HK1098357A - Combinations for hcv treatment - Google Patents

Description

Combination for HCV treatment

Technical Field

The present invention relates to the combination of a hepatitis c virus NS3/4A protease inhibitor and a cytochrome P450 monooxygenase inhibitor. The combination interferes with the life cycle of the hepatitis c virus and is therefore useful as an antiviral therapy. Thus, the combination may be used to treat or prevent hepatitis c infection in a patient. The invention also relates to compositions, kits and pharmaceutical packs comprising the combination. The invention also relates to methods of preparing such combinations, compositions, kits and packages.

Background

Hepatitis C Virus (HCV) infection is an urgent human medical problem to be solved. HCV is thought to be responsible for most non-A, non-B Hepatitis and has been estimated to have a 3% serum prevalence in the global population [ A. Alberti et al, "Natural History of Hepatitis C,")J. Hepatology，31.，(Suppl.1)，pp.17-24(1999)]. Nearly four million people may have been infected in The United States alone [ M.J. Alter et al, "The epidemic 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.Heatopology, 31., (Suppl.1), pp.88-91(1999)]。

Upon first exposure to HCV, only about 20% of infected individuals develop acute clinical hepatitis, while infections of others appear to resolve spontaneously. However, in almost 70% of cases, The virus establishes a Chronic infection lasting for decades [ S.Iward, "The Natural course of viral Hepatitis,")FEMS Microbiology Reviews，14，pp.201-204(1994)；D.Lavanchy，″Global Surveillance and Controlof Hepatitis C，″ J.Viral Hepatitis，6，pp.35-47(1999)]. This often leads to recurrent and progressive worsening liver inflammation, often leading to more severe disease states such as cirrhosis and hepatocellular Carcinoma [ m.c. kew, "hepatotis C and hepatocellular carcinosoma",FEMS Microbiology Reviews，14，pp.211-220(1994)；I.Saito et.al.，″Hepatitis C Virus Infectionis Associated with the Development of HepatocellularCarcinoma，″ Proc.Natl.Acad.Sci.USA，87，pp.6547-6549(1990)]. UnfortunatelyThat is, there is no generally effective treatment that can impair the progression of chronic HCV.

The HCV genome encodes a 3010-3033 amino acid polyprotein [ Q.L.Choo, et. al., "Genetic Organization and division of the Hepatitis CVirus.")Proc.Natl.Acad.Sci.USA88, pp.2451-2455 (1991); kato et al, "Molecular Cloning of the Human Hepatitis C viral genome From Japanese tissues 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 HepatitisC Virus Genome Isolated From Human Carriers，″ J.Virol.，65，pp.1105-1113(1991)]. The HCV Nonstructural (NS) proteins are postulated to provide the necessary catalytic mechanism for viral replication. NS proteins are derived from proteolytic cleavage of polyproteins [ R.Bartenschlager et al, "" Nonstructural Protein 3 of the hepatitis C viruses enzymes a spring-Type Protein requiring for cleavage at the NS3/4and NS4/5 junctins ""J.Virol.，67，pp.3835-3844(1993)；A.Grakoui et.al.，″Characterization of theHepatitis C Virus-Encoded Serine Proteinase：Determination ofProteinase-Dependent Polyprotein Cleavage Sites，″ J.Virol.，67，pp.2832-2843(1993)；A.Grakoui et.al.，″Expression andIdentification of Hepatitis C Virus Polyprotein CleavageProducts，″ J.Virol.，67，pp.1385-1395(1993)；L.Tomei et.al.，″NS3 is a serine protease required for processing of hepatitisC virus polyprotein，″ J.Virol.，67，pp.4017-4026(1993)]。

HCV NS protein 3(NS3) contains serine protease activity that contributes to the processing of most viral enzymes and is therefore considered essential for viral replication and infectivity. Mutations in the NS3 protease of yellow fever virus are known to reduce the infectivity of the virus [ Chambers, T.J.et.al., "infection of the N-terminal Domain of Nonstructural protein NS3 From Yellow Fever Virus is a Serine Protease Responsiblefor Site-Specific Cleavages in the Viral Polyprotein″， Proc. Natl.Acad.Sci.USA，87，pp.8898-8902(1990)]. The first 181 amino acids of NS3 (1027-1207 residues of the viral polyprotein) have been shown to contain the Serine protease domain of NS3 at all four downstream sites of the processing of the HCV polyprotein [ C.Lin et al, "Hepatitis C Virus NS3 spring protease: Trans-CleavageRequirements and Processing Kinetics ", J.Virol., 68, pp.8147-8157(1994)]。

There is currently no satisfactory anti-HCV agent or treatment. 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 viruses: an Overview of Current applications and ProgressDDT4, pp.518-29 (1999); mordapour et al, "Current and Evalling therapeutics for Heapatitis C,", et alEur.J.Gastroenterol.Hepatol.11, pp.1199-1202 (1999); janssen et al, "Suicide Associated with Alfa-interference therapy for Viral Hepatitis,"J.Hepatol.21, pp.241-243 (1994); renault et al, "Side Effects of AlphaInterferonSeminars in Liver Disease，9，pp.273-277.(1989)]Long-term remission was induced in only a fraction of cases (-25%) [ O.Weiland, "interference Therapy in viral Hepatitis C Virus Infection",FEMS Microbiol.Rev.，14，pp.279-288(1994)]. Recently pegylated forms of interferon (PEG-INTRON) have been introduced^®And PEGASYS^®) And ribavirin in combination therapy with pegylated interferon (REBETROL)^®) This resulted in only modest increases in remission rates and only partial reductions in side effects. Moreover, the prospects for effective anti-HCV vaccines remain uncertain.

The HCV NS3 serine protease and its related cofactor NS4A contribute to the processing of all viral enzymes and are therefore considered essential for viral replication. This processing appears to be similar to that performed by the human immunodeficiency virus aspartyl protease, which is also involved in the processing of HIV protease inhibitors by viral enzymes, which inhibit the processing of viral proteins and are potent human antiviral agents, suggesting that interference with this phase of the viral life cycle may result in a therapeutically active agent. Therefore, it is an attractive target for drug development.

Some drugs are known to be metabolized by cytochrome P450 enzymes. Such metabolism often results in drugs with undesirable pharmacokinetic profiles (e.g., reduced blood levels, reduced half-life). Conversely, inhibition of drug metabolism may result in an improvement in the pharmacokinetic profile of the drug. This approach has been reported as a method of improving the pharmacokinetics of certain drugs (see, e.g., U.S. Pat. No. 6,037,157; D.E. Kempf et alAntimicrob.Agents Chemother.41, pp.654-660 (1997)). However, no method for improving the pharmacokinetics of the hepatitis C virus NS3/4A protease inhibitor has been reported.

Thus, there is a need for compositions and therapeutic combinations that improve the pharmacokinetics of hepatitis c NS3/4A viral protease inhibitors. Such compositions, combinations and methods should be useful for anti-HCV therapy.

Disclosure of Invention

The present invention relates to the combination of a hepatitis C virus NS3/4A protease inhibitor and a cytochrome P450 monooxygenase inhibitor.

The present invention also relates to a method of treating an HCV infection in a patient by administering a combination according to the present invention.

The present invention provides compositions, kits and pharmaceutical packages comprising a hepatitis C virus NS3/4A protease inhibitor and a cytochrome P450 monooxygenase inhibitor. The invention also provides methods of making such combinations, kits and packages.

Detailed description of the invention

The present invention provides methods for improving the pharmacokinetics of hepatitis C NS3/4A protease inhibitors. The advantage of improving the pharmacokinetics of drugs is recognized in the art (US 2004/0091527; US 2004/0152625; US 2004/0091527). Such an improvement may result in an increase in blood levels of the drug. More importantly with respect to HCV therapy, the improved effect may result in an increased concentration of protease inhibitors in the liver.

In one embodiment, the invention provides a method of improving the pharmacokinetics of a hepatitis c NS3/4A protease inhibitor by co-administering the protease inhibitor in combination with a cytochrome P450 ("CYP") inhibitor. Applicants have demonstrated that hepatitis C NS3/4A protease inhibitors are metabolized by cytochrome P450 enzymes, more specifically the 3A4 isozyme. Applicants have also demonstrated that this metabolic effect is reduced in the presence of cytochrome P450 inhibitors. By combining the NS3/4A protease inhibitor and the CYP inhibitor, the present invention reduces the metabolic effects of the protease inhibitor. The pharmacokinetics of the protease inhibitor is thereby improved.

Accordingly, the present invention provides therapeutic combinations of a cytochrome P450 monooxygenase inhibitor ("CYP inhibitor") and a hepatitis C NS3/4A protease inhibitor. In one aspect, the invention relates to the co-administration of an NS3/4A protease inhibitor and a CYP inhibitor.

One embodiment of the present invention provides a method for increasing the bioavailability of a hepatitis C virus NS3/4A protease inhibitor in a patient comprising administering to the patient a hepatitis C NS3/4A viral protease inhibitor in combination with a cytochrome P450 monooxygenase inhibitor.

Another embodiment of the present invention provides a method of increasing blood levels or liver concentrations of a hepatitis C virus NS3/4A protease inhibitor in a patient comprising administering to the patient a hepatitis C NS3/4A viral protease inhibitor in combination with a cytochrome P450 monooxygenase inhibitor.

Another embodiment of the present invention provides a method of treating a patient infected with hepatitis C virus comprising administering to the patient a) an inhibitor of hepatitis C virus NS3/4A protease; and b) a cytochrome P450 monooxygenase inhibitor.

The cytochrome P450 monooxygenase inhibitors useful in the present invention are expected to inhibit the metabolism of NS3/4A protease inhibitor compounds. Thus, the cytochrome P450 monooxygenase inhibitor will be present in an amount effective to inhibit the metabolic effects of the protease inhibitor. Thus, the CYP inhibitor is administered in an amount such that: the bioavailability of the protease inhibitor is increased compared to the bioavailability in the absence of the CYP inhibitor.

In these embodiments, the inhibitor is preferably administered in a therapeutically effective amount. The compounds used in the present invention are expected to inhibit HCV by inhibiting the NS3/4A protease. Thus, the combination of CYP inhibitor and protease inhibitor is preferably co-administered in an amount sufficient to produce antiviral activity.

It will be appreciated that, as the present invention relates to a combination of compounds, the specific amount of each compound may depend on the specific amount of the other compound in the combination. For example, administration of the CYP inhibitor and the protease inhibitor in the methods of the invention results in an improvement in the pharmacokinetics of the protease inhibitor compared to the pharmacokinetics of the protease inhibitor administered in the absence of the CYP inhibitor. Thus, lower amounts of protease inhibitor in the presence of the CYP inhibitor will produce an equivalent in vivo effect than in the absence of the CYP inhibitor.

In the methods of the invention, the amount of CYP inhibitor administered is sufficient to improve the pharmacokinetics of the protease inhibitor compared to the pharmacokinetics of the protease inhibitor in the absence of the CYP inhibitor. In certain embodiments, the amount of CYP inhibitor administered is sufficient to increase blood levels of the protease inhibitor or increase liver concentrations of the protease inhibitor. Thus, it is advantageous in the methods of the invention to use a lower dose of protease inhibitor (relative to the administration of the protease inhibitor alone).

In addition to treating a patient infected with hepatitis C, the methods of the present invention may also be used to prevent infection of a patient with hepatitis C. Accordingly, one embodiment of the present invention provides a method of preventing hepatitis C virus infection in a patient comprising administering to the patient a) an inhibitor of hepatitis C virus NS3/4A protease; and b) a cytochrome P450 monooxygenase inhibitor.

As the skilled artisan will recognize, if the methods of the invention are used to prophylactically treat a patient, and the patient has been infected with hepatitis C virus, then the methods can treat the infection. Accordingly, one embodiment of the present invention provides a combination of a hepatitis C virus NS3/4A protease inhibitor and a cytochrome P450 monooxygenase inhibitor, wherein the combination of inhibitors is present in a therapeutically effective amount for treating or preventing a hepatitis C infection in a subject.

The methods of the present invention may employ any hepatitis C NS3/4A protease inhibitor. The ability of a compound to inhibit hepatitis c protease can be determined by methods known in the art and/or by the methods provided herein. Examples of such inhibitors include, but are not limited to, compounds identified as inhibitors in such assays and inhibitors in the following references: WOO3/087092, WO 03/006490, WO 03/064456, WO 03/064416, WO03/035060, WO 02/060926, WO 02/079234, WO 02/48116, WO 02/48157, WO 00/31129, WO 02/18369, WO 02/08256, WO 02/08244, WO 02/08198, WO 02/08187, WO 01/81325, WO 01/77113, WO 01/74768, WO 01/64678, WO 01/07407, WO 00/59929, WO 00/09588, WO 00/09543, WO 99/64442, WO 99/50230, WO 99/38888, WO 99/07734, WO 99/07733, WO 98/46630, WO 98/46630, WO 98/22496, WO 98/17679, WO 97/43310, US 6,018,020, US 5,990,276, US 5,866,684, US 20030008828, US 20020177725, US20020016442, US 20020016294, m.lllinas-Brunet et al,Bioorg. Med.Chem.Lett.8, pp.1713-18 (1998); w. han et al,Bioorg. Med.Chem.Lett.10, 711-13 (2000); r. Dunsdon et al, Bioorg. Med.Chem.Lett.10, pp.1571-79 (2000); rlinas-Brunet et al,Bioorg.Med.Chem.Lett.10, pp.2267-70 (2000); and s.laplane et al,Bioorg.Med.Chem.Lett.10, pp.2271-74(2000) (which are incorporated herein by reference as described below). In certain embodiments, the inhibitor is selected from the compounds of WO 03/087092, WO 02/18369, or WO 98/17679. In a specific embodiment, the inhibitor is VX-950.

VX-950 is a competitive, reversible peptidomimetic (peptidomimetic) NS3/4A protease inhibitor with steady state binding constant (ki)^*) At 3nM (WO 02/018369).

Thus, in addition to the NS3/4A protease inhibitor described above, the NS3/4A protease inhibitor used in the methods, processes, combinations, compositions, packages, and kits of the present invention is VX-950.

Preferred compounds for use in the present invention are those wherein the compound is sufficiently stable to permit manufacture and administration to a mammal using methods known in the art. Typically, such compounds are stable for at least one week at a temperature of 40 ℃ or less in the absence of moisture or other chemically reactive conditions.

VX-950 (and other compounds employed in accordance with the present invention) may contain one or more asymmetric carbon atoms and thus racemates may exist with racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. All such isomeric forms of these compounds are expressly included in the present invention. Each stereochemically yielding carbon may be of the R or S configuration. The D-and L-isomers of the N-propyl side chain of VX-950 are specifically included within the present invention.

Any CYP inhibitor that improves the pharmacokinetics of the relevant NS3/4A protease can be used in the methods of the invention. Such CYP inhibitors include, but are not limited to, ritonavir (WO94/14436), ketoconazole, oleandomycin, 4-methylpyrazole, cyclosporin, clomerthiazol, 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, oleandomycin, 4-methylpyrazole, cyclosporin, and chlormethiazole.

Methods for determining the ability of a compound to inhibit cytochrome P450 monooxygenase activity are known (see U.S. Pat. No. 6,037,157 and Yun, et alDrug Metabolism & DispositionVol.21, pp.403-407 (1993)). For example, the compound to be evaluated can be incubated with 0.1, 0.5, and 1.0mg protein/ml or other suitable concentrations of human liver microsomes (e.g., commercially available, pool-identified liver microsomes) in the presence of an NADPH-producing system for 0, 5, 10, 20, and 30 minutes or other suitable times. Control incubations can be performed in triplicate for 0 and 30 minutes in the absence of liver microsomes. The sample may be analyzed for the presence of compounds. Incubation conditions that produce a linear rate of metabolism of the compound will be used as a guide for further studies.

A typical experiment will determine the metabolic kinetics (K) of a compound_mAnd V_max). The rate of disappearance of the compound can be determined by analyzing the data according to Michaelis-Menten kinetics using Lineweaver-Burk, Eadie-Hofstee, or nonlinear regression analysis.

Metabolic inhibition experiments can then be performed. For example, the compound (at a concentration, ≦ K) may be administered in the presence or absence of a CYP inhibitor (e.g., ritonavir) under conditions as determined above_m) Incubate with pooled human liver microsomes. As will be appreciated, the control incubation should contain the same concentration of organic solvent as the incubation with the CYP inhibitor. The concentration of the compound in the sample can be quantified and the rate of disappearance of the parent compound can be determined, and the rate expressed as a controlPercentage of activity.

Methods for evaluating the effect of co-administration of NS3/4A protease inhibitor and a CYP inhibitor in a subject are also known (US 2004/0028755). Any such method can be used in the present invention to determine the effect of the combination on pharmacokinetics. A subject may then be selected that would benefit from treatment according to the present invention. In particular, subjects that metabolize NS3/4A protease inhibitor may be selected for treatment according to the present invention. Subjects that metabolize NS3/4A protease inhibitor extensively or at least to a greater extent than other subjects would be preferred subjects for treatment according to the invention.

The CYP inhibitor used in the present invention may be an inhibitor of only one isoenzyme or more than one isoenzyme. If a CYP inhibitor inhibits multiple isozymes, the inhibitor may nonetheless inhibit one isozyme more selectively than another isozyme. Any such CYP inhibitor may be used in the methods of the invention.

Accordingly, one embodiment of the present invention provides methods of administering a CYP3a4 inhibitor and an NS3/4A protease inhibitor. Another embodiment of the invention provides a method of administering an inhibitor of isoenzyme 3a4(CYP3a4), isoenzyme 2C19(CYP2C19), isoenzyme 2D6(CYP2D6), isoenzyme 1a2(CYP1a2), isoenzyme 2C9(CYP2C9), or isoenzyme 2E1(CYP2E 1). In embodiments where the protease inhibitor is VX-950 (or a stereoisomer thereof), the CYP inhibitor preferably inhibits CYP3A 4.

As will be appreciated, CYP3a4 activity is widely observed in humans. Thus, embodiments of the invention directed to inhibiting isoenzyme 3a4 are expected to be applicable to a wide range of patients.

The methods herein relate to the administration of a combination (co-administration) of a hepatitis C virus NS3/4A protease inhibitor and a cytochrome P450 monooxygenase inhibitor. Such administration may be referred to as co-administration. Co-administration includes administering each inhibitor in the same dosage form or in different dosage forms. When administered in different dosage forms, the inhibitors may be administered at different times, in any order.

Accordingly, the present invention provides methods of administering a CYP inhibitor in conjunction with a hepatitis c virus NS3/4A protease inhibitor, either in the same dosage form or in separate dosage forms.

If the CYP inhibitor and the protease inhibitor are administered in separate dosage forms, each inhibitor may be administered at approximately the same time. Alternatively, the CYP inhibitor can be administered at any time period before and after administration of the protease inhibitor. That is, the CYP inhibitor can be administered before, together with, or after the NS3/4A protease inhibitor. The time period of administration should be such that the CYP inhibitor affects the metabolism of the protease inhibitor. For example, if the protease inhibitor is administered first, the CYP inhibitor should be administered before the protease inhibitor is metabolized and/or excreted (e.g., within the half-life of the protease inhibitor).

The methods of the present invention may also involve the administration of another component comprising an additional agent selected from an immunomodulator, an antiviral, an HCV protease inhibitor, an inhibitor of another target in the HCV life cycle, a cytochrome P-450 inhibitor, or a combination thereof.

Thus, in another embodiment, the invention provides a method comprising administering an NS3/4A protease inhibitor, a CYP inhibitor, and another antiviral agent (preferably an anti-HCV agent). Such antiviral agents include, but are not limited to, immunomodulators, such as alpha-, beta-and gamma-interferons, pegylated interferon-alpha compounds and thymosins; other antiviral agents, such as ribavirin, amantadine, and telbivudine; other hepatitis c protease inhibitors (NS2-NS3 inhibitors and NS3-NS4A inhibitors); inhibitors of other targets in the HCV life cycle, including helicase, polymerase, and metalloprotease inhibitors; inhibitors of internal ribosome entry; broadspectrum 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, WO98/40381, WO 00/56331 and mycophenolic acid and derivatives thereof, including but not limited to VX-497, VX-148 and/or VX-944); or a combination of any of the above.

Other drugs (e.g., non-immunomodulatory or immunomodulatory compounds) that may be used in combination with the compounds of the invention include, but are not limited to, those specified in WO 02/18369, which is incorporated herein by reference (see, e.g., pages 9-22 and lines 274, 4 to 276, 11).

Other drugs include, but are not limited to, PEG-INTRON^®(Pegylated interferon alpha-2 b, available from Schering Corporation, Kenilworth, N.J.); INTRON-A^®(interferon alpha-2 b, available from Schering Corporation, Kenilworth, N.J.); ribavirin (1- β -D-ribofuranosyl-1H-1, 2, 4-triazole-3-amide, available from ICNPharmaceuticals, inc., Costa Mesa, CA, described in the Merck Index, entry 8365, 12 th edition); REBETROL^®(Schering Corporation，Kenilworth，NJ)，COPEGASUS^®(Hoffmann-La Roche，Nutley，NJ)，PEGASYS^®(pegylated interferon alpha-2a, available from Hoffmann-La Roche, Nutley, NJ); ROFERON^®(recombinant interferon alpha-2a, available from Hoffmann-La Roche, Nutley, NJ); BEREFOR^®(interferon alpha 2, available from Boehringer ingelheim pharmaceutical, inc., ridgfield, CT); sumiferon^®(purified blends of natural alpha interferons, such as Sumiferon, available from Sumitomo, Japan); WELLFERON^®(interferon α n1, available from Glaxo Wellcome ltd., Great Britain); ALFERON^®(a mixture of natural alpha interferons, manufactured by Interferon Sciences, available from Purdue Frederick co., CT); an alpha-interferon; natural alpha interferon 2 a; natural interferon-alpha 2 b; pegylated alpha interferon 2a or 2 b; synonymous (consensus) interferon alpha (Amgen, inc., Newbury Park, CA); viraferon^®；INFERGEN^®；REBETRON^®(Schering Plough, interferon-. alpha.2B + ribavirin); pegylated Interferon alpha (Reddy, K.R. et al, "efficiency and Safety of Pegylated (40-kd) Interferon alpha-2aComparedwith Interferon alpha-2a in Noncirrhotic Patients with ChronicHepatitis C”，( Hepatology33, pp.433-438 (2001)); synonymous interferons (Kao, J.H., et al, "Efficacy of Consensus Interferon approach of viral Hepatitis",J.Gastroenterol.Hepatol.15, pp.1418-1423 (2000); a lymphoblastoid or "native" 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， 19Pp.103-112 (1999); interleukin 6(Davis et al, "Future Options for the management of Hepatitis C.")Seminars in Liver Disease19, pp.103-112 (1999); interleukin 12(Davis, G.L. et al, "Future operation for the Management of Hepatitis C.")Seminars in Liver Disease19, pp.103-112 (1999); and compounds that enhance the formation of type 1 helper T cell responses (Davis et al, "Future Options for the Management of hepatitis C.")Seminars in Liver Disease19, pp.103-112 (1999)). Also included are compounds that stimulate Interferon synthesis in cells (Tazulakhova, E.B., et al, "Russian experiment in Screening, analysis, and clinical application of Novel interference indicatorsJ.Interferon Cytokine Res.21pp.65-73), including but not limited to double stranded RNA, alone or in combination with tobramycin, and imiquimod (3M Pharmaceuticals; sauder, d.n. "immunomodulation and pharmacological Properties of immiquodJ.Am.Acad.Dermatol.，43pp.S6-11(2000))。

As recognized by the skilled artisan, the protease inhibitor and CYP inhibitor will preferably be administered orally. Interferons are not usually administered orally. Nevertheless, the methods or combinations of the present invention are not limited herein to any particular dosage form or regimen. Thus, each of the components of the combination according to the invention may be administered separately, together or in any combination thereof. As recognized by the skilled artisan, the dose of interferon is typically quantified in IU (e.g., from about 4 million IU to about 12 million IU).

If the additional drug is selected from another CYP inhibitor, the method will therefore employ two or more CYP inhibitors. Each component may be administered in one or more dosage forms. Each dosage form can be administered to a patient in any order.

The NS3/4A protease inhibitor, the CYP inhibitor, and any additional drugs may be formulated in separate dosage forms. Alternatively, the NS3/4A protease inhibitor, the CYP inhibitor, and any additional drugs may be formulated together in any combination in order to reduce the number of times a dosage form is administered to a patient. For example, the NS3/4A protease inhibitor may be formulated in one dosage form, and the CYP inhibitor and additional drugs may be formulated together in another dosage form. Any of the divided dosage forms may be administered at the same time or at different times. It will be appreciated that the CYP inhibitor should be administered over a period of time such that the CYP inhibitor is enabled to reduce the metabolic effects of the NS3/4A protease inhibitor (or additional drug (s)).

Accordingly, another embodiment of the present invention provides a composition comprising an NS3/4A protease inhibitor or a pharmaceutically acceptable salt thereof and a CYP inhibitor or a pharmaceutically acceptable salt thereof. According to a preferred embodiment, the NS3/4A protease inhibitor is present in an amount effective to reduce viral load in the sample or patient, wherein the virus encodes the NS3/4A serine protease essential to the viral life cycle, and the composition further comprises a pharmaceutically acceptable carrier. Alternatively, the compositions of the present invention comprise an additional drug as described herein. Each component may be present in a separate composition, in a combined composition, or in a single composition.

If pharmaceutically acceptable salts of the compounds are employed in these compositions, these salts are preferably derived from inorganic or organic acids and bases. The acid salt includes: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. Basic 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 salt, N-methyl-D-glucamine salt; and salts of amino acids such as arginine, lysine, and the like.

Also, the basic nitrogen-containing groups may be quaternized with agents such as lower alkyl halides, e.g., methyl, ethyl, propyl, and butyl chlorides, 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 bromide and phenethyl bromide, among others. Thereby obtaining a water or oil soluble or dispersible product.

The compounds used in the compositions and methods of the present invention may also be modified by the addition of appropriate functionality to enhance selective biological properties. Such modifications are known in the art and include increasing the biological permeability into a given biological system (e.g., blood, lymphatic system, central nervous system), increasing oral bioavailability, increasing solubility for administration by injection, altering metabolism, and altering excretion rates.

Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., phosphates, glycine, sorbic acid, potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate), polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and lanolin.

According to a preferred embodiment, the compositions of the present invention are formulated for administration to mammals, and more particularly to humans.

Such pharmaceutical compositions of the invention (and compositions for use in the methods, combinations, kits and packages of the invention) may be administered orally, parenterally, sublingually, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the composition is administered orally or intravenously. More preferably, the composition is administered orally.

Sterile injectable forms of the compositions of the present invention may be aqueous or oleaginous suspensions. 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 carriers 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 oily solutions or suspensions may also contain a long chain alcohol diluent or dispersant, for example, carboxymethyl cellulose or similar dispersing agents, which are commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. For formulation purposes, 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.

In the compositions of the present invention, and in accordance with the present invention (i.e., the compositions used in the methods, kits, combinations or packages of the present invention), the NS3/4A protease inhibitor, the CYP inhibitor, and any optional additional drugs should be present at dosage levels between about 10 and 100%, more preferably between about 10 and 80%, of the usual dosages administered in monotherapy regimens.

The pharmaceutical compositions of the present invention may be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, pills, powders, granules, aqueous suspensions or solutions. In the case of tablets for oral use, commonly used carriers include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in capsule dosage form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral administration, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. Acceptable liquid dosage forms include emulsions, solutions, suspensions, syrups and elixirs.

Alternatively, the pharmaceutical compositions of the present invention may be administered rectally in the form of suppositories. They may be prepared by mixing the drug with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of the present invention may also be administered topically, particularly when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, skin or lower intestinal tract. Suitable topical formulations can be readily prepared according to each of these regions or organs.

Topical administration to the lower intestinal tract may be carried out as a rectal suppository (see above) or as a suitable enema. Topical transdermal patches may also be used.

For topical administration, the pharmaceutical compositions may be formulated in a suitable ointment containing the active ingredient 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 ingredient 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.

For ophthalmic purposes, 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, with or without preservatives such as benzalkonium chloride. Alternatively, for ophthalmic use, the pharmaceutical compositions may be formulated as ointments, such as petrolatum.

The pharmaceutical compositions according to the invention may also be administered by means of nasal aerosols or inhalants. 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.

The pharmaceutical compositions may also be administered in the form of liposomes, as is recognized in the art.

Preferred are pharmaceutical compositions according to the invention formulated for oral administration.

The dosage levels useful for the prevention and treatment of HCV-mediated diseases are from about 0.01 to about 100mg/kg body weight per day of NS3/4A protease inhibitor, preferably from about 0.5 to about 75mg/kg body weight per day. For CYP inhibitors, typical dosage levels are from about 0.001 to about 200mg/kg body weight per day. More typical dosage levels are from about 0.1 to about 50mg/kg or from about 1.1 to about 25mg/kg per day. Typically, the pharmaceutical composition according to the 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 form a single dosage form will vary depending upon the host treated and the particular mode of administration. Typical formulations will contain from about 5% to about 95% active compound (w/w). Preferably, such formulations contain from about 20% to about 80% active compound.

If necessary, maintenance doses of the compounds, compositions or combinations of the present invention can be administered as the condition of the patient improves. Subsequently, the dosage or frequency of administration, or both, can be reduced, as a function of the symptoms, to a level that allows the improved condition to be maintained, and treatment should be discontinued when the symptoms have been alleviated to the desired level. However, once any recurrence of disease symptoms has occurred, the patient may need to receive intermittent treatment on a long-term basis.

It will also be understood that the specific dose and 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, the judgment of the attending physician, and the severity of the particular disease undergoing therapy. The amount of active ingredient will also depend on the presence or absence and nature of the additional antiviral agent in the particular compound and composition.

For a preferred dosage form of ritonavir, see U.S. Pat. No. 6,037,157 and references cited therein: U.S. Pat. No. 5,484,801, U.S. patent application 08/402,690 and international patent applications WO 95/07696 and WO 95/09614.

According to another embodiment, the present invention provides a method of treating a patient infected with a virus characterized by the virally encoded serine protease NS3/4A, which protease is essential for the life cycle of the virus, by administering to said patient a pharmaceutically acceptable composition of the present invention. Preferably, the methods of the invention are used to treat a patient suffering from HCV infection. Such treatment may completely eradicate the viral infection or reduce its severity. More preferably, the patient is a human.

In yet another embodiment, the present invention provides a method of pretreating a biological substance for administration to a patient, comprising the step of contacting the biological substance with a pharmaceutically acceptable composition comprising a compound of the present invention. Such biological substances include, but are not limited to, blood and its components, such as plasma, platelets, subpopulations of blood cells, and the like; organs such as kidney, liver, heart, lung, etc.; sperm and eggs; bone marrow and its components; and other fluids to be infused into the patient, such as saline, glucose, and the like.

The present invention also provides a method of making a composition comprising a hepatitis C virus NS3/4A protease inhibitor and a cytochrome P450 monooxygenase inhibitor, said method comprising the step of combining a hepatitis C virus NS3/4A protease inhibitor and a cytochrome P450 monooxygenase inhibitor. Another embodiment of the present invention provides such a method wherein the composition comprises one or more additional agents as described herein.

The invention also provides therapeutic combinations comprising a hepatitis C virus NS3/4A protease inhibitor and a cytochrome P450 monooxygenase inhibitor. In another embodiment of the present invention, the therapeutic combination further comprises one or more additional agents as described herein.

The pharmaceutical composition may also be prescribed to the patient in a "patient pack" containing all the courses of treatment in a single package, usually a blister pack. Patient packs have the advantage over traditional prescriptions, where pharmacists dispense a patient's supply of medication from a bulk supply, that the patient always sees the package insert contained in the patient pack, which is often lost in the case of traditional prescriptions. The inclusion of package inserts has been shown to improve patient compliance with physician guidance.

It will be appreciated that the combination of the invention may be administered by means of a single patient pack or a patient pack of each formulation, and that inclusion of instructions in the package insert as to the correct use of the invention by the patient is an additional feature of the invention which may be desirable.

A further aspect of the invention is a package comprising at least one NS3/4A protease inhibitor of the invention in combination with a CYP inhibitor and information leaflet instructing the use of the combination of the invention. In another embodiment of the present invention, the pharmaceutical package further comprises one or more additional medicaments as described herein. The one or more additional medicaments may be provided in the same package or in separate packages.

Another aspect of the invention relates to a packaged patient kit for use in the treatment of HCV infection or prevention of HCV infection, comprising: one or more pharmaceutical formulations of each pharmaceutical component; a container for holding a pharmaceutical formulation during storage and prior to administration; and instructions for administering the drug in a manner effective to treat or prevent HCV infection.

Accordingly, the present invention provides a kit for the simultaneous or sequential administration of an NS3/4A protease inhibitor and a CYP inhibitor (and optionally additional drugs) or derivatives thereof, prepared in a conventional manner. Typically, such a kit will contain a combination of each inhibitor and optionally additional drugs in a pharmaceutically acceptable carrier (and in one or more pharmaceutical preparations), along with written instructions for simultaneous or sequential administration.

In another embodiment, a packaged pharmaceutical kit is provided comprising one or more dosage forms for self-administration; container means containing the dosage form during storage and prior to use, preferably sealed; and instructions for administering the drug to the patient. The instructions are typically written instructions, labels on the package insert and/or other components for the kit, and the dosage form is as described herein. Each dosage form may be contained individually, within a metal foil-plastic laminate, each dosage form isolated from each other in a single cell or blister, or the dosage forms may be contained in a single container, such as a plastic bottle. The kits of the present invention will also generally include means for packaging the individual kit components, i.e., dosage forms, containment devices and written instructions for use. Such packaging means may take the form of paperboard or carton, plastic or foil bags and the like.

While certain exemplary embodiments are depicted and described below, it will be appreciated that the compounds of the invention may be prepared according to the methods generally described above using appropriate starting materials that are generally available to those of ordinary skill in the art.

In order that the invention may be more fully understood, the following preparation and test examples are provided. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

Example 1

HCV replicon cell assay protocol

Cells containing Hepatitis C Virus (HCV) replicons were maintained in DMEM (Medium A) containing 10% Fetal Bovine Serum (FBS), 0.25mg/ml G418, and appropriate additives.

On day 1, replicon monolayers were trypsinized: the EDTA mixture was treated, removed, and then diluted with Medium A to a final concentration of 100,000 cells/ml. Mu.l of the dilution containing 10,000 cells was plated into each well of a 96-well tissue culture plate and cultured overnight in a tissue culture incubator at 37 ℃.

On day 2, 100% DMSO solutions of compounds were serially diluted in DMEM (medium B) containing 2% FBS, 0.5% DMSO with appropriate additives. The final concentration of DMSO was maintained at 0.5% throughout the dilution series.

The medium on the replicon monolayer was removed and then medium B containing various concentrations of the compound was added. Medium B without any compound was added to the other wells as a control without compound.

Cells were incubated with compound or 0.5% DMSO in medium B for 48 hours at 37 ℃ in a tissue culture incubator. At the end of the 48 hour incubation period, the medium was removed and the replicon monolayers were washed once with PBS and stored at-80 ℃ prior to RNA extraction.

The culture plates containing the treated replicon monolayers were thawed and a fixed amount of another RNA virus, such as Bovine Viral Diarrhea Virus (BVDV), was added to the cells in each well. RNA extraction reagents (e.g. from RNeasy kit) are added immediately to the cells to avoid degradation of RNA. Total RNA was extracted according to the manufacturer's instructions and slightly modified to improve extraction efficiency and consistency. Finally, total cellular RNA, including HCV replicon RNA, was eluted and stored at-80 ℃ until further processing.

And establishing a Taqman real-time RT-PCR quantitative determination method by utilizing two sets of specific primers and probes. One set was for HCV and the other was for BVDV. Total RNA extract from treated HCV replicon cells was added to the PCR reaction to quantify HCV and BVDV RNA within the same PCR well. Experimental failures were labeled and excluded based on BVDV RNA levels in each well. HCV RNA levels in each well were calculated according to a standard curve obtained in the same PCR plate. The percentage of inhibition or reduction of HCV RNA levels by compound treatment was calculated, using DMSO or no compound control as 0% inhibition. IC50 (the concentration at which HCV RNA levels were observed to be 50% inhibited) was calculated from the titration curve for any given compound.

Example 2

HCV Ki assay protocol

HPLC Microbore method for separating 5AB substrate and product

Substrate: NH (NH)₂-Glu-Asp-Val-Val-(α)Abu-Cys-Ser-Met-Ser-Tyr-COOH

Stock solutions of 20mM 5AB (or concentration of you chose) were prepared in DMSO w/0.2M DTT. Stored in aliquots at-20 ℃.

Buffer solution: 50mM HEPES, pH 7.8; 20% of glycerol; 100mM NaCl.

The total assay volume was 100. mu.l.

	X1(μl)	Concentration in assay
			Buffer solution	86.5	See above
5mM KK4A	0.5	25μM
			1M DTT	0.5	5mM
DMSO or inhibitors	2.5	2.5％v/v
			50μM tNS3	0.05	25nM
250 μ M5 AB (original)	20	25μM

Combining the buffer with KK4A, DTT and tNS 3; 78 μ l of each solution was dispensed into wells of a 96-well plate and incubated at 30 ℃ for-5-10 minutes.

To each well was added 2.5 μ l of a DMSO solution of the appropriate concentration of test compound (DMSO only control) and incubated at room temperature for 15 minutes.

The reaction was initiated by adding 20. mu.l of 250. mu.M 5AB substrate (25. mu.M concentration equal to or slightly below the Km of 5 AB) and incubated for 20 minutes at 30 ℃.

The reaction was stopped by adding 25. mu.l of 10% TFA.

Transfer 120 μ l aliquots to HPLC vials.

The SMSY product was isolated from the substrate and KK4A by the following method.

Microbore separation method

The instrument is used: agilent 1100

Degasser G1322A

Binary pump G1312A

Autosampler G1313A

Column thermostatic chamber G1316A

Diode array detector G1315A

Column:

phenomenex Jupiter; 5 micron C18; 300 angstroms; 150 x 2 mm; P/O00F-4053-B0

Keeping the column constant temperature: 40 deg.C

Sample introduction volume: 100 μ l

Solvent a ═ HPLC grade water + 0.1% TFA

Solvent B ═ HPLC grade acetonitrile + 0.1% TFA

Time (min)	％B	Flow rate (ml/min)	Maximum pressure
				0	5	0.2	400
12	60	0.2	400
				13	100	0.2	400
16	100	0.2	400
				17	5	0.2	400

End time: 17min

Time after operation: for 10min

Example 3

Metabolic stability of NS3/4A protease inhibitors

Interactions of ritonavir in VX-950 metabolism

Human liver microsomes were used to study the metabolism and interaction of VX-950 with ritonavir. The initial incubation was performed in 0.1M phosphate buffer (pH 7.4) containing 1mM EDTA, NADPH, 1. mu.M VX-950 and 0.1, 0.5 or 1.0mg microsomal protein/mL for each time point. Due to the non-linear metabolic rate, additional incubations were performed at two concentrations of VX-950(0.5 and 1. mu.M) for 30 minutes, containing 0.25 or 0.5mg microsomal protein/mL.

Due to the apparently rapid metabolism of VX-950 in human liver microsomes, the kinetics of VX-950 metabolism (Vmax and Km) were determined using a protein concentration of 0.25mg/mL and an incubation time of 2 minutes. A single concentration of VX-950 (0.25. mu.M) and human liver microsomes (0.25mg microsomes protein/mL) was incubated with various concentrations of ritonavir (0 to 100. mu.M) for 2 minutes and the interaction of ritonavir with VX-950 metabolism was determined. Addition of ritonavir at concentrations up to 3 μ M produced an inhibitory effect on VX-950 metabolism. At higher concentrations of ritonavir (10 to 100. mu.M), an increase in the metabolism of VX-950 was observed.

In summary, VX-950 was rapidly metabolized in human liver microsomes at concentrations used in this study (e.g., 73% @ 2. mu.M 60min, 7% @ 20. mu.M; or 86% @ 2. mu.M 120min, 21% @ 20. mu.M). Other NS3/4A protease inhibitors showed similar results.

Ritonavir has been shown to inhibit the metabolism of VX-950. However, following interaction with ritonavir at high concentrations, activation of VX-950 metabolism occurs. The mechanism of this increased metabolism of VX-950 in the presence of ritonavir is unclear. Without being limited by theory, this increase may be the result of combining both compounds at the same active site, or abrogation of CYP3a4 activity by ritonavir may result in VX-950 being metabolized by other uninhibited CYP450 enzymes.

All documents cited herein are incorporated herein by reference.

While we have described a number of embodiments of this invention, it is apparent that our basic examples can be altered to provide other embodiments that employ the compounds and methods of the invention. It is, therefore, to be understood that the scope of the invention is defined by the appended claims rather than the specific embodiments exemplified above.

Claims (22)

1. A pharmaceutical composition comprising a hepatitis c virus NS3/4A protease inhibitor or a pharmaceutically acceptable salt thereof, a cytochrome P450 monooxygenase inhibitor or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

2. The composition of claim 1 wherein the hepatitis c virus NS3/4A protease inhibitor is selected from the group consisting of compounds in WO 98/17679, WO 02/18369, and WO 03/087092.

3. The composition according to claim 2, wherein said hepatitis c virus NS3/4A protease inhibitor is VX-950 or a stereoisomer thereof.

4. The composition according to claim 2, wherein said hepatitis c virus NS3/4A protease inhibitor is VX-950.

5. The composition according to any one of claims 1 to 4, wherein the inhibitor of the cytochrome P450 is an inhibitor of the isoenzyme 3A4(CYP3A4), isoenzyme 2C19(CYP2C19), isoenzyme 2D6(CYP2D6), isoenzyme 1A2(CYP1A2), isoenzyme 2C9(CYP2C9) or isoenzyme 2E1(CYP2E 1).

6. The composition according to any one of claims 1 to 4, wherein the inhibitor of the cytochromes P450 is ritonavir, ketoconazole, oleandomycin, 4-methylpyrazole, cyclosporin or chlormezole.

7. The composition according to any one of claims 1-4, wherein the inhibitor of the cytochrome P450 is an inhibitor of CYP3A 4.

8. The composition according to any one of claims 1-4, wherein the inhibitor of the cytochrome P450 is ritonavir.

9. A method of increasing the bioavailability of a hepatitis c virus NS3/4A protease inhibitor in a patient, comprising administering to the patient a composition according to any one of claims 1-8.

10. A method for treating or preventing a hepatitis C virus infection in a subject, comprising administering to the subject a hepatitis C virus NS3/4A protease inhibitor, or a pharmaceutically acceptable salt thereof, and a cytochrome P450 monooxygenase inhibitor, or a pharmaceutically acceptable salt thereof.

11. A method of increasing the bioavailability of a hepatitis C virus NS3/4A protease inhibitor in a subject, comprising administering to the subject a hepatitis C virus NS3/4A protease inhibitor or a pharmaceutically acceptable salt thereof and a cytochrome P450 monooxygenase inhibitor or a pharmaceutically acceptable salt thereof.

12. A method of increasing the hepatic concentration of a hepatitis C virus NS3/4A protease inhibitor in a subject, comprising administering to the subject a hepatitis C virus NS3/4A protease inhibitor or a pharmaceutically acceptable salt thereof and a cytochrome P450 monooxygenase inhibitor or a pharmaceutically acceptable salt thereof.

13. A method of increasing blood levels of a hepatitis C virus NS3/4A protease inhibitor in a subject, comprising administering to the subject a hepatitis C virus NS3/4A protease inhibitor or a pharmaceutically acceptable salt thereof and a cytochrome P450 monooxygenase inhibitor or a pharmaceutically acceptable salt thereof.

14. A method according to any one of claims 10 to 13 wherein the hepatitis c virus NS3/4A protease inhibitor and the cytochrome P450 monooxygenase inhibitor are in separate dosage forms.

15. The method according to claim 14, wherein said separate dosage forms are administered substantially simultaneously.

16. The method according to any one of claims 10-14, wherein said compound and said cytochrome P450 monooxygenase inhibitor are in one dosage form.

17. The method according to any one of claims 9-13, wherein said method comprises administering an additional agent selected from an immunomodulatory agent, an antiviral agent, another HCV NS3/4A protease inhibitor, an inhibitor of a target other than NS3/4A protease in the HCV life cycle, an internal ribosome entry inhibitor, a broad-spectrum viral inhibitor, another cytochrome P450 inhibitor, or a combination thereof.

18. The method of claim 17, wherein the immunomodulator is alpha-, beta-or gamma-interferon or thymosin; the antiviral agent is ribavirin, amantadine, or terbufidil; inhibitors of another target in the HCV life cycle are inhibitors of HCV helicase, polymerase or metalloprotease.

19. A method of making a composition comprising a hepatitis c virus NS3/4A protease inhibitor and a cytochrome P450 monooxygenase inhibitor comprising the step of combining said hepatitis c virus NS3/4A protease inhibitor with said cytochrome P450 monooxygenase inhibitor.

20. A therapeutic combination comprising a hepatitis C virus NS3/4A protease inhibitor and a cytochrome P450 monooxygenase inhibitor.

21. A pharmaceutical package comprising a hepatitis c virus NS3/4A protease inhibitor, a cytochrome P450 monooxygenase inhibitor, and information instructions containing instructions for use of the inhibitor.

22. A kit comprising a hepatitis c virus NS3/4A protease inhibitor and a cytochrome P450 monooxygenase inhibitor.