In another embodiment, the patient is a treatment naive
patient.
[0042] In another embodiment, the patient is non-responsive to
interferon monotherapy.
[0043] In another embodiment, the patient is non-responsive to
combination therapy using ribaviron and an interferon.
[0044] In another aspect, the invention provides a method of
reducing HCV-RNA levels in a patient in need thereof, comprising
the step of administering to said patient a therapeutic combination
of the present invention.
[0045] In an embodiment of the invention, the HCV-RNA levels in the
patient are reduced to a less than detectable level.
[0046] In another aspect, the invention provides a pharmaceutical
regimen comprising administering to a patient in need thereof a
therapeutic combination of the present invention until the HCV-RNA
level in the patient is below a detectable level.
Formulations, Administrations, and Uses
[0047] If pharmaceutically acceptable salts of the compounds of
this invention are utilized in the therapeutic combinations, those
salts are preferably derived from inorganic or organic acids and
bases. Included among such acid salts are the following: acetate,
adipate, alginate, aspartate, benzoate, benzene sulfonate,
bisulfate, butyrate, citrate, camphorate, camphor sulfonate,
cyclopentane-propionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,
hydroiodide, 2 hydroxyethanesulfonate, lactate, maleate,
methanesulfonate, 2 naphthalenesulfonate, nicotinate, oxalate,
pamoate, pectinate, persulfate, 3 phenyl propionate, picrate,
pivalate, propionate, succinate, tartrate, thiocyanate, tosylate
and undecanoate. Base salts include ammonium salts, alkali metal
salts, such as sodium and potassium salts, alkaline earth metal
salts, such as calcium and magnesium salts, salts with organic
bases, such as dicyclohexylamine salts, N methyl D glucamine, and
salts with amino acids such as arginine, lysine, and so forth.
[0048] Also, the basic nitrogen containing groups may be
quaternized with such agents as lower alkyl halides, such as
methyl, ethyl, propyl, and butyl chloride, bromides and iodides;
dialkyl sulfates, such as dimethyl, diethyl, dibutyl and diamyl
sulfates, long chain halides such as decyl, lauryl, myristyl and
stearyl chlorides, bromides and iodides, aralkyl halides, such as
benzyl and phenethyl bromides and others. Water or oil soluble or
dispersible products are thereby obtained.
[0049] The compounds utilized in the compositions and methods of
this invention may also be modified by appending appropriate
functionalities to enhance selective biological properties. Such
modifications are known in the art and include those which increase
biological penetration into a given biological system (e.g., blood,
lymphatic system, central nervous system), increase oral
availability, increase solubility to allow administration by
injection, alter metabolism and alter rate of excretion.
[0050] Pharmaceutically acceptable carriers that may be used in
these compositions include, but are not limited to, ion exchangers,
alumina, aluminum stearate, lecithin, serum proteins, such as human
serum albumin, buffer substances such as phosphates, glycine,
sorbic acid, potassium sorbate, partial glyceride mixtures of
saturated vegetable fatty acids, water, salts or electrolytes, such
as protamine sulfate, disodium hydrogen phosphate, potassium
hydrogen phosphate, sodium chloride, zinc salts, colloidal silica,
magnesium trisilicate, polyvinyl pyrrolidone, cellulose based
substances, polyethylene glycol, sodium carboxymethylcellulose,
polyacrylates, waxes, polyethylene polyoxypropylene block polymers,
polyethylene glycol and wool fat.
[0051] The therapeutic combinations of this invention are
formulated for pharmaceutical administration to a mammal. In one
embodiment said mammal is a human being.
[0052] Such pharmaceutical compositions of the present invention
may be administered orally, parenterally, by inhalation spray,
topically, rectally, nasally, buccally, vaginally or via an
implanted reservoir. The term "parenteral" as used herein includes
subcutaneous, intravenous, intramuscular, intra articular, intra
synovial, intrasternal, intrathecal, intrahepatic, intralesional
and intracranial injection or infusion techniques. Preferably, the
compositions are administered orally or intravenously.
[0053] Sterile injectable forms of the compositions of this
invention may be aqueous or oleaginous suspension. These
suspensions may be formulated according to techniques known in the
art using suitable dispersing or wetting agents and suspending
agents. The sterile injectable preparation may also be a sterile
injectable solution or suspension in a non toxic parenterally
acceptable diluent or solvent, for example as a solution in
1,3-butanediol. Among the acceptable vehicles and solvents that may
be employed are water, Ringer's solution and isotonic sodium
chloride solution. In addition, sterile, fixed oils are
conventionally employed as a solvent or suspending medium. For this
purpose, any bland fixed oil may be employed including synthetic
mono- or diglycerides. Fatty acids, such as oleic acid and its
glyceride derivatives are useful in the preparation of injectables,
as are natural pharmaceutically-acceptable oils, such as olive oil
or castor oil, especially in their polyoxyethylated versions. These
oil solutions or suspensions may also contain a long-chain alcohol
diluent or dispersant, such as carboxymethyl cellulose or similar
dispersing agents which are commonly used in the formulation of
pharmaceutically acceptable dosage forms including emulsions and
suspensions. Other commonly used surfactants, such as Tweens, Spans
and other emulsifying agents or bioavailability enhancers which are
commonly used in the manufacture of pharmaceutically acceptable
solid, liquid, or other dosage forms may also be used for the
purposes of formulation.
[0054] In one embodiment, dosage levels of between about 0.01 and
about 100 mg/kg body weight per day of VX-950 are useful in a
combination therapy for the prevention and treatment of antiviral,
particularly anti-HCV mediated disease. In another embodiment,
dosage levels of between about 0.5 and about 75 mg/kg body weight
per day of VX-950 are useful in a combination therapy for the
prevention and treatment of antiviral, particularly anti-HCV
mediated disease. Typically, the pharmaceutical compositions of
this invention will be administered from about 1 to about 5 times
per day or alternatively, as a continuous infusion. Such
administration can be used as a chronic or acute therapy. The
amount of active ingredient that may be combined with the carrier
materials to produce a single dosage form will vary depending upon
the host treated and the particular mode of administration. A
typical preparation will contain from about 5% to about 95% active
compound (w/w). In one embodiment, such preparations contain from
about 20% to about 80% active compound.
[0055] In one embodiment, dosage levels of between about 0.01 and
about 100 mg/kg body weight per day of the polymerase inhibitor
compounds described herein are useful in a combination for the
prevention and treatment of antiviral, particularly anti-HCV
mediated disease. In another embodiment, dosage levels of between
about 0.5 and about 75 mg/kg body weight per day of the polymerase
inhibitor compounds described herein are useful in a combination
therapy for the prevention and treatment of antiviral, particularly
anti-HCV mediated disease. Typically, the pharmaceutical
compositions of this invention will be administered from about 1 to
about 5 times per day or alternatively, as a continuous infusion.
Such administration can be used as a chronic or acute therapy. The
amount of active ingredient that may be combined with the carrier
materials to produce a single dosage form will vary depending upon
the host treated and the particular mode of administration. A
typical preparation will contain from about 5% to about 95% active
compound (w/w). In one embodiment, such preparations contain from
about 20% to about 80% active compound.
[0056] The pharmaceutical compositions of this invention may be
orally administered in any orally acceptable dosage form including,
but not limited to, capsules, tablets, aqueous suspensions or
solutions. In the case of tablets for oral use, carriers that are
commonly used include lactose and corn starch. Lubricating agents,
such as magnesium stearate, are also typically added. For oral
administration in a capsule form, useful diluents include lactose
and dried cornstarch. When aqueous suspensions are required for
oral use, the active ingredient is combined with emulsifying and
suspending agents. If desired, certain sweetening, flavoring or
coloring agents may also be added.
[0057] Alternatively, the pharmaceutical compositions of this
invention may be administered in the form of suppositories for
rectal administration. These may be prepared by mixing the agent
with a suitable non-irritating excipient which is solid at room
temperature but liquid at rectal temperature and therefore will
melt in the rectum to release the drug. Such materials include
cocoa butter, beeswax and polyethylene glycols.
[0058] The pharmaceutical compositions of this invention may also
be administered topically, especially when the target of treatment
includes areas or organs readily accessible by topical application,
including diseases of the eye, the skin, or the lower intestinal
tract. Suitable topical formulations are readily prepared for each
of these areas or organs.
[0059] Topical application for the lower intestinal tract may be
effected in a rectal suppository formulation (see above) or in a
suitable enema formulation. Topically transdermal patches may also
be used.
[0060] For topical applications, the pharmaceutical compositions
may be formulated in a suitable ointment containing the active
component suspended or dissolved in one or more carriers. Carriers
for topical administration of the compounds of this invention
include, but are not limited to, mineral oil, liquid petrolatum,
white petrolatum, propylene glycol, polyoxyethylene,
polyoxypropylene compound, emulsifying wax and water.
Alternatively, the pharmaceutical compositions may be formulated in
a suitable lotion or cream containing the active components
suspended or dissolved in one or more pharmaceutically acceptable
carriers. Suitable carriers include, but are not limited to,
mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters
wax, cetearyl alcohol, 2 octyldodecanol, benzyl alcohol and
water.
[0061] For ophthalmic use, the pharmaceutical compositions may be
formulated as micronized suspensions in isotonic, pH adjusted
sterile saline, or, preferably, as solutions in isotonic, pH
adjusted sterile saline, either with our without a preservative
such as benzylalkonium chloride. Alternatively, for ophthalmic
uses, the pharmaceutical compositions may be formulated in an
ointment such as petrolatum.
[0062] The pharmaceutical compositions of this invention may also
be administered by nasal aerosol or inhalation. Such compositions
are prepared according to techniques well known in the art of
pharmaceutical formulation and may be prepared as solutions in
saline, employing benzyl alcohol or other suitable preservatives,
absorption promoters to enhance bioavailability, fluorocarbons,
and/or other conventional solubilizing or dispersing agents.
[0063] The therapeutic combination of this invention may
additionally comprise another anti-viral agent, preferably an
anti-HCV agent. Such anti-viral agents include, but are not limited
to, immunomodulatory agents, such as alpha, beta, and
gamma-interferons, pegylated derivatized interferon-alpha
compounds, and thymosin; other anti-viral agents, such as
ribavirin, amantadine, and telbivudine; other inhibitors of
hepatitis C proteases (NS2-NS3 inhibitors and NS3-NS4A inhibitors);
inhibitors of other targets in the HCV life cycle, including
helicase and other polymerase inhibitors; inhibitors of internal
ribosome entry; broad-spectrum viral inhibitors, such as IMPDH
inhibitors (e.g., compounds of U.S. Pat. Nos. 5,807,876, 6,498,178,
6,344,465, 6,054,472, WO 97/40028, WO 98/40381, WO 00/56331, and
mycophenolic acid and derivatives thereof, and including, but not
limited to VX-497, VX-148, and/or VX-944); or combinations of any
of the above. See also W. Markland et al., Antimicrobial &
Antiviral Chemotherapy, 44, p. 859 (2000) and U.S. Pat. No.
6,541,496.
##STR00009##
[0064] According to another aspect, the present invention provides
a method of treating HCV infection or alleviating one or more
symptoms thereof, in a patient, comprising administering a
composition comprising a polymerase inhibitor of Formula I, II or
III. In one embodiment, the patient is infected by an HCV variant.
In another embodiment, the composition further comprises
VX-950.
[0065] According to another aspect, the present invention provides
a method of treating HCV infection or alleviating one or more
symptoms thereof, in a patient, comprising administering a
composition comprising a polymerase inhibitor of Formula I, II, or
III, and VX-950. In one embodiment, the patient is infected by an
HCV variant.
[0066] According to another aspect, the present invention provides
a method of eliminating or reducing HCV infection in a cell,
comprising administering a composition comprising a polymerase
inhibitor of Formula I, II or III. In one embodiment, the cell
contains an HCV variant. In another embodiment, the composition
further comprises VX-950.
[0067] According to another aspect, the present invention provides
a method of eliminating or reducing HCV infection in a cell,
comprising administering a composition comprising a polymerase
inhibitor of Formula I, II or III and VX-950. In one embodiment,
the cell contains an HCV variant.
[0068] The following definitions are used herein (with trademarks
referring to products available as of this application's filing
date):
[0069] "Peg-Intron" means PEG-INTRON.RTM., peginteferon alfa-2b,
available from Schering Corporation, Kenilworth, N.J.;
[0070] "Intron" means INTRON-A.RTM., interferon alfa-2b available
from Schering Corporation, Kenilworth, N.J.;
[0071] "ribavirin" means ribavirin
1-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, available
from ICN Pharmaceuticals, Inc., Costa Mesa, Calif.; described in
the Merck Index, entry 8365, Twelfth Edition; also available as
REBETROL.RTM. from Schering Corporation, Kenilworth, N.J., or as
COPEGASUS.RTM. from Hoffmann-La Roche, Nutley, N.J.;
[0072] "Pegasys" means PEGASYS.RTM., peginterferon alfa-2a
available Hoffmann-La Roche, Nutley, N.J.;
[0073] "Roferon" mean ROFERON.RTM., recombinant interferon alfa-2a
available from Hoffmann-La Roche, Nutley, N.J.;
[0074] "Berefor" means BEREFOR.RTM., interferon alfa 2 available
from Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield,
Conn.;
[0075] SUMIFERON.RTM., a purified blend of natural alpha
interferons such as Sumiferon available from Sumitomo, Japan;
[0076] WELLFERON.RTM., interferon alpha n1 available from Glaxo
Wellcome LTd., Great Britain; and
[0077] ALFERON.RTM., a mixture of natural alpha interferons made by
Interferon Sciences, and available from Purdue Frederick Co.,
CT.
[0078] The term "interferon" as used herein means a member of a
family of highly homologous species-specific proteins that inhibit
viral replication and cellular proliferation, and modulate immune
response, such as interferon alpha, interferon beta, or interferon
gamma. The Merck Index, entry 5015, Twelfth Edition.
[0079] The therapeutic combination of the present invention may
utilize natural alpha interferon 2a. Or, the therapeutic
combination of the present invention may utilize natural alpha
interferon 2b. The therapeutic combination of the present invention
may utilize recombinant alpha interferon 2a or 2b. Additionally,
the invention may utilize pegylated alpha interferon 2a or 2b.
Interferons suitable for the present invention include:
[0080] (a) INTRON-A.RTM. (interferon-alpha 2B, Schering
Plough),
[0081] (b) PEG-INTRON.RTM.,
[0082] (c) PEGASYS.RTM.,
[0083] (d) ROFERON.RTM.,
[0084] (e) BEREFOR.RTM.,
[0085] (f) SUMIFERON.RTM.,
[0086] (g) WELLFERON.RTM.,
[0087] (h) consensus alpha interferon available from Amgen, Inc.,
Newbury Park, Calif.,
[0088] (i) ALFERON.RTM.;
[0089] (j) VIRAFERON.RTM.;
[0090] (k) INFERGEN.RTM.; and
[0091] (l) ALBUFERON.TM..
[0092] As is recognized by skilled practitioners, protease and
polymerase inhibitors would be preferably administered orally.
Interferon is not typically administered orally. Nevertheless,
nothing herein limits the methods or combinations of this invention
to any specific dosage forms or regime. Thus, each component of a
combination according to this invention may be administered
separately, together, or in any combination thereof.
[0093] In one embodiment, the protease inhibitor and polymerase
inhibitor are administered in separate dosage forms. In one
embodiment, any additional agent is administered as part of a
single dosage form with the protease inhibitor or as a separate
dosage form. As this invention involves a combination of compounds,
the specific amounts of each compound may be dependent on the
specific amounts of each other compound in the combination. As
recognized by skilled practitioners, dosages of interferon are
typically measured in IU (e.g., about 4 million IU to about 12
million IU).
[0094] Accordingly, agents (whether acting as an immunomodulatory
agent or otherwise) that may be used in combination with a compound
of this invention include, but are not limited to, Albuferon.TM.
(albumin-Interferon alpha) available from Human Genome Sciences;
PEG-INTRON.RTM. (peginterferon alfa-2b, available from Schering
Corporation, Kenilworth, N.J.); INTRON-A.RTM., (interferon alfa-2b
available from Schering Corporation, Kenilworth, N.J.); ribavirin
1-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, available
from ICN Pharmaceuticals, Inc., Costa Mesa, Calif.; described in
the Merck Index, entry 8365, Twelfth Edition); REBETROL.RTM.
(Schering Corporation, Kenilworth, N.J.), COPEGUS.RTM. (Hoffmann-La
Roche, Nutley, N.J.); PEGASYS.RTM. (peginterferon alfa-2a available
Hoffmann-La Roche, Nutley, N.J.); ROFERON.RTM. (recombinant
interferon alfa-2a available from Hoffmann-La Roche, Nutley, N.J.);
BEREFOR.RTM. (interferon alfa 2 available from Boehringer Ingelheim
Pharmaceutical, Inc., Ridgefield, Conn.); SUMIFERON.RTM. (a
purified blend of natural alpha interferons such as Sumiferon
available from Sumitomo, Japan); WELLFERON.RTM. (interferon alpha
n1 available from Glaxo Wellcome Ltd., Great Britain); ALFERON.RTM.
(a mixture of natural alpha interferons made by Interferon
Sciences, and available from Purdue Frederick Co., CT);
alpha-interferon; natural alpha interferon 2a; natural alpha
interferon 2b; pegylated alpha interferon 2a or 2b; consensus alpha
interferon (Amgen, Inc., Newbury Park, Calif.); VIRAFERON.RTM.;
INFERGEN.RTM.; REBETRON.RTM. (Schering Plough, Interferon-alpha
2B+Ribavirin); pegylated interferon alpha (Reddy, K. R. et al.
"Efficacy and Safety of Pegylated (40-kd) Interferon alpha-2a
Compared with Interferon alpha-2a in Noncirrhotic Patients with
Chronic Hepatitis C (Hepatology, 33, pp. 433-438 (2001); consensus
interferon (Kao J. H., et al., "Efficacy of Consensus Interferon in
the Treatment of Chronic Hepatitis," J. Gastroenterol. Hepatol.,
15, pp. 1418-1423 (2000); lymphoblastoid or "natural" interferon;
interferon tau (Clayette, P. et al., "IFN-tau, A New Interferon
Type I with Antiretroviral activity," Pathol. Biol., (Paris) 47,
pp. 553-559 (1999); interleukin-2 (Davis, G. L. et al., "Future
Options for the Management of Hepatitis C," Seminars in Liver
Disease, 19, pp. 103-112 (1999); Interleukin-6 (Davis et al.
"Future Options for the Management of Hepatitis C," Seminars in
Liver Disease, 19, pp. 103-112 (1999); interleukin-12 (Davis, G. L.
et al., "Future Options for the Management of Hepatitis C,"
Seminars in Liver Disease, 19, pp. 103-112 (1999); and compounds
that enhance the development of type 1 helper T cell response
(Davis et al., "Future Options for the Management of Hepatitis C,"
Seminars in Liver Disease, 19, pp. 103-112 (1999)). Also included
are compounds that stimulate the synthesis of interferon in cells
(Tazulakhova, E. B. et al., "Russian Experience in Screening,
analysis, and Clinical Application of Novel Interferon Inducers,"
J. Interferon Cytokine Res., 21 pp. 65-73) including, but are not
limited to, double stranded RNA, alone or in combination with
tobramycin, and Imiquimod (3M Pharmaceuticals; Sauder, D. N.
"Immunomodulatory and Pharmacologic Properties of Imiquimod," J.
Am. Acad. Dermatol., 43 pp. S6-11 (2000)).
[0095] Compounds that stimulate the synthesis of interferon in
cells (Tazulakhova, E. B. et al., "Russian Experience in Screening,
analysis, and Clinical Application of Novel Interferon Inducers,"
J. Interferon Cytokine Res., 21, pp. 65-73) include, but are not
limited to, double stranded RNA, alone or in combination with
tobramycin, and Imiquimod (3M Pharmaceuticals; Sauder, D. N.
"Immunomodulatory and Pharmacologic Properties of Imiquimod," J.
Am. Acad. Dermatol., 43 pp. S6-11 (2000)).
[0096] Other non-immunomodulatory or immunomodulatory compounds may
be used in combination with a compound of this invention including,
but not limited to, those specified in WO 02/18369, which is
incorporated herein by reference (see, e.g., page 273, lines 9-22
and page 274, line 4 to page 276, line 11).
[0097] This invention may also involve administering a cytochrome
P450 monooxygenase inhibitor. CYP inhibitors may be useful in
increasing liver concentrations and/or increasing blood levels of
compounds that are inhibited by CYP.
[0098] If an embodiment of this invention involves a CYP inhibitor,
any CYP inhibitor that improves the pharmacokinetics of the
relevant NS3/4A protease may be used in a method of this invention.
These CYP inhibitors include, but are not limited to, ritonavir (WO
94/14436), ketoconazole, troleandomycin, 4-methyl pyrazole,
cyclosporin, clomethiazole, cimetidine, itraconazole, fluconazole,
miconazole, fluvoxamine, fluoxetine, nefazodone, sertraline,
indinavir, nelfinavir, amprenavir, fosamprenavir, saquinavir,
lopinavir, delavirdine, erythromycin, VX-944, and VX-497. Preferred
CYP inhibitors include ritonavir, ketoconazole, troleandomycin,
4-methyl pyrazole, cyclosporin, and clomethiazole. For preferred
dosage forms of ritonavir, see U.S. Pat. No. 6,037, 157, and the
documents cited therein: U.S. Pat. No. 5,484,801, U.S. patent
Application Ser. No. 08/402,690, and International Applications WO
95/07696 and WO 95/09614).
[0099] Methods for measuring the ability of a compound to inhibit
cytochrome P450 monooxygenase activity are known (see U.S. Pat. No.
6,037,157, and Yun et al., Drug Metabolism & Disposition, 21,
pp. 403-407 (1993).
[0100] Upon improvement of a patient's condition, a maintenance
dose of a compound, composition or combination of this invention
may be administered, if necessary. Subsequently, the dosage or
frequency of administration, or both, may be reduced, as a function
of the symptoms, to a level at which the improved condition is
retained when the symptoms have been alleviated to the desired
level, treatment should cease. Patients may, however, require
intermittent treatment on a long-term basis upon any recurrence of
disease symptoms.
[0101] It should also be understood that a specific dosage and
treatment regimen for any particular patient will depend upon a
variety of factors, including the activity of the specific compound
employed, the age, body weight, general health, sex, diet, time of
administration, rate of excretion, drug combination, and the
judgment of the treating physician and the severity of the
particular disease being treated. The amount of active ingredients
will also depend upon the particular described compound and the
presence or absence and the nature of the additional anti-viral
agent in the composition.
[0102] The invention provides a method for treating a patient
infected with a virus characterized by a virally encoded serine
protease that is necessary for the life cycle of the virus by
administering to said patient a pharmaceutically acceptable
composition of this invention. In one embodiment, the methods of
this invention are used to treat a patient suffering from an HCV
infection. Such treatment may completely eradicate the viral
infection or reduce the severity thereof. In another embodiment,
the patient is a human being.
[0103] The methods of this invention may additionally comprise the
step of administering to said patient an anti-viral agent,
preferably an anti-HCV agent. Such anti-viral agents include, but
are not limited to, immunomodulatory agents, such as alpha-, beta-,
and gamma-interferons, pegylated derivatized interferon-.alpha.
compounds, and thymosin; other anti-viral agents, such as
ribavirin, amantadine, and telbivudine; other inhibitors of
hepatitis C proteases (NS2-NS3 inhibitors and NS3-NS4A inhibitors);
inhibitors of other targets in the HCV life cycle, including but
not limited to helicase and other polymerase inhibitors; inhibitors
of internal ribosome entry; broad-spectrum viral inhibitors, such
as IMPDH inhibitors (e.g., VX-497 and other IMPDH inhibitors
disclosed in U.S. Pat. Nos. 5,807,876 and 6,498,178, mycophenolic
acid and derivatives thereof); inhibitors of cytochrome P-450, such
as ritonavir, or combinations of any of the above.
[0104] Such additional agent may be administered to said patient as
part of a single dosage form comprising both a compound of this
invention and an additional anti-viral agent. Alternatively, the
additional agent may be administered separately from the compound
of this invention, as part of a multiple dosage form, wherein said
additional agent is administered prior to, together with or
following a composition comprising a compound of this
invention.
[0105] The present invention may also be utilized as a method of
pre-treating a biological substance intended for administration to
a patient comprising the step of contacting said biological
substance with a pharmaceutically acceptable composition comprising
a compound of this invention. Such biological substances include,
but are not limited to, blood and components thereof such as
plasma, platelets, subpopulations of blood cells and the like;
organs such as kidney, liver, heart, lung, etc; sperm and ova; bone
marrow and components thereof, and other fluids to be infused into
a patient such as saline, dextrose, etc.
[0106] The invention may also be utilized as a method of treating
materials that may potentially come into contact with a virus
characterized by a virally encoded serine protease necessary for
its life cycle. This method comprises the step of contacting said
material with a compound according to the invention. Such materials
include, but are not limited to, surgical instruments and garments
(e.g. clothes, gloves, aprons, gowns, masks, eyeglasses, footwear,
etc.); laboratory instruments and garments (e.g. clothes, gloves,
aprons, gowns, masks, eyeglasses, footwear, etc.); blood collection
apparatuses and materials; and invasive devices, such as, for
example, shunts and stents.
[0107] All references cited within this document are incorporated
herein by reference.
IV. PREPARATIONS AND EXAMPLES
[0108] In order that the invention described herein may be more
fully understood, the following examples are set forth. It should
be understood that these examples are for illustrative purposes
only and are not to be construed as limiting this invention in any
manner.
VI. Assays for Detecting and Measuring Inhibition Properties of
Compounds
[0109] A. HCV Replicon Cells
[0110] Huh-7 cells were propagated in Dulbecco's modified Eagle's
medium (DMEM, JRH Biosciences, Lenexa, Kans.) supplemented with 10%
heat-inactivated FBS (fetal bovine serum), 2 mM L-glutamine, and
nonessential amino acids (JRH). The cells were transfected with an
in vitro transcribed HCV replicon RNA identical to replicon
I377neo/NS3-3'/wt as described by Lohmann et al. (1999). Stable
cell clones were selected and maintained in the presence of 250
.mu.g/mL G418 (Invitrogen, Carlsbad, Calif.). One of the clones,
24-2, was used as the wild-type in the subsequent HCV replicon
assays for combination studies. The HCV NS3 protease variant
replicons were constructed in the background of the mADE wild-type
replicon as described previously [Chao Lin et al., "In Vitro
Resistance Studies of Hepatitis C Virus Serine Protease Inhibitors,
VX-950 and BILN 2061: Structural Analysis Indicates Different
Resistance Mechanisms," Journal of Biological Chemistry, 279(17);
pp. 17508-17514 (2004)]. The replicon cells were propagated in DMEM
supplemented with 10% FBS, 2 mM L-glutamine, nonessential amino
acids, and 250 .mu.g/mL G418. The cells were split twice per week
in fresh media upon reaching confluence. There are approximately
200-300 copies of HCV RNA per 24-2 replicon cell.
[0111] HCV replicon RNA from cells was measured using the
Quantigene Discover XL kit (Panomics Inc., Fremont Calif.) as per
the manufacturer's instructions. Briefly, compound-treated replicon
cells were lysed and immobilized on to capture plates using HCV
specific oligonucleotides (designed based on the 5' UTR region of
HCV 1b genome sequence AJ238799 in the GenBank database) over night
and the relative amounts of captured RNA was measured using
oligonucleotide probe sets as per the manufacturer's
instructions.
[0112] B. 2-Day HCV Replicon IC.sub.50 Assay
[0113] On the day prior to the assay, 10,000 replicon cells were
plated per well of a 96-well plate and allowed to attach and grow
overnight in DMEM (Invitrogen, Carlsbad, Calif.) supplemented with
10% heat-inactivated FBS (JRH Biosciences, Lenexa, Kans.), 2 mM
L-glutamine (Invitrogen), nonessential amino acids (Invitrogen) and
250 .mu.g/ml G418 (Invitrogen). Next day, the medium was removed
and antiviral agents that were serially diluted in DMEM containing
2% FBS and 0.5% DMSO (Sigma Chemical Co., St. Louis, Mo.) without
G418, was added. The replicon cells were incubated with the
antiviral agents for 48 h. HCV replicon RNA from cells was measured
using the Quantigene Discover XL kit (Panomics Inc., Fremont
Calif.) as per the manufacturer's instructions. Briefly,
compound-treated replicon cells were lysed and immobilized onto
capture plates using HCV specific oligonucleotides overnight and
the relative amounts of captured RNA was measured using
oligonucleotide probe sets as per the manufacturer's instructions.
Unless indicated otherwise, each data point represents the average
of three replicates. The IC.sub.50 is the concentration of the
compound at which the HCV replicon RNA level in cells is reduced by
50% as compared to the untreated replicon cell controls. To monitor
the effect of compounds on cell proliferation or cell viability,
replicon cells were treated with serially diluted compounds for 48
h, after which cell viability was determined using a CellTiter Glo
assay (Promega, Madison, Wis.). Each CC.sub.50 is derived from at
least two replicates and is the concentration of the compound at
which the number of viable cells is reduced by 50% as compared to
untreated cell controls. The IC.sub.50 and CC.sub.50 was determined
using 4-parameter curve fitting in the SoftMax Pro program
(Molecular Devices, Sunnyvale, Calif.).
[0114] C. Synergy and Antagonism Analysis
[0115] The effects of drug-drug combinations were evaluated using
the Bliss independence model [W. R. Greco et al., "The search for
synergy: a critical review from a response surface perspective,"
Pharmacol. Rev., 47, pp. 331-385 (1995)]. The experimental data
were analyzed by using MacSynergy, a three-dimensional analytical
method developed by Prichard and Shipman [M. N. Prichard and C.
Shipman, Jr., "A three-dimensional model to analyze drug-drug
interactions," Antivir. Res., 14, pp. 181-205 (1990)]. In this
model, the theoretical additive effect is calculated from the
dose-response curves of individual compounds by the equation
Z=X+Y(1-X), where X and Y represent the inhibition produced by drug
1 alone and drug 2 alone, respectively, and Z represents the effect
produced by the combination of drug 1 and drug 2. The theoretical
additive surface is subtracted from the actual experimental
surface, resulting in a surface that would appear as a horizontal
plane at 0% inhibition if the combination were merely additive. Any
peak above this plane would indicate synergy, whereas any
depression below it would indicate antagonism. The 95% confidence
intervals for the experimental dose-response surface are used to
evaluate the data statistically. The volume of the peak or
depression is calculated to quantify the overall synergy or
antagonism produced.
[0116] Using the assays above, therapeutic combinations of the
present invention are determined to be useful HCV replication
inhibitors.
Example 1
[0117] Combinations of VX-950 and polymerase inhibitors were tested
in a checkerboard format in a 2-day Replicon assay with bDNA
quantitation. The results are shown in Table 6. The combination of
VX-950 and the polymerase inhibitor of formula I resulted in an
additive to modestly synergistic effect. The combination of VX-950
and the polymerase inhibitor of formula II resulted in an additive
to modestly synergistic effect. The combination of VX-950 and the
polymerase inhibitor of formula III resulted in an additive
effect.
TABLE-US-00001 TABLE 6 2-day Replicon Assay with bDNA quantitation
Polymerase Protease Polymerase Checkerboard VX-950 Inhibitor Log
Inhibitor Inhibitor Combo Result IC.sub.50 IC.sub.50 Synergy Volume
VX-950 Formula I Additive/Modest 0.42 .mu.M 7.21 .mu.M 21.95 3.15
Synergy VX-950 Formula II Additive/Modest 0.34 .mu.M 1.04 .mu.M
45.13 6.48 Synergy VX-950 Formula III Additive 0.31 .mu.M 0.70
.mu.M 0.37 0.05
Example 2
[0118] The sensitivity of NS3 protease variants to ribavirin and
interferon was assessed in a 2-Day HCV Replicon IC.sub.50 Assay.
The variants are as follows: at position 36, the wild-type valine
was mutated to an alanine (V36A) or a methionine (V36M), the
wild-type threonine at position 54 was mutated to an alanine
(T54A), the wild-type arginine at position 155 was mutated to
lysine (R155K), the wild-type arginine at position 155 was mutated
to threonine (R155T) and the wild-type arginine at position 155 was
mutated to methionine (R155M). As shown in Table 7, the results
indicate the sensitivity of the variants to ribavirin and
interferon. The fold change is the ratio of NS3 protease mutant
IC50 to that of the Wild type IC50 for the same inhibitor. The
sensitivity of replicons with the wild-type alanine at position 156
mutated to threonine (A156T) and the wild-type alanine at position
156 mutated to valine (A156V) is comparable to the wild-type (Lin,
et al., 2005, supra).
TABLE-US-00002 TABLE 7 2-Day HCV Replicon IC.sub.50 Assay of NS3
variants and ribavirin or interferon. Replicons Other Inhibitors
Protease domain IFN-.alpha. (units/ml) Ribavirin (.mu.M) Con1 Avg.
IC.sub.50 Fold Avg. IC.sub.50 Fold Seq. Changes (uM) Change (uM)
Change Wild Type -- 11.6 .+-. 1.1 1.0 57.8 .+-. 17.6 1.0 (mADE) Val
36 V36M 11.3 .+-. 5.9 1.0 32.9 .+-. 17.8 0.6 V36A 10.3 .+-. 6.0 0.9
43.1 .+-. 21.3 0.7 Arg 155 R155K 15.2 .+-. 12.3 1.3 37.2 .+-. 17
0.6 R155T 4.8 .+-. 3.3 0.4 32.4 .+-. 17.7 0.6 R155M 4.9 .+-. 1.0
0.4 38.9 .+-. 4.7 0.7 Val 36/Arg V36M- 10.1 .+-. 5.9 0.9 40.6 .+-.
6.1 0.7 155 R155K V36M- 3.1 .+-. 0.2 0.3 36.4 .+-. 1.3 0.6 R155T
V36A- 6.8 .+-. 0.5 0.6 35.8 .+-. 2.2 0.6 R155K V36A- 3.9 .+-. 2.1
0.3 41.7 .+-. 21.6 0.7 R155T Thr 54 T54A 3.9 .+-. 0.5 0.3 21.7 .+-.
11.1 0.4
Example 3
[0119] The sensitivity of NS3 protease variants to VX-950
[Sarrazin, et al., "Dynamic Hepatitis C Virus Genotypic and
Phenotypic Changes in Patients Treated With the Protease Inhibitor
Telaprevir," Gastroenterology 132, pp. 1767-1777 (2007), which is
incorporated herein by reference in its entirety] and the
polymerase inhibitors of Formula I, II and III was assessed in a
2-Day HCV Replicon IC.sub.50 Assay. The variants are as follows: at
position 36, the wild-type valine was mutated to an alanine (V36A)
or a methionine (V36M), the wild-type threonine at position 54 was
mutated to an alanine (T54A), the wild-type arginine at position
155 was mutated to lysine (R155K), the wild-type alanine at
position 156 was mutated to threonine (A156T) and the wild-type
alanine at position 156 was mutated to valine (A156V). The variant,
V36M+R155K, having two mutations was also tested. As shown in Table
8, the results indicate the sensitivity of the variants to VX-950
and to Formula I, II and III. The fold change is the ratio of NS3
protease mutant IC50 to that of the Wild type IC50 for the same
inhibitor.
TABLE-US-00003 TABLE 8 2-Day HCV Replicon IC.sub.50 Assay of NS3
variants and VX-950, Formula I, II and III Replicon HCV Protease
Inhibitor HCV Polymerase Inhibitors Protease VX-950 Formula I
Formula II Formula III Domain Avg. IC.sub.50 .+-. Fold Avg.
IC.sub.50 .+-. Fold Avg. IC50 .+-. Fold Avg. IC.sub.50 .+-. Fold
Changes STDEV Change STDEV Change STDEV Change STDEV Change Wild
Type 0.46 .+-. 0.101 1 .+-. 0.2 1.343 .+-. 0.473 1.0 0.347 .+-.
0.108 1.0 0.313 .+-. 0.032 1.0 V36M 3.38 .+-. 0.775 7.3 .+-. 1.7
1.589 .+-. 0.361 1.2 0.36 .+-. 0.164 1.0 0.227 .+-. 0.064 0.7 V36A
3.567 .+-. 1.05 7.7 .+-. 2.3 2.148 .+-. 0.874 1.6 0.571 .+-. 0.093
1.6 0.369 .+-. 0.066 1.2 R155K 3.587 .+-. 0.283 7.8 .+-. 0.6 2.371
.+-. 0.678 1.8 0.45 .+-. 0.023 1.3 0.39 .+-. 0.065 1.2 V36M + R155K
30.61 .+-. 0.637 .gtoreq.65.2 2.716 .+-. 1.384 2.0 0.578 .+-. 0.331
1.7 0.419 .+-. 0.197 1.3 T54A 3.043 .+-. 0.819 6.6 .+-. 1.8 1.976
.+-. 0.388 1.5 0.564 .+-. 0.111 1.6 0.337 .+-. 0.045 1.1 A156T
>30* >65.2 1.27 .+-. 0.185 0.9 0.326 .+-. 0.003 0.9 0.306
.+-. 0.01 1.0 A156V >30* >65.2 1.313 .+-. 0.216 1.0 0.264
.+-. 0.045 0.8 0.297 .+-. 0.066 0.9
Other Embodiments
[0120] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages and modifications are
within the scope of the following claims.
* * * * *