HK1148962A - Dose forms comprising vx-950 and their dosage regimen - Google Patents

Description

Dosage forms comprising VX-950 and dosing regimens therefor

Cross-referencing

This application claims priority from U.S. application No. 60/931,108 filed on 21/5/2008 and U.S. application No. 60/994,430 filed on 19/9/2008, the contents of which are hereby incorporated by reference in their entirety.

Field of the invention

The present invention relates to methods for treating hepatitis c virus infection.

Background

Infection by the hepatitis c virus ("HCV") is a very acute human medical problem. HCV is recognized as the causative agent of most non-A, non-B hepatitis with an estimated global human seropositivity of 3% (see, e.g., A. Alberti et al, "Natural History of hepatitis C,") "J.Hepatology31 (appendix 1), 17-24 (1999)). Nearly four million individuals may be infected in the United States alone (see, e.g., m.j. alter et al, "the epidemic of Viral Hepatitis in the United States,Gastroenterol. Clin.North Am.，23，437-455(1994)；M.J.Alter，“Hepatitis C VirusInfection in the United States，”J.Hepatology31 (appendix 1), 88-91 (1999)).

In the population infected with HCV, 20-25% clear the virus after acute infection, but 75-80% will develop chronic hepatitis C infection. (see for example the introduction of,Frontiers in Viral Hepatitisedit RF Schinazi, J-P Sommadossi and CM Rice, page xi, Elsevier (2003)). This often leads to Hepatitis recurrence and worsening, often leading to more severe disease states (e.g., cirrhosis and Hepatocellular Carcinoma) (see, e.g., m.c. kew, "Hepatitis C and Hepatocellular carcinosa",FEMS Microbiology Reviews14, 211-220 (1994); saito et al, "Hepatitis C Virus Infection Associated with the Development of hepatocellular Carcinoma" with the Development of the hepatocellular Carcinoma, "Hepatitis C Virus Infection"Proc.Natl.Acad.Sci.USA,87, 6547-6549(1990)). Unfortunately, there is no widely effective therapy for attenuating the course of chronic HCV.

The HCV genome encodes a 3010-3033 amino acid polyprotein (see, e.g., Q.L.Choo et al, "Genetic Organization and Diversityof the Hepatitis C Virus (genetic organization and diversity of Hepatitis C)') "Proc.Natl.Acad.Sci.USA88, 2451-2455 (1991); kato et al, "Molecular Cloning of the human hepatitis C Virus Genome From Japanese Patents with Non-A, Non-BHEPTIS (Molecular Cloning of the human hepatitis C Genome of Japanese Patients with Non-A, Non-B hepatitis)," "Molecular Cloning of the human hepatitis C Virus Genome with Non-A, Non-B hepatitis"Proc.Natl.Acad.Sci.USA87, 9524-9528 (1990); takamizawa et al, "Structure and Organization of the Hepatitis C Virus From Human Cariers (structures and tissues of Hepatitis C Virus Isolated From Human Carriers)," Structure and Organization of the Hepatitis C Virus "J.Virol.,65, 1105-1113(1991)). It is speculated that the non-structural (NS) protein of HCV provides the necessary catalytic structure for viral replication (catalyticcatalytic). NS proteins are derived by protease Cleavage of polyproteins (see, e.g., R.Bartenschlager et al, "non structural Protein 3 of the Hepatitis CVirus a series-Type Protein requiring for Cleavage at NS3/4 and NS4/5 junctins (non structural Protein 3 of Hepatitis C virus Encodes a Serine-Type protease Required for Cleavage at the junction of NS3/4 and NS 4/5),") "J.Virol.67, 3835-3844 (1993); grakoui et al, "Characterization of the Hepatitis CVirus-Encoded spring protease: determination of protease-Dependent Polyprotein Cleavage Sites (characterization of the serine protease encoded by the hepatitis C Virus: Determination of the protease-Dependent Polyprotein Cleavage Sites) ',', and "J. Virol.67, 2832-; grakoui et al, "Expression and differentiation of Hepatitis C Virus Polyprotein Cleavage Products (Expression and identification of Hepatitis C Polyprotein Cleavage Products)", and "J.Virol.67, 1385-; tomei et al, "NS 3 is a serine protease required for processing of hepatitis C virus polyprotein" (NS3 is a serine protease required for processing hepatitis C virus polyprotein) ",J.Virol.，67，4017-4026(1993))。

HCV NS protein 3(NS3) has serine protease activity, which contributes to the processing of most virusesThe enzyme, and therefore the protein, is considered to be extremely important for viral replication and infectivity. It is known that mutation of the NS3 Protease of Yellow Fever Virus reduces the infectivity of the Virus (see, e.g., Chambers, T.J., et al, "Evidence that the N-terminal domain of Nonstructural Protein NS3 From Yellow wine Virus viral Protein responsibles for Site-Specific cleavage of the N-terminal domain of the Nonstructural Protein NS3 From Yellow Fever Virus is Evidence of a serine Protease Responsible for Site-Specific cleavage of viral polyproteins)",Proc. Natl.Acad.Sci.USA,87, 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, which processes all four downstream sites of the HCV polyprotein (see, e.g., C.Lin et al, "Hepatitis C Virus NS3 spring protein enzymes: Trans-Cleavage Requirements and Processing Kinetics),J.Virol.，68，8147-8157(1994))。

the HCV NS3 serine protease and its associated cofactor NS4A, contribute to the processing of all viral enzymes and are therefore considered to be extremely important for viral replication. This process appears to be similar to that performed by the human immunodeficiency virus aspartyl protease, which is also associated with the processing of viral enzymes. HIV protease inhibitors that inhibit viral protein processing are effective antiviral drugs in humans, suggesting that disruption of this stage of the viral life cycle results in the production of therapeutically effective drugs. This is therefore an interesting target for drug discovery.

There is currently no satisfactory drug or therapy against HCV. Until recently, the only established therapy for HCV disease was interferon treatment. The first approved therapy for HCV infection was standard (non-pegylated) interferon alpha therapy. However, interferons have serious side effects (see, e.g., M.A. Wlaker et al, "Heapatitis C viruses: An Overview of Current applications and Progress (type C)Hepatitis virus: summary of current methods and developments) ','.DDT4, 518-29 (1999); mordapour et al, "Current and EvalVing therapeutics for Hepatitis C (present and progress in Hepatitis C therapy),".Eur.J.Gastroenterol.Hepatol.11, 1199-1202 (1999); "Suicide Associated with Alfa-interference therapy for Viral Hepatitis hepatis (for interferon-alpha-Associated spontaneous inactivation therapy of Chronic Hepatitis Virus),".J.Hepatol.21, 241-; renault et al, "Side Effects of Alpha Interferon"Seminars in Liver Disease9, 273-, (1989)), and Interferon alpha monotherapy causes long-term remission in only a small (about 25%) fraction of cases (see, e.g., o.weiland, "Interferon Therapy in Chronic Hepatitis C Infection",FEMS Microbiol.Rev.,14, 279-288(1994)). The addition of ribavirin (ribavirin) to the treatment regimen slightly increased the response rate. More recently pegylated forms of interferon (PEG-And) It also results in only a slight improvement in the remission rate and only partial alleviation of side effects. Depending on prognostic factors (e.g., HCV genotype and manifestation of initial response to treatment), the current standard of care (standard of care) is a treatment regimen that lasts for 24-48 weeks. Furthermore, the prospect of an effective vaccine against HCV remains uncertain.

Thus, there is a need for anti-HCV therapy and suitable dosing regimens for anti-HCV compounds.

HCV and other diseases and disorders are associated with liver damage. There is also a need for therapies and appropriate dosing regimens for the treatment of liver damage.

Summary of The Invention

The present invention provides a therapy for hepatitis c virus infection. The invention further provides for the prevention of clinical sequelae of hepatitis c virus infection.

The invention also provides therapies for liver damage and hepatitis.

Brief Description of Drawings

Fig. 1A and 1B depict graphs of mean concentration versus time (example 3) made as a function of dose level.

Figures 2A-2D depict the derived pharmacokinetic parameters. The lines in the boxes represent the median values, while the boxes represent the limits of the median value of the data (example 3).

FIG. 3 depicts the concentration of HCV RNA in plasma (IU/mL) in a study lasting 14 days (example 5).

FIG. 4 depicts the change in concentration of HCV RNA from baseline (IU/mL) in a 14 day-long study (example 5).

FIG. 5 depicts the change in HCV RNA concentration (IU/mL) from baseline in individual subjects in the 750mg q8h dose group over a 14 day duration study (example 5).

FIG. 6 depicts the mean +/-SEM of neopterin, ALT (alanine aminotransferase) and HCVRNA in all dose groups. The symbols used in all 3 dose groups and placebo in figure 6 are shown below: mean ALT ± SEM vs baseline (top 4 lines with open symbols), mean plasma level of neopterin ± SEM vs baseline (middle 4 lines with open symbols), and mean plasma loading of HCV RNA ± SEM vs baseline (lower 4 lines, filled symbols). The patient was treated with VX-950 for 14 days. The transient increase in mean ALT levels on day 12 was artificially generated for the 450mg q8h group (5 of 10 samples were lost with a median of 38U/l, ranging from 25 to 125U/l) (example 5).

FIG. 7 depicts the mean +/-SEM of neopterin in all groups. Mean plasma levels of neopterin ± SEM at day 7, day 14, pre-treatment, for all 3 dose groups and placebo. It should be noted that the mean reduction of neopterin was greatest in the 750mg q8h dose group, which was greatest before treatment and lowest on the 14 th day. The 750mg q8h dose group showed a significant reduction in neopterin at day 14 compared to baseline and placebo (unpaired two-tail test, Mann-Whitney test). The horizontal dashed line at 7.7nmol/l represents ULN (example 5).

FIGS. 8, 9 and 10 depict that in vitro cleavage of TRIF (TLR3 adaptor protein) by HCV NS3/4A protease is inhibited by VX-950.

FIG. 8 (containing the adaptor toll-IL1 receptor domain inducing IFN-. beta.TRIF or TICAM-1) depicts a schematic illustration of TRIF, showing the individual protein binding domains of TRIF. Hcv ns3 protease cleaves TRIF at Cys 372 to form two fragments: Δ C340 and Δ N372 (modified from Li et al, 2005, Proc. nat' l.Acad. Sci. USA, 102, 2992-.

FIG. 9 depicts the kinetic properties of the HCV NS3 protease on TRIF cleavage. The in vitro transcription/translation product of the coupled TRIF protein (as substrate) was labeled with 35S methionine, incubated with 6. mu.M tNS3 protease at various time points (ranging from 0 to 240 minutes), and subjected to SDS-PAGE. The gel was exposed to light using a photoimaging machine to quantify the cleavage products. The quantification of the Δ N372 cleavage product as a function of time is shown in the figure.

FIG. 10 depicts NS3 protease-dependent TRIF cleavage and the inhibitory effect of VX-950 on TRIF cleavage. The in vitro transcription/translation product of the coupled TRIF protein labeled with 35S methionine (as substrate) was incubated with tNS3 protease at increasing concentrations in the range of 0-4. mu.M in the presence (circles) or absence (squares) of 10. mu.M VX-950, followed by SDS-PAGE and exposure to a photoimaging instrument. Quantification of Δ N372 cleavage products is shown in the figure.

FIG. 11 depicts in vitroPhenotypic characterization of VX-950 resistant mutants. In vitro enzyme reaction assay (Ki) or two-day replicon assay (IC)₅₀) The A156V/T mutation confers increased resistance to VX-950 compared to the wild-type protease. The Kcat/Km ratios of the mutants relative to the wild-type enzyme are shown in the table (modified from Li et al, J.biol.chem., 280, 36784-36791, 2005).

FIG. 12 shows cleavage of HCV 4A/B substrate by the A156V/T mutant relative to Wild Type (WT) NS3 protease: in vitro transcription/translation products of inactivated HCV mutant protease coupled with a 35S methionine tag fused to SEAP proteins (4A/B linker between the two) as substrate were incubated with varying amounts of Wild Type (WT) (square) tNS3 protease or a156V/T (triangle or circle) tNS3 protease in the range of 0.008 μ M to 6 μ M followed by SDS-PAGE and exposure to photoimaging. Quantification of Δ N372 cleavage products is shown in the figure.

FIG. 13 depicts cleavage of TRIF substrate by the A156V/T mutant relative to wild-type (WT) NS3 protease. In vitro transcription/translation products of coupled TRIF labeled with 35S methionine (as substrate) were incubated with varying amounts of Wild Type (WT) (square) tNS3 protease or A156V/T (triangle and circle) tNS3 protease in the range of 0.008. mu.M to 6. mu.M, followed by SDS-PAGE and exposure to photoimaging. Quantification of Δ N372 cleavage products is shown in the figure.

FIG. 14 depicts the mean values of HCV RNA, neopterin and ALT at baseline, day 7 and day 14 (example 5).

FIG. 15 shows the inhibition of IFN-. beta.promoter activity by HCV protease in Huh7 cells stimulated with Sendai virus. Huh7 cells were co-transfected with a plasmid expressing a luciferase gene under the control of an IFN- β promoter, together with a protease plasmid of Wild Type (WT) or inactivated Mutant (MT), followed by stimulation with Sendai virus (SeV). The fold activation of the luciferase gene compared to the control not induced with Sendai virus is shown in the figure.

FIG. 16 shows that treatment with VX-950 overcomes the inhibitory effect of HCV protease on the Sendai virus-stimulated IFN- β promoter activity. Huh7 cells were co-transfected with a plasmid expressing a luciferase gene under the control of the IFN- β promoter, together with a protease plasmid of Wild Type (WT) or inactivated Mutant (MT). These cells were treated with DMSO (control) or 10. mu.M VX-950. Cells were stimulated with Sendai virus (SeV) and luciferase activity was detected 16 hours after infection. The fold activation of the luciferase gene compared to the control not induced with Sendai virus is shown in the figure.

Fig. 17 shows that VX-950 treatment resulted in a reduction of HCV RNA in patients who had previously not responded to HCV treatment (fig. 17A) and in the initial treatment patients (fig. 17B). Median HCV RNA levels for the patients in each treatment regimen are shown in the figure. The concentration of HCV RNA in plasma was determined using the RocheCOBAS TaqMan HCV/HPS assay.

FIG. 18 depicts the phenotypic characteristics of VX-950-resistant mutants for various treatment regimens described herein.

Figure 19 shows the evaluation of the duration of treatment in the presence of the wild type and resistant mutants of figure 90.

FIG. 20 shows a graph of treatment duration evaluated at high potency Peg-IFN/RBV.

FIG. 21 shows a graph of treatment duration evaluated at low potency Peg-IFN/RBV.

FIG. 22 shows the recurrence of the virus after 8-12 weeks of treatment.

FIG. 23 shows the treatment duration of the SVR evaluated.

FIG. 24 shows a timeline of a study including daily administration of placebo and Peg-IFN; VX-950; or VX-950 and Peg-IFN for 14 days; followed by a follow-up over a period of 48 weeks to assess the time of administration of Peg-IFN and RBV.

Fig. 25 shows the rapid antiviral response of subjects during the 14 day dosing period of treatment with VX-950. Typically, HCV RNA levels in these treated subjects are reduced by at least 2log upon completion of the dosing regimen₁₀And is incorporated inIn some cases at least 4log reduction₁₀。

FIG. 26 shows the levels of individual HCV RNA in subjects over the 14-day dosing period of treatment with HCV/Peg-IFN-2 α. Typically, HCV RNA levels in these treated subjects are reduced by at least 3log upon completion of the dosing regimen₁₀And in some cases at least 4log reduction₁₀。

Detailed Description

The present invention relates to specific dosages and dosing regimens for administering VX-950. VX-950 (also known as Telaprevir) is a competitive reversible peptidomimetic NS3/4A protease inhibitor with a steady state binding constant (ki;) of 7 nM. See, for example, WO 02/018369.

For the purposes of the present invention, the compound "VX-950" referred to herein includes pharmaceutically acceptable salts, prodrugs and solvates thereof.

The phrase "one or more pharmaceutically acceptable salts" of VX-950, as used herein, refers to salts of VX-950 that are safe and effective for treating HCV infection. Pharmaceutically acceptable salts include salts of acidic or basic groups present in VX-950. Pharmaceutically acceptable acid addition salts include, but are not limited to: hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulphonate, ethanesulphonate, benzenesulphonate, p-toluenesulphonate and pamoate. VX-950 can also form pharmaceutically acceptable salts with various amino acids, and the use of these amino acid salts is also within the scope of the present invention. Suitable basic salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts. For a review of pharmaceutically acceptable salts see Berge et al, j.pharm.sci., 66, 1-19(1977), the contents of which are incorporated herein by reference.

The phrase "pharmaceutically acceptable prodrug" of VX-950, as used herein, refers to a compound that can be converted under physiological conditions or by solvolysis to VX-950 or a pharmaceutically acceptable salt of VX-950 before exhibiting its pharmacological effects in the treatment of HCV infection. Typically, prodrugs are formulated for the purpose of: increased chemical stability, increased patient acceptance and compliance, increased bioavailability, prolonged duration of action, increased organ selectivity, improved formulation (e.g., increased aqueous solubility), or reduced side effects (e.g., toxicity). Pharmaceutically acceptable prodrugs thereof can be readily prepared from VX-950 using methods known in the art, for example, see methods in the following references: burger's Medicinal Chemistry and Drug Chemistry, Vol.1, 172-. See also Bertolini et al, j.med.chem., 40, 2011-2016 (1997); shan et al, J.Pharm.Sci., 86(7), 765-767 (1997); bagshawe, Drug Dev. Res., 34, 220-; bodor, Advances in Drug Res., 13, 224-; bundgaard, Design produgs, Elsevier Press (1985) and Larsen, Design and application of produgs, Drug Design and Development (edited by Krogsgaard-Larsen et al), Harwood Academic Publishers (1991).

The phrase "pharmaceutically acceptable solvate" of VX-950, as used herein, refers to a pharmaceutically acceptable solvate form of VX-950 that comprises one or more solvent molecules and retains the biological effectiveness of VX-950. Examples of solvates include VX-950 in combination with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine.

In the case where the salt, prodrug or solvate of VX-950 is a solid, one skilled in the art will understand that these salts, prodrugs and solvates can exist in different crystalline or amorphous forms, and the use of all such salts, prodrugs and solvates is also within the scope of the present invention.

VX-950 can contain one or more asymmetric carbon atoms and therefore can exist as racemates and racemic mixtures, individual enantiomers, diastereomeric mixtures and individual diastereomers. All such isomeric forms of these compounds are expressly included in the present invention. Each stereogenic carbon may be in the R or S configuration. The D-and L-isomers at the N-propyl side chain of VX-950 are expressly included within the scope of the present invention.

A single dose of VX-950 has been tested in humans and found to be well tolerated (example 3). The incidence or severity of adverse events did not increase with dosage of VX-950. No adverse events were considered severe (grade 3 or 4). The more common and serious adverse events are adverse events of the skin (e.g., rashes and itching), followed by gastrointestinal adverse events and anemia. There was no clinically significant change in baseline experimental values for hematological or clinical chemistry parameters. There were no clinically significant changes in physical examination, vital signs or electrocardiograms in all subjects examined.

Applicants found that wild-type HCV was cleared by VX-950 within 10 weeks. And VX-950 resistant variants (IC) against HCV₅₀Increased by 7-20 fold) can be cleared by follow-up on Peg-IFN/RBV treatment regimen for 10-24 weeks.

Assays were performed to examine the pharmacokinetic profile of VX-950. This data is shown in fig. 1 and 2.

Liver exposure to VX-950 was predicted based on the integrated preclinical and clinical data (liver exposure to VX-950). The predicted human liver exposure was combined with the results of the VX-950 replicon assay (repliconassay) and infectious virus assay to determine the dose that was expected to be well tolerated and produce a therapeutic benefit. The predicted mean liver concentration values over the dose range studied are replicon test IC₉₀Up to 57-fold and replicon test IC₅₀Up to 113 times.

These results indicate that the dosage regimen of applicants 'invention will result in hepatic concentrations of VX-950 that are much higher than the IC's detected in non-clinical trials₅₀And IC₉₀。

Accordingly, one embodiment of the present invention provides pharmaceutical compositions each comprising VX-950 and a pharmaceutically acceptable carrier. The amount of VX-950 in these pharmaceutical compositions can be about 100mg to about 1500mg, about 300mg to about 1250mg, about 450mg, about 750mg, or about 1250 mg. Each of these pharmaceutical compositions may be administered, for example, once, twice or three times daily. Each of these compositions may be in one or more dosage forms (e.g., ampoules, capsules, creams, emulsions, liquids, granules, drops, injections, suspensions, tablets, powders). Each of these pharmaceutical compositions may be administered by one or more routes (e.g., oral, infusion, injection, topical or parenteral) according to the degree of suitability recognized by those skilled in the art and according to the dosage form.

Another aspect of the invention provides a method for treating or preventing an HCV infection in a patient, comprising administering VX-950 to the patient.

In some embodiments, the amount of VX-950 is at least about 300mg (e.g., at least about 450mg, at least about 500mg, at least about 750mg, at least about 1250mg, or at least about 1500 mg). In some embodiments, the amount of VX-950 is no more than about 1500mg (e.g., no more than about 1250mg, no more than about 750mg, no more than about 450mg, no more than about 500mg, or no more than about 300 mg).

It is understood that the lower and upper limits mentioned above may be combined to provide a dosage range for administration of VX-950. For example, in some embodiments, VX-950 is administered in an amount of about 300mg to about 1250mg or about 300mg to about 1500 mg.

In some embodiments, VX-950 is administered in an amount of about 450mg, about 500mg, about 600mg, about 750mg, about 1000mg, or about 1250 mg.

In the methods of the invention, a specific amount of VX-950 can be administered, for example, once a day, twice a day (e.g., BID; q12h), or three times a day (e.g., TID; q8 h). In addition, VX-950 can be administered with or without food.

VX-950 has been tested in humans and found to effectively inhibit HCV replication, significantly reduce HCV RNA levels, and inhibit the virus so that the viral RNA is undetectable.

In 8 subjects who received 750mg of VX-950 per 8 hours (q8h), 4 HCV RNA levels were below the detection limit (LLQ 30 IU/mL), and 2 HCV RNA levels were below the detection limit (LLD 10 IU/mL) in these 4 subjects.

Detection of HCV RNA can be performed, for example, using the Roche COBASS TaqMan HCV/HPS assay (available from Roche molecular Diagnostics). Subjects (or patients) receiving 750mg of VX-950 every 8 hours for 14 days had a more than 4log reduction in the median HCV-RNA value at the end of treatment₁₀(i.e., a 10000-fold reduction). At the end of the 14 day treatment, more than a 2log reduction in median HCV-RNA was observed for each of the other two VX-950 dose groups₁₀. Each subject receiving VX-950 had more than a 2log reduction in HCV-RNA within the first three days of treatment₁₀While 26 of 28 subjects treated with VX-950 had a 3log reduction in HCV-RNA within the first three days of treatment₁₀. See example 5 and FIGS. 3-5.

It was demonstrated that the viral load of plasma in patients treated with VX-950 dropped rapidly and returned slowly to baseline HCV RNA levels after dosing ended. Specifically, the rate of return to the baseline HCV RNA level after the end of treatment is slower than the rate of HCV RNA decline during treatment. These results, together with the achievement of undetectable HCV RNA levels, indicate the effectiveness of VX-950 as a monotherapy.

Accordingly, the present invention provides a method for treating a patient infected with HCV, the method comprising administering VX-950 to the patient in the following manner: (a) about 450mg each time, 3 times daily, every 8 hours; (b) about 750mg each time, 3 times daily, every 8 hours; (c) about 1250mg each time, 2 times daily, every 12 hours; or (d) about 1250mg once every 8 hours 3 times daily.

Another aspect of the invention provides methods for treating a patient infected with HCV by administering VX-950 such that the HCV RNA level in the patient after administration is at least about 2log lower than the level prior to the treatment₁₀(e.g., at least about 4 log)₁₀)。

Yet another aspect of the invention provides a method of administering treatment to a patient infected with HCV by administering VX-950 such that the patient's viral RNA level is reduced to and maintained at an undetectable level until a "sustained viral response" is achieved. The term "sustained viral response" as used herein means that viral RNA levels are not detectable at 24 weeks after completion of administration (or completion of administration of VX-950).

The method of the invention with 750mg VX950 every 8 hours effectively resulted in higher trough levels. The term "trough level" as used herein refers to the blood concentration just before the next dose, or the minimum drug concentration between two doses. It is important to maintain drug levels above a certain concentration to maintain proper inhibition of viral replication, particularly in viral diseases. Advantageously, a dosing regimen of about 750mg of VX950 three times a day, once every 8 hours, was found to result in the highest trough level of VX 950.

Thus in a preferred embodiment, the present invention provides a method for administering VX-950 to a patient in need thereof, which comprises administering about 750mg of the compound three times daily, once every 8 hours.

It will be appreciated that a flexible administration schedule is advantageous. Thus, in another embodiment of the invention, it is optionally administered with food 3 times daily but not every 8 hours. In certain embodiments, VX-950 is administered in conjunction with food.

The invention also provides a method for providing VX-950 to a patient in need thereof, comprising administering to the patient an oral dose of a composition comprising VX-950, wherein the dose is such that upon administration it is affectedProvides a mean plasma concentration (C) of VX-950 of at least about 750ng/mL_{Flat plate Are all made of}). In some embodiments, C of VX-950_AverageAbout 1000ng/mL or about 1250 ng/mL. In some embodiments, the dose consists essentially of about 750mg VX-950. In some embodiments, C is obtained or achieved within 3 hours (e.g., 2 hours or 1 hour) of administration of VX-950_Average. In some embodiments, C of VX-950_AverageFor more than about 24 hours (e.g., about 5 or 12 weeks).

Another aspect of the invention provides a method for treating an HCV infection in a patient by administering at least one dosage form comprising VX-950 over a 24 hour period such that a minimum VX-950 plasma trough level of about 750ng/mL is maintained over the 24 hour period.

In some embodiments, the dosage form is administered such that a minimum VX-950 plasma trough level of about 800ng/mL (e.g., about 900ng/mL or about 1000ng/mL) is maintained over a 24 hour period.

In certain preferred embodiments, a therapeutically effective blood level is obtained and maintained at a trough level. These methods are particularly useful for treating a human having an HCV infection by administering a formulation of VX-950 such that a minimum VX-950 plasma trough level of about 750, 800, 900 or 1000ng/mL is maintained over a 24 hour period. Without being bound by theory, a trough level greater than about 1500ng/mL is not considered necessary for the present invention. Accordingly, valley levels of about 750, 800, 900, 1000ng/mL to about 1500ng/mL (particularly 1000 to about 1500) are within the scope of the invention.

Also provided is a dosage form for delivering VX-950 to a human, wherein the dosage form comprises VX-950 that maintains the following VX-950 plasma trough levels over a 24 hour period when the dosage form is administered at least once over a 24 hour period: at least about 750ng/mL, 800ng/mL, 900ng/mL, or 1000ng/mL to about 1500ng/mL (particularly 1000ng/mL to about 1500 ng/mL).

Ideally, while the methods of the present invention relate to treating a patient infected with HCV, the methods relate to achieving therapy relatively quicklyAn effective plasma concentration of VX-950, and then maintaining this trough level to achieve an effective therapeutic response. The effective therapeutic response is preferably one or both of: a) achieving a sustained virologic response; and b) undetectable plasma HCV RNA for at least 12 weeks (12 weeks or longer). As used herein, "undetectable" HCV RNA means by currently commercially available assays (preferably Roche COBASTaqMan)^TMHCV/HPS assay) detected less than 10IU/mL of HCV RNA.

By administering a loading dose to a patient, the blood level can be lowered relatively rapidly. In one embodiment, the loading dose is about 1250mg VX-950.

In certain dosage forms of the invention, the dosage form (other than the dosage form used to administer the loading dose) contains about 750mg VX-950, and the dosage form is administered every 8 hours (i.e., q8 h).

In certain embodiments, the VX-950 dosage form is administered every 8 hours.

In certain embodiments, the duration of treatment with VX-950 is shorter than the current standard of care.

In certain embodiments, VX-950 is administered for less than about 12 weeks (or less than 12 weeks).

In certain embodiments, VX-950 is administered for about 8-12 weeks (or 8-12 weeks).

In certain embodiments, VX-950 is administered for about 10 weeks (or 10 weeks).

As shown in fig. 90-93, modeling data indicates that administration of VX-950 can clear wild-type virus within about 10 weeks.

In certain embodiments, VX-950 is administered for less than about 10 weeks.

In certain embodiments, VX-950 is administered for about 2 weeks.

Applicants have demonstrated that patients treated with VX-950 reached SVR for 2 weeks.

In other embodiments, VX-950 is administered for less than about 8 weeks (or about 8 weeks or 8 weeks), less than about 6 weeks (or about 6 weeks or 6 weeks), or less than about 4 weeks (or about 4 weeks or 4 weeks).

In certain embodiments, the methods of the invention relate to treating a patient infected with genotype 1 hepatitis c virus. Genotype 1 HCV infection is the most refractory strain of HCV and the most prevalent strain in the united states.

Applicants also demonstrated that administration of VX-950 reduced the levels of neopterin and ALT in vivo (see fig. 6, 7, and 14). AST (aspartate aminotransferase) levels are also reduced when VX-950 is administered. ALT is an enzyme present in hepatocytes; ALT infiltrates into the blood from cells when liver cells are damaged or inflamed. Blood ALT levels can be used as markers of hepatitis or liver damage. See Tatyana Yashina& J.Sanders Sevall，“Hepatitis C Virus”in Use and Interpretation of Laboratory Tests in GastroenterologyEdited by James B.Peter, page 127, (1998) and Andres T.Blei, "Liver and Biliary track" inLaboratory MedicineEdited by d.a. noe and Robert c.rock, chapter 19, page 363 (1994).

Neopterin (6-d-erythrosyl-trihydroxypropylpteridine) is a pteridine derivative produced in the metabolism of Guanosine Triphosphate (GTP). Neopterin is a marker of inflammation, produced primarily by activation of monocytes and macrophages by interferon gamma or interferon alpha. Levels of neopterin tend to be elevated in chronic HCV infection (Quiroga et al, Dig Dis Sci., 39 (11): 2485-2496, 1994). The plasma levels of neopterin in healthy individuals are desirably between 3.1 and 7.7 nmol/L.

Thus, applicants examined the change in serum concentration of neopterin (as a marker of monocyte/macrophage activity) upon administration of the (HCV) NS 3.4A protease inhibitor. As described herein, VX-950 was administered to 34 patients infected with genotype 1 HCV for a period of 14 days in a randomized, double-blind, placebo-controlled, multiple dose study (table 1). The patient received VX-950450 mg q8h (10) in 750mg q8h (8 people), 1250mg q12h (10 people) or placebo (6 people). Can be used for quantitative competitive ELISA before treatment, at the 7 th, 14 th and 10 th follow-up daysNeopterin, Brahms, Hennigsdorf, Germany) measures the concentration of Neopterin in serum. The Lower Limit of Detection (LLD) was 2 nmol/L. By real-time PCR during the study (TaqMan HCV Test; linear dynamic range of 3.0 x 10¹-2.0×10⁸HCV RNA IU/mL; the LLD of HCV RNA is 10 IU/mL; roche Diagnostics, branch burg, NJ) estimates HCV RNA at frequent intervals.

At least a 2-log reduction in viral load per patient was demonstrated in all dose groups during VX-950 dosing₁₀. In the 750mg q8h dose group, the mean HCV RNA decreased by 3.6log on day 3₁₀4.3log reduction on day 14₁₀. The greatest effect was observed on days 3-7 in the dose groups of 450mg q8h and 1250mg q12h, followed by an increase in the average viral load on days 7-14. The mean viral load was increased in all dose groups during the follow-up. Advantageously, both treatment-naive and treatment-naive patients of HCV benefit from the methods of the invention. As depicted in fig. 17A and 17B, both treatment-naive and treatment-naive patients respond to VX-950. For the avoidance of doubt, patients treatable according to the methods of the invention include those who have not attempted or failed HCV therapy, including non-responsive, recovery, relapse and rebound (breakthrough).

There were 23 of the 34 patients with elevated baseline neopterin (mean 9.33 nmol/L; regular Upper Limit (ULN)7.7 nmol/L). In the 750mg dose group, neopterin decreased significantly on day 14 compared to baseline and placebo (750mg q8h dose group baseline versus day 14 10.48 ± 0.84nmol/L versus day 7.32 ± 0.48nmol/L P ═ 0.0104, mann-whitney test; 750mg q8h dose group versus day 14 placebo 7.32 ± 0.48nmol/L versus day 9.81 ± 1.36nmol/L P ═ 0.0036, unpaired two-tailed T test). Only the mean level of neopterin at day 14 in the 750mg dose group was within the normative values (fig. 7 and 14). The reduction in mean neopterin levels was less in the 450mg q8h dose group and the 1250mg q12h dose group (fig. 6, 7, and 14). Mean neopterin levels in the placebo group did not change (fig. 6 and 7). Mean neopterin levels were elevated in all dose groups during the follow-up visit.

Serum alanine Aminotransferase (ALT) levels can be measured by commercially available methods. Mean ALT levels above baseline were reduced when administered in all groups (fig. 6). In the follow-up, mean ALT levels were elevated for all dose groups and returned to baseline levels.

Although HCV RNA rose after day 7 in the 450mg dose group and the 1250mg dose group, neopterin, particularly ALT, continued to decrease. Changes in mean concentrations of neopterin during administration of VX-950 were correlated with decreased HCV RNA and ALT levels. Mean neopterin concentrations dropped most at day 14 in the 750mg q8h dose group. This group was also the dose group with the greatest reduction in HCV RNA at day 14. In the 450mg dose group and the 1250mg dose group, the levels of ALT and neopterin decreased with increasing HCV RNA levels after day 7. These data suggest that inhibition of HCV replication by VX-950 results in a significant reduction in systemic inflammatory activity associated with viral infection.

VX-950 also improved elevated ALT levels in animal models (see WO 2005/025517). In particular, expression of WT-HCV protease-SEAP in SCID mice results in elevated ALT levels, which can be improved by treatment with VX-950. Expression of WT-HCV protease alone in SCID mice also resulted in time-dependent and dose-dependent elevation of ALT levels.

Accordingly, the present invention provides methods for reducing (including normalizing) ALT levels in a patient. The method comprises administering to a patient in need thereof a therapeutically effective amount of VX-950 (e.g., about 1350mg per day, about 2250mg per day, or about 2500mg per day). The patients may be infected or not infected with HCV.

In some embodiments, about 450mg of VX-950 is administered daily, or about 750mg is administered every 8 hours, or about 1250mg of VX-950 is administered every 12 hours.

Another aspect of the invention provides methods for treating or preventing one or more of the following in an HCV positive or HCV negative patient: liver damage, hepatitis, steatosis, fatty liver, NAFLD, NASH, alcoholic steatosis or Reye's syndrome (Reye' ssyndrome).

Methods for protecting the liver of an HCV positive or HCV negative patient are also within the scope of the present invention.

Applicants have also demonstrated that VX-950 blocks immune evasion in vitro.

In Huh7 cells infected with Sendai virus, VX-950 restored IFN β -dependent gene expression. In the presence of the WT HCV protein, the IFN β promoter activity is reduced in response to stimulation by Sendai virus. VX-950 overcomes the WT HCV protein-mediated inhibition of IFN beta promoter activation. See fig. 15 and 16.

Furthermore, NS3/4A is known to be involved in the escape of innate defenses (through, for example, the TRIF-dependent mechanisms (and processing of viral polyproteins)). This immune evasion results in a persistent infection of the virus. Therefore, compounds that inhibit both viral polyprotein processing and innate defense evasion at the same time are desirable. Advantageously, VX-950 has been shown to inhibit both. In particular, VX-950 inhibits the in vitro cleavage of TRIF, which is a TLR3 adaptor protein. Fig. 8-10.

Without being bound by theory, modeling suggests that VX-950 inhibits the cleavage of TRIF by the NS3 protease. TRIF binds to the non-major side of the protease active site of NS 3. VX-950 binds to the non-primary side of the active site, as does TRIF, thereby blocking cleavage of TRIF.

Two variants of VX-950 virus, A156T and A156V, have been shown to exhibit reduced ability to cleave either TRIF or 4A/4B (see C.Lin et al, J.biol.chem., (8/2005)). Because of their poor adaptability, these viral variants are ineffective for both viral polyprotein processing and viral persistent infection. Without being bound by theory, this is related to the steric hindrance of a156V affecting binding to the 4A/4B and TRIF substrates. See fig. 11-13.

This suggests that VX-950 is acting as both a direct antiviral and an immune evasion inhibitor. Accordingly, the present invention also provides methods of inhibiting HCV protease-mediated evasion of host defenses.

These results, together with the in vivo data disclosed herein, demonstrate the effectiveness of VX-950 as a monotherapy.

The amount of VX-950 of the invention is administered in a single dosage form or in multiple dosage forms. If administered in separate dosage forms, each dosage form is administered at approximately the same time. For the avoidance of doubt, for dosing regimens requiring more than one time per day, one or more tablets or one or more doses may be administered per day (e.g. three times per day, 1 tablet per time, or three times per day, 3 tablets per time). Most embodiments of the invention will employ at least 2 tablets per dose.

The skilled artisan will appreciate that if the methods of the invention are used to prophylactically treat a patient, the methods can treat infection when the patient is infected with hepatitis c virus. Accordingly, one embodiment of the present invention provides a method for treating or preventing hepatitis c in a patient.

In addition to treating patients infected with hepatitis C, the methods of the invention can be used to prevent patients from hepatitis C infection. Accordingly, one embodiment of the present invention provides a method for preventing infection of a patient with hepatitis c virus comprising administering to the patient a composition or dosage form of the present invention.

The method of the present invention may further involve administering an additional component comprising an additional agent selected from the group consisting of: an immunomodulator; an antiviral drug; HCV protease inhibitors (except VX 950); inhibitors of additional targets in the HCV life cycle (except for the NS3/4 protease); internal ribosome entry inhibitors, broad-spectrum viral inhibitors; or a cytochrome P-450 inhibitor; or a combination thereof. The additional agent is also selected from inhibitors of viral entry into cells.

Thus, in another embodiment, the invention provides a method comprising administering VX-950 and an additional antiviral drug (preferably an anti-HCV drug). Such antiviral drugs include, but are not limited to: immunomodulators (e.g., alpha, beta and gamma interferons or thymosins, polyethylene glycol derivatized interferon alpha compounds, and thymosins); other antiviral drugs (e.g., ribavirin, amantadine, and telbivudine); other inhibitors of hepatitis c protease (NS2-NS3 inhibitors and NS3-NS4A inhibitors); inhibitors of other targets in the HCV life cycle (including helicase, polymerase and metalloprotease inhibitors); an internal ribosome entry inhibitor; broad spectrum viral inhibitors such as IMPDH inhibitors (e.g., compounds described in U.S. Pat. Nos. 5,807,876, 6,498,178, 6,344,465 and 6,054,472; and compounds described in PCT publications WO 97/40028, WO 98/40381 and WO 00/56331; mycophenolic acid and derivatives thereof; and including but not limited to VX-497, VX-148 and VX-944); or any combination thereof.

Other drugs (e.g., non-immunomodulatory compounds or immunomodulatory compounds) may be used in combination with the compounds of the invention, including but not limited to those detailed in WO02/18369, which is incorporated herein by reference (see, e.g., page 273, lines 9-22 and page 274, lines 4-276, 11, the disclosure of which is expressly incorporated herein by reference).

Other drugs also include those described in various published U.S. patent applications. These published applications provide additional teachings of compounds and methods that can be used in conjunction with VX-950 in the methods of the invention, particularly for the treatment of hepatitis. It is contemplated that any such methods and compositions can be used in conjunction with the methods and compositions of the present invention. For the sake of brevity, the disclosure of these applications refers to the reference to publication numbers, but it should be noted that the disclosure of especially compounds is specifically incorporated herein by reference. Examples of such publications include the following U.S. patent application nos.: US 20040058982, US 20050192212, US 20050080005, US 20050062522, US 20050020503, US 20040229818, US20040229817, US 20040224900, US 20040186125, US 20040171626, US 20040110747, US 20040072788, US 20040067901, US20030191067, US 20030187018, US 20030186895, US 20030181363, US 20020147160, US 20040082574, US 20050192212, US20050187192, US 20050187165, US 20050049220, and US 20050222236.

Other drugs also include, but are not limited to: albuferon^TM(albumin-interferon α), available from Human Genome Sciences; PEG-(Pegylated interferon alpha-2 b, available from Schering Corporation, Kenilworth, N.J.); INTRON-Interferon alpha-2 b, available from Schering Corporation, Kenilworth, NJ); ribavirin (1- β -D-ribofuranosyl-1H-1, 2, 4-triazole-3-carboxamide, available from ICN Pharmaceuticals, Inc., Costa Mesa, Calif.; see Merck Index, entry 8365, twelfth edition);(ScheringCorporation，Kenilworth，NJ)；(Hoffmann-La Roche，Nutley，NJ)；(Pegylated interferon alpha-2a, available from Hoffmann-LaRoche, Nutley, NJ);(recombinant interferon alpha-2a, available from Hoffmann-La Roche, Nutley, NJ);(interferon alpha 2, available from Boehringer Ingelheim Pharmaceutical, inc., ridgfield, CT);(a mixture of purified natural alpha interferons (e.g., Sumiferon), available from Sumitomo, Japan);(interferon α n1, available from GlaxoWellcome ltd., Great Britain);(a mixture of natural alpha interferons produced by Interferon Sciences, available from Purdue Frederick Co., CT); an alpha interferon; natural interferon-alpha 2 a; natural interferon-alpha 2 b; pegylated alpha interferon 2a or 2 b; consensus alpha interferon (Amgen, inc., Newbury Park, CA);(Schering Plough, interferon alpha 2B + ribavirin); pegylated Interferon alpha (Reddy, K.R. et al, "efficiency and safety of Pegylated (40-kd) Interferon alpha-2a Complex with Interferon alpha-2a in Noncirrototic Patients with Chronic Hepatitis C (Efficacy versus safety of Pegylated (40-kd) Interferon alpha-2a and Interferon alpha-2a in non-cirrhotic Chronic Hepatitis C Patients)," Pegylated Interferon alpha-2a, and "Hepatology33, 433-438 (2001); composite interferon(Kao, J.H. et al, "efficiency of Consensus InterferonInterhe Treatment of Chronic HepatitisJ.Gastroenterol.Hepatol.15, 1418-; lymphoblastoid or "native" interferon; interferon tau (Clayette, P., et al, "IFN-tau, A New Interferon Type I with Antiretroviral activity")Pathol.Biol.(Paris)47, 553-; interleukin-2 (Davis, G.L., et al, "Future Options for the Management of hepatitis C.")Seminars in Liver Disease， 19103-112 (1999); interleukin-6 (Davis et al, "Future Options for the management of Hepatitis C,") "Seminars in Liver Disease19, 103-112 (1999); interleukin-12 (Davis, G.L., et al, "Future Options for the management of Hepatitis C.")Seminars in Liver Disease19, 103-112 (1999); and compounds that enhance the development of type 1 helper T cell responses (Davis et al, "Future Options for the Management of Hepatitis C,") "Seminars in Liver Disease,19, 103-112(1999)). Also included are compounds that stimulate interferon synthesis in cells (Tazulakhova, E.B., et al, "Russian Exception in screening, analysis, and Clinical Application of Novel Interferon Inducers")J.Interferon Cytokine Res.2165-73), including but not limited to: double-stranded RNA alone or in combination with tobramycin (tobramycin) and Imiquimod (Imiquimod) (3M Pharmaceuticals; Sauder, D.N. 'Immunomomodulatoryand pharmacological Properties of Imiquimod'J.Am.Acad.Dermatol.43S 6-11 (2000). See also WO02/18369, especially page 272, line 15 to page 273, line 8, the disclosure of which is hereby specifically incorporated by reference.

The skilled artisan recognizes that VX-950 is preferably administered orally. Although oral administration forms are under development, interferon is typically not administered orally. However, the methods or combinations of the present invention are not limited herein to any particular dosage form or regimen. Thus, the components of the combination of the invention may be administered separately, together or in any combination thereof. The skilled artisan recognizes that the dose of interferon is typically measured in IU (e.g., about 4 million IU to about 2 million IU). Interferons may also be administered in micrograms. For example, Peg-Intron has a standard dose of 1.0-1.5 μ g/kg/week and Pegasys 180 μ g/week.

In certain aspects, the method comprises administering the drug in two phases (an initial phase and a second phase). For example, the initial phase may be a period of less than about 12 or 24 weeks, while the second phase may be greater than or equal to about 12 weeks, e.g., the second phase may be 12-36 weeks. In certain embodiments, the second phase is 12 weeks. In a further embodiment, the second phase is 36 weeks. In certain embodiments, the initial phase and the second phase total about 24-48 weeks (e.g., 24, 36, or 48 weeks). In certain embodiments, the duration of the initial and second phases may be the same.

VX-950 can be administered during the initial phase, the second phase, or both. In some embodiments, VX-950 is administered only during the initial phase. When VX-950 is administered only during the initial phase, VX-950 can be administered alone or in combination with other drugs and one or more drugs are administered during the second phase. The additional agent may be one or more antiviral agents, one or more additional agents, or a combination thereof, as described herein. In some embodiments, the particular drug administered in the initial phase and the second phase is the same.

In some embodiments, the method comprises administering VX-950 for two weeks (initial phase), followed by administration of a combination of peginterferon alfa-2 a (Peg-IFN) and Ribavirin (RBV) for 22 weeks (second phase). In other embodiments, the method comprises administering VX-950 for two weeks (initial phase), followed by administration of a combination of Peg-IFN and RBV for 46 weeks (second phase).

In additional embodiments, the method comprises administering VX-950 in combination with Peg-IFN for two weeks (initial phase), followed by administration of a combination of Peg-IFN and RBV for 22 weeks (second phase). In other embodiments, the method comprises administering VX-950 in combination with Peg-IFN for two weeks (initial phase), followed by administration of a combination of Peg-IFN and RBV for 46 weeks (second phase).

In additional embodiments, the method comprises administering VX-950 in combination with Peg-IFN and RBV for two weeks (initial phase), followed by administration of the combination of Peg-IFN and RBV for 22 weeks (second phase). In other embodiments, the method comprises administering VX-950 in combination with Peg-IFN and RBV for two weeks (initial phase), followed by administration of the combination of Peg-IFN and RBV for 46 weeks (second phase).

In some embodiments, the method comprises administering VX-950 for four weeks (initial phase), followed by administration of a combination of Peg-IFN and RBV for 20 weeks (second phase). In other embodiments, the method comprises administering VX-950 for four weeks (initial phase), followed by administration of a combination of Peg-IFN and RBV for 44 weeks (second phase).

In yet a further embodiment, the method comprises administering VX-950 in combination with Peg-IFN for four weeks (initial phase), followed by administration of a combination of Peg-IFN and RBV for 20 weeks (second phase). In other embodiments, the method comprises administering VX-950 in combination with Peg-IFN for four weeks (initial phase), followed by administration of a combination of Peg-IFN and RBV for 44 weeks (second phase).

In additional embodiments, the method comprises administering VX-950 in combination with Peg-IFN and RBV for four weeks (initial phase), followed by administration of the combination of Peg-IFN and RBV for 20 weeks (second phase). In other embodiments, the method comprises administering VX-950 in combination with Peg-IFN and RBV for four weeks (initial phase), followed by administration of the combination of Peg-IFN and RBV for 44 weeks (second phase).

In some embodiments, any of the initial stages described above may be performed for about 12 weeks and the second stage may be performed for about 12 weeks. Alternatively, the initial phase may be performed for about 12 weeks and the second phase may be performed for about 24 weeks. In further aspects, the initial phase may be performed for about 12 weeks and the second phase may be performed for about 36 weeks.

In some embodiments, any of the foregoing initial stages may be performed for about 8 weeks and the second stage may be performed for about 16 weeks. Alternatively, the initial phase may be performed for about 8 weeks and the second phase may be performed for about 28 weeks. In further aspects, the initial phase may be performed for about 8 weeks and the second phase may be performed for about 40 weeks.

In some embodiments, the method comprises administering VX-950 in combination with Peg-IFN for less than 48 weeks. For example, the method comprises administering VX-950 in combination with Peg-IFN for less than 24 weeks.

In some embodiments, the method comprises administering VX-950 in combination with Peg-IFN and RBV for less than 48 weeks. For example, the method comprises administering VX-950 in combination with Peg-IFN and RBV for less than 24 weeks.

In some embodiments, the methods of the invention comprise administering VX-950 for about 2 weeks (or 2 weeks), followed by administration of Peg-IFN and ribavirin for about 22 weeks (or 22 weeks) or about 46 weeks (or 46 weeks).

Modeling data also indicates that VX-950 resistant mutants (e.g., V36A/M, T54A, R155K/T, A156SA156V/T, V36A/M-R155K/T and V36A/M-A156V/T) can be cleared primarily by administration of PEG-IFN and ribavirin for about 10-24 weeks (or 8-26 weeks) after VX-950 treatment (see FIGS. 18-21). Some of these regimens show a reduction in treatment time compared to current standard of care treatment regimens lasting 24-48 weeks.

In some embodiments, the methods of the invention are capable of achieving an RVR at week 4 and achieving a state that remains undetectable at week 12.

As shown in fig. 22, viral relapse 8-12 weeks after VX-950 treatment was associated with VX-950 resistant mutants and the relapse rate was essentially the same for 24 weeks or 48 weeks of treatment, particularly in subjects who exhibited a good initial response to treatment.

As shown in FIG. 23, treatment with VX-950, PEG-IFN and RBV for 12 weeks (and possibly as low as 8 weeks) appeared to be sufficient to eliminate wild-type virus.

Thus, the invention also provides methods for administering VX-950 in combination with an interferon. In certain embodiments, the interferon is administered for about 10 weeks (or 10 weeks), about 12 weeks (or 12 weeks), about 14 weeks (or 14 weeks). Ribavirin is also optionally administered in whole or in part of a regimen, including but not limited to a complete regimen.

In some embodiments, the methods of the invention comprise administering a combination of VX-950 and Peg-IFN for about 12 weeks (or 12 weeks).

In some embodiments, the methods of the invention comprise administering a combination of VX-950 and Peg-IFN for about 12 + -4 weeks (e.g., 8, 12, or 16 weeks).

In one embodiment, the methods of the invention comprise administering a combination of VX-950 and Peg-IFN for about 24 weeks (or 24 weeks).

In one embodiment, the methods of the invention comprise administering a combination of VX-950 and Peg-IFN for about 24 + -4 weeks (e.g., 20, 24, or 28 weeks).

For the avoidance of doubt, it is to be understood that the present invention includes, but is not limited to, the following arrangements: comprising administering VX-950 and interferon for about 8 weeks (or 8 weeks), followed by interferon for about 16 weeks (or 16 weeks), for a total treatment regimen of about 24 weeks (or 24 weeks). The following solutions are also provided: comprising administering VX-950 and interferon for about 12 weeks (or 12 weeks), followed by administration of interferon for about 12 weeks (or 12 weeks), for a total treatment regimen of about 24 weeks (or 24 weeks). The regimen optionally administers ribavirin in a whole or partial regimen, including but not limited to administering ribavirin in a complete regimen of about 24 weeks (or 24 weeks).

In one embodiment, the methods of the invention comprise administering a combination of VX-950, Peg-IFN and ribavirin for about 12 weeks (or 12 weeks).

In one embodiment, the methods of the invention comprise administering a combination of VX-950, Peg-IFN and ribavirin for about 12 weeks (or 12 weeks), followed by administration of Peg-IFN and ribavirin for about 12 weeks (or 12 weeks).

In one embodiment, the methods of the invention comprise administering a combination of VX-950, Peg-IFN and ribavirin for about 12 weeks (or 12 weeks), followed by administration of Peg-IFN and ribavirin for about 36 weeks (or 36 weeks).

In one embodiment, the methods of the invention comprise administering a combination of VX-950, Peg-IFN and ribavirin for about 24 weeks (or 24 weeks), followed by administration of PEGIFN and ribavirin for about 24 weeks (or 24 weeks).

In some embodiments, the method comprises providing a loading dose of VX-950(1250mg) followed by administration of 750mg q8h VX-950 plus a combination of Peg-IFN and ribavirin.

Cytochrome P450 monooxygenase ("CYP") inhibitors useful in conjunction with the present invention are expected to inhibit the metabolism of VX-950. Thus, the cytochrome P450 monooxygenase inhibitor should be in an amount effective to inhibit the metabolic effect of VX-950. Thus, the CYP is administered in an amount such that the bioavailability of VX-950, or contact with VX-950, is increased relative to VX-950 in the absence of the CYP inhibitor. CYP inhibitors include, but are not limited to: ritonavir (ritonavir) (WO 94/14436), ketoconazole (ketoconazole), oleandomycin (troleandomycin), 4-methylpyrazole, cyclosporine (cyclosporine), clomeprazole (clomethiazole), cimetidine (cimetidine), itraconazole (itraconazole), fluconazole (fluconazole), miconazole (miconazole), fluvoxamine (fluvoxamine), fluoxetine (fluxetine), nefazodone (nefazodone), sertraline (sertraline), indinavir (indinavir), nelfinavir (nelfinavir), amprenavir (amprenavir), furametprenavir (fosamprenavir 944), saquinavir (saquinavir), lopinavir (pinloivir), dithiin (acetylhridine), erythromycin (vxymycin-497 and vxymycin. Preferred CYP inhibitors are ritonavir, ketoconazole, oleandomycin, 4-methylpyrazole, cyclosporin and clomeprazole.

Methods for testing the ability of compounds to inhibit cytochrome P50 monooxygenase activity are known (see U.S. Pat. No. 6,037,157 and Yun et al, drug metabolism & displacement, 21, 403-. Methods for evaluating the effect of VX-950 and a CYP inhibitor co-administration on a subject are also known (US 2004/0028755). Any such method can be used in conjunction with the present invention to detect the pharmacokinetic effects of the combination.

One embodiment of the invention provides a method for administering an inhibitor of CYP3a4 and VX-950.

The methods herein may comprise administering or co-administering the following: a) a combination of VX-950 and an additional agent; or b) more than one dosage form of VX-950. Co-administration includes administration of each inhibitor in the same dosage form or in different dosage forms. When administered in different dosage forms, the inhibitor may be administered at different times, including any time period near the same time or near the administration of the other dosage forms. The individual dosage forms may be administered in any order. That is, any dosage form may be administered before, simultaneously with, or after the other dosage form.

VX-950 and any additional drugs can be formulated in separate dosage forms. Alternatively, VX-950 and any additional drugs can be formulated together in any combination in order to reduce the number of dosage forms administered to a patient. Any of the individual dosage forms may be administered simultaneously or at different times. It will be appreciated that the dosage form should be administered over a certain period of time so that its biological effect is beneficial.

In accordance with the regimens and dosage forms of the invention, VX-950 is present in an amount effective to reduce the viral load in a sample or patient, wherein the virus encodes the NS3/4A serine protease enzyme essential to the life cycle of the virus (or is present in an amount effective to carry out the methods of the invention), and a pharmaceutically acceptable carrier. Alternatively, the compositions of the present invention comprise additional agents as described herein. Each component may be present in separate compositions, combined compositions, or separate compositions.

If pharmaceutically acceptable salts of the compounds are used in these compositions, these salts are preferably derived from inorganic or organic acids and bases. Included among such acid salts are the following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, 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 amine salts, alkali metal salts (e.g., sodium and potassium salts), alkaline earth metal salts (e.g., calcium and magnesium salts), salts with organic bases (e.g., dicyclohexylamine salts, N-methyl-D-glucamine), and salts with amino acids (e.g., arginine, lysine, etc.).

In addition, the nitrogen-containing basic groups may be quaternized with the following agents: such as lower halogenated hydrocarbons (e.g., chlorine, bromine and methyl iodide, ethane, propane and butane), dialkyl sulfates (e.g., dimethyl sulfate, diethyl sulfate, dibutyl sulfate and diamyl sulfate), long chain halides (e.g., chlorine, bromine and decane iodide, dodecane, tetradecane and octadecane), halogenated aralkanes (e.g., benzyl bromide and phenethyl bromide), and the like. Water-soluble, fat-soluble or dispersible products are thereby obtained.

The compounds employed in the compositions and methods of the present invention may also be modified by the attachment of suitable functional groups to enhance selective biological properties. Such modifications are known in the art and include the following: increasing the biological permeability into a given biological system (e.g., blood, lymphatic system, central nervous system), increasing oral availability, increasing solubility to allow administration by injection, altering metabolism, and altering excretion rates.

Pharmaceutically acceptable carriers that can be used in these compositions include, but are not limited to: ion exchangers, aluminum oxide, 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, such as protamine sulfate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block copolymers, polyethylene glycol and wool fat.

According to a preferred embodiment, the composition of the invention is formulated for administration to a mammal, in particular a human, in the form of a medicament.

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, intranasally, buccally, vaginally or via an implantable reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional or intracranial injection or infusion techniques. Preferably the composition is administered orally or intravenously. More preferably, the composition is administered orally.

The compositions of the present invention in sterile injectable form may be in the form of aqueous or oily suspensions. These suspensions may be formulated according to the 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 parenterally-acceptable non-toxic diluent or solvent, for example as a solution in 1, 3-butanediol. Acceptable solvents and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, non-volatile oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including mono-or diglycerides. Fatty acids (e.g. oleic acid and its glyceride derivatives) are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils (e.g. 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 conventionally used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. For formulation purposes, other commonly used surfactants (e.g., tweens, spans) and other emulsifiers or bioavailability enhancers may also be used, which are commonly used in the preparation of pharmaceutically acceptable solid, liquid, or other dosage forms.

In the compositions of the present invention comprising VX-950 and an additional agent, VX-950 and the additional agent are present at a dosage level of about 10-100%, more preferably about 10-80% of the dosage normally administered in a monotherapy regimen.

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 administration, carriers which are commonly used include lactose and corn starch. Lubricating agents (e.g., magnesium stearate) are also typically added. For oral administration in the form of a capsule, useful diluents include lactose and dried corn starch. 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. Acceptable liquid dosage forms include emulsions, solutions, suspensions, syrups, and elixirs.

Alternatively, the pharmaceutical compositions of the present invention may be administered in the form of suppositories for rectal administration. These suppositories can 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 and release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of the invention may also be administered topically, particularly when the therapeutic target includes areas or organs accessible by topical application (including diseases of the eye, skin or lower intestinal tract). Suitable topical formulations are readily prepared for use in various of the areas or organs.

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

Applicants have demonstrated that VX-950 is bioavailable when administered orally. Thus, preferred pharmaceutical compositions of the present invention are formulated for oral administration.

For CYP inhibitors, dosage levels of about 0.001 to about 200mg/kg body weight per day will be typical. More typically at a dosage level of from about 0.1 to about 50mg/kg body weight per day or from about 1.1 to about 25mg/kg body weight per day.

Preferred dosages of ritonavir are described in U.S. patent No. 6,037,157 and the following references cited herein: U.S. Pat. No. 5,484,801, U.S. Pat. No. 08/402,690, and PCT publications WO 95/07696 and WO 95/09614.

Administration in connection with the present invention may be used as a chronic treatment or an acute treatment. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. A typical formulation will contain from about 5% to about 95% active compound by weight. Preferably, such formulations contain from about 20% to about 80% of the active compound.

If desired, a maintenance dose of a compound, composition or combination of the invention can be administered in improving the condition of the patient. Thus, the dose or frequency of administration, or both, may be reduced to a level that allows the improved condition to be maintained, depending on the symptoms, and treatment should be discontinued when the symptoms have been alleviated to a desired level. However, patients may require long-term intermittent treatment when there is any recurrence of disease symptoms.

It will also be understood that the particular dose and treatment regimen for any particular patient will depend upon a variety of factors including: the activity of the particular compound used, age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the judgment of the treating physician and the severity of the particular disease being treated, pre-treatment history, co-morbidities or concomitant medications, baseline viral load, race, duration of disease, liver function status, degree of liver fibrosis/cirrhosis, and the purpose of treatment (elimination of virus or clearance of virus in the circulation per transplant). The amount of active ingredient also depends on the particular compound, whether or not additional antiviral drugs are present in the composition, and the nature of the additional antiviral drugs.

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

Preferably the dosages herein are for use in vivo. Nevertheless, this is not meant to limit the application of the amount of VX-950 for any purpose. In another embodiment of the present invention, there is provided a method of pretreating a biological substance intended for administration to a patient, the method 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 (e.g., plasma, platelets, subpopulations of blood cells, etc.); organs (e.g., kidney, liver, heart, lung, etc.); sperm and eggs; bone marrow and its components; and other fluids (e.g., saline, glucose, etc.) infused to the patient.

The present invention also provides a method for preparing a composition comprising VX-950, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle, comprising the step of combining VX-950, or a pharmaceutically acceptable salt thereof, with a pharmaceutically acceptable carrier, adjuvant, or vehicle, wherein the amount of VX-950 in the composition is in accordance with any one of the embodiments of the invention. An alternative embodiment of the invention provides a method wherein the composition comprises one or more additional agents as described herein.

The present invention also provides a treatment regimen comprising a dosage of VX-950 or a pharmaceutically acceptable salt thereof as disclosed herein. In an alternative embodiment of the invention, the treatment regimen further comprises one or more additional agents as described herein.

The pharmaceutical composition may also be administered to the patient in the form of a "personal pack" containing the entire course of treatment in individual packages, usually blister packs. Personal bags have advantages over traditional prescriptions, where a pharmacist divides a medication from a bulk package into individual packages, enabling a patient to always access the package insert placed in the individual bag, which is often missing from traditional prescriptions. It has been shown that the placement of the package insert improves patient compliance with the physician's instructions.

It will be appreciated that the administration of the combination of the invention by means of a separate personal bag or personal kit for each formulation, containing packaging instructions instructing the patient on the correct use of the invention, is an additional feature required by the invention.

Another aspect of the invention is a package comprising VX-950 (in a dosage amount according to the invention) and information describing instructions for the use of the combination of the invention. Any of the compositions, dosage forms, treatment regimens, or other embodiments of the invention may be presented in a pharmaceutical package. In an alternative embodiment of the 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 kit for use in treating or preventing HCV infection (or in a further method of the invention) in a patient, the packaged kit comprising: single or multiple pharmaceutical formulations of each pharmaceutical component; a container for holding one or more pharmaceutical agents during storage and prior to administration and instructions for administering the pharmaceutical agents in a manner effective to treat or prevent HCV infection.

Accordingly, the present invention provides kits for the simultaneous or sequential administration of a dose of VX-950 (and optionally an additional drug). Typically, such kits will comprise, for example, a composition of each compound and optionally one or more additional agents in a pharmaceutically acceptable carrier (and one or more pharmaceutical preparations) and written instructions for simultaneous or sequential administration.

In another embodiment, there is provided a kit of parts comprising: one or more dosage forms for self-administration; containers (container means) for storing the dosage forms (preferably sealed) prior to storage and use; and instructions for administering the drug to the patient. The instructions will generally be written on the package insert, label, and/or other components of the kit, and the one or more dosage forms are as described herein. Each dosage form may be stored separately, such as in a layered sheet of metal foil-plastic with each dosage form separated from the other dosage forms in separate compartments or blisters, or the dosage forms may be stored in a single container (such as a plastic bottle). The kits of the present invention also typically include means for packaging the individual kit components (i.e., dosage form, container, and written instructions for use). Such packaging means may take the form of paper or carton, plastic or foil bags and the like.

The kits of the invention may encompass any aspect of the invention, such as any component, dosage form, treatment regimen, or pharmaceutical package.

The packages and kits of the invention optionally comprise a plurality of compositions or dosage forms. Thus, the invention shall encompass packages and kits containing one composition or more than one composition.

While certain exemplary embodiments are illustrated and described below, it will be appreciated that the compounds of the invention can be prepared according to the aforementioned conventional methods using suitable starting materials that are generally available to those skilled in the art.

All cited documents are incorporated herein by reference.

In order to provide a more thorough understanding of the present invention, preparation examples and test examples are given below. 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 supplements.

On the first day, with pancreatin: the replicon cells in monolayer were treated with the mixture of EDTA, and after removal, the cells were diluted with medium A to a final concentration of 100000 cells/mL. 10000. mu.L of medium containing 10000 cells were inoculated into each well of a 96-well tissue culture plate, followed by overnight culture in a tissue culture chamber at 37 ℃.

On day 2, 100% DMSO serial dilutions containing compound VX-950 were made in DMEM ("Medium B") containing 2% FBS, 0.5% DMSO and appropriate supplements to obtain solutions containing varying concentrations of VX-950. The final concentration of DMSO was maintained at 0.5% in each serial dilution.

Medium a in the replicon cells of the monolayer was removed and then medium B containing various concentrations of VX950 was added. Medium B without any compound was added to the other wells as a control.

Cells were incubated with medium B containing VX-950 solution or control for 48 hours at 37 ℃ in a tissue culture incubator. At the end of the 48 hour incubation period, medium B was removed, and the monolayer replicon cells were washed once with PBS and stored at-80 ℃ prior to RNA extraction.

The plates containing the treated monolayer of replicon cells were thawed and a quantity of another RNA virus, 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 with modifications to improve the efficiency and consistency of extraction. Finally, the total intracellular RNA containing HCV replicon RNA was eluted and stored at-80 ℃ for further processing.

Two sets of specific primers and probes are used to establish Taqman real-time RT-PCR quantitative determination. One set of primers and probes was used for HCV and the other set was used for BVDV. The total RNA extract from the treated HCV replicon cells was added to the PCR reaction to quantitatively determine both HCV RNA and BVDB RNA in the same PCR well. Failed experiments were labeled and eliminated based on the level of BVDV RNA in each well. The level of HCV RNA in each well was calculated according to a standard curve run on the same PCR plate. Percentage inhibition or reduction of HCV RNA levels resulting from treatment with VX-950 was calculated using DMSO or a control without VX-950 as inhibition 0%. IC was calculated from titration curves of arbitrary VX-950 concentration₅₀(concentration of VX-950 at which 50% inhibition of HCV RNA levels was observed).

The results of the replicon assay showed that VX-950 had significant inhibitory activity, its IC₅₀About 240ng/mL and IC₉₀About 476 ng/mL.

Example 2: HCV Ki assay protocol

HPLC microwell method for separating substrates and products of 5AB

The substrates used in this study were:

NH₂-Glu-Asp-Val-Val-(α)Abu-Cys-Ser-Met-Ser-Tyr-COOH。SEQ ID NO：1

a20 mM 5AB stock solution in DMSO was prepared with 0.2M DTT and stored in aliquots at-20 ℃. A buffer pH 7.8 containing 50mM HEPES, 100mM NaCl and 20% glycerol was prepared.

100 μ L of assay solution was prepared according to the following table:

	X1(μL)	concentration in assay
			Buffer solution	86.5	See above
5mM KK4A	0.5	25μM
			1M DTT	0.5	5mM
DMSO or VX-950	2.5	2.5% by volume
			50μM tNS3	0.05	25nM
250 μ M5 AB (initial reaction)	20	25μM

Buffer, KK4A, DTT and tNS3 were combined, dispensed in 78 μ L portions into wells of a 96-well plate, and then incubated at 30 ℃ for 5-10 minutes.

mu.L of VX-950 at an appropriate concentration was dissolved in DMSO (DMSO alone was used as a control) and added to each well, followed by incubation at room temperature for 15 minutes.

The reaction was started by adding 20. mu.L of 250. mu.M 5AB substrate (25. mu.M concentration corresponding to or slightly below the Km of 5 AB). The reaction mixture was incubated at 30 ℃ for 20 minutes, the reaction was stopped by adding 25. mu.L of 10% TFA and the mixture (in 120. mu.L aliquots) was transferred to an HPLC vial for analysis.

The SMSY product was separated from the substrate and KK4A by the following method:

the instrument comprises the following steps: agilent 1100

Degassing instrument G1322A

Binary pump G1312A

Automatic sampler G1313A

Column oven G1316A

Diode array detector G1315A

Column:

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

Column temperature: 40C

Sample introduction volume: 100 μ L

Solvent a ═ HPLC gradient of water + 0.1% TFA

Solvent B ═ HPLC gradient acetonitrile + 0.1% TFA

Time (minutes)	％B	Flow rate (mL/min)	The 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: 17 is divided into

Time after operation: 10 minutes.

Example 3: tolerability and pharmacokinetic Studies

VX-950 was detected in a randomized, double-blind, placebo-controlled, single dose escalation study. 25 healthy male volunteers were enrolled and received multiple single doses of VX-950 (3 doses of VX-950 taken at increasing dose levels at least 7 days apart) and one placebo dose per person.

Dosages of 25mg-1250mg were evaluated. A dose escalation plan is used that combines dose multiplication with a modified Fibonacci (Fibonacci) such that the escalation ratio is greater in the low dose range and smaller in the high dose range. The results show that VX-950 is well tolerated at all dose levels. No serious adverse events were reported in the study, and no increase in adverse events was observed as the dose level increased.

Pharmacokinetic analysis was performed using statistical kinetic methods. Fig. 1A and 1B illustrate the average concentration-time profiles. Selected deduced pharmacokinetic parameters are shown in figures 2A-2D. Pharmacokinetic analysis showed t of VX-950 uptake_maxThe median was 3 hours. Less than 2% of unchanged VX-950 is excreted in urine, suggesting that the drug is mainly cleared via metabolic pathways.

Example 4: infectious virus assay

Infectious virus assay demonstrated IC for VX-950₅₀196 ng/mL.

Example 5: therapeutic Effect of VX-950

VX-950 was tested in a randomized, placebo-controlled, multi-dose, single-blind (clamped) dose escalation study conducted in 24 healthy subjects and 36 hepatitis c positive subjects.

24 healthy subjects were divided into 3 groups of 8 subjects each. In each group 6 subjects received VX-950 and 2 subjects received placebo. VX-950 was administered to healthy subjects for 5 consecutive days at 450mg, 750mg, or 1250mg q8 h. Healthy subjects were between 18-65 years of age (including 18 and 65 years of age) and were hepatitis b, hepatitis c and HIV negative. The male body mass index is 18.5-29.0kg/m²(including 18.5 and 29.0kg/m²). Body mass index of womenIs 18.5-32.5kg/m²(including 18.5 and 32.5 kg/m)²)。

Hepatitis C (genotype 1) positive subjects were divided into 3 groups of 12 subjects, each receiving 450mg q8h, 750mg q8h, or 1250mg q12h VX-950 for 14 consecutive days. In each group 10 subjects received VX-950 and 2 subjects received placebo. 2 subjects in the 750mg group were withdrawn prior to dosing. All other 34 subjects completed the study.

Studies have shown that VX-950 is well tolerated at all dose levels and no serious adverse events were reported in the study; mild and moderate adverse events were reported. Of the HCV positive subjects receiving placebo, the 450mg q8h, 750mg q8h and 1250mg q12h groups, 33.2%, 10%, 12.5% and 30% were primary treatments, respectively.

HCV positive subjects were tested after treatment to monitor the return of HCV RNA levels to baseline.

TABLE 1 Baseline characteristics of the subjects

Samples from 4 patients were classified as genotype 1 because the assay did not determine whether they were genotype 1a or genotype 1 b.

Abbreviations: BMI (body mass index); HCV (hepatitis c virus); q8h (every 8 hours); q12h (every 12 hours); SD (standard deviation).

Study of VX04-950- "101" with respect to baseline HCV RNA changes

TABLE 2 maximum variation of HCV RNA species

Values were number of people (%): q8h, every 8 hours; q12h, every 12 hours

Example 6: VX-950 preparation

Oral dosage formulations were prepared as follows. VX-950 and povidone K29/32 were dissolved in methylene chloride and sodium lauryl sulfate was added to and dispersed in the VX-950 solution to form a homogeneous suspension. The suspension was spray dried using an inlet temperature of 90 ℃ and an outlet temperature of 56 ℃ and the product was collected from the cyclone. The spray-dried suspension was dried in a fluidized bed at 75 ℃ for 8 hours. The resulting powder was pre-measured in a glass vial and suspended in water (30mL) immediately prior to administration to the subject. Each vial was washed with 3 aliquots of water alone during the dosing period, the total volume of water being 90 mL.

Example 7: validation of VX-950 in human plasma

Assays for detecting VX-950 concentration in human plasma are performed by methods well known in the art, see, e.g., Wasley, a. et al, Semin Liver dis, 20: 1-16, 2000; alter, h.j. et al, semin. liver dis, 20: 17-35, 2000; brown, r.s.jr. et al, Liver transform, 9: chapters 10-13, 2003; defrncesco, r. et al, Nature, 436 (7053): 953-960, 2005; bowen, d.g., et al, j.hepato1, 42: 408 417, 2005; hofnagle, j.h., Hepatology, 36: chapter 21-29, 2002, Brown, r.s.jr. et al, Nature, 436 (7053): 973-978, 2005; and Chisari, f.v., Nature, 436 (7053): 930-932, 2005.

Specifically, the following VX-950 solutions were prepared and stored in sealed borosilicate tubing (11.5mL) at-20 ℃:

storage solution: 2-propanol containing 961. mu.g/mL VX-950 (10.0mL)

Dilution of stock solution 1: 2-propanol containing 96.1. mu.g/mL VX-950 (5.00mL)

Dilution stock solution 2: 2-propanol containing 9.61. mu.g/mL VX-950 (10.0mL)

Dilution stock solution 3: 2-propanol containing 0.961. mu.g/mL VX-950 (10.0mL)

An internal standard stock solution containing 1.00mg/mL compound 1 (an analog structurally close to VX-950) in 5.00mL 2-propanol was prepared and stored at-20 ℃ in a sealed borosilicate tube (11.5 mL). A working solution containing the same Compound 1 was prepared containing 300ng/mL Compound 1 in 100mL acetonitrile and stored in a sealed borosilicate tube (100mL) at-20 ℃.

Sample preparation: 100 μ L of plasma and 100 μ L of internal standard working solution (or acetonitrile as blank sample) were added to the extraction tube. After vortex mixing for 30 seconds, 500. mu.L of toluene was added and extraction was performed by vortex mixing for 30 seconds. After centrifugation at 3000rpm for 5 minutes at 4 ℃, the aqueous layer was frozen in a mixture of acetone and dry ice, and the organic layer was transferred to another extraction tube. 50 μ L of 2, 2-dimethoxypropane was added at about 30 ℃ and the sample was evaporated to dryness under nitrogen. The residue was redissolved in 300. mu.L of a mixture of heptane and acetone (90: 10, vol.) [ or a mixture of heptane and THF (80: 20, vol.) ] by vortex mixing for 60 seconds. Samples were transferred to injection vials and 60 μ L aliquots of the samples were injected into the chromatography system and analyzed under the following chromatographic conditions:

mobile phase: (isocratic elution) heptane/acetone/methanol (80: 19: 1, volume ratio)

The composition solvent is as follows: acetonitrile/acetone/methanol/formic acid (40: 60: 1, volume ratio)

Column temperature: -1 ℃ C

Flow rate: 1.00 mL/min (including 0.750 mL/min of mobile phase and 0.250 mL/min of component solvent, all transferred to detector)

Sample introduction volume: 60 μ L

Temperature of the automatic sampler: 3 deg.C

Example 8: combination therapy of VX-950

Combination treatment of VX-950 was performed to test the safety and antiviral response of VX-950. Specifically, the study included 12 primary treatment patients infected with genotype 1 HCV. All patients received VX-950(750mg q8h), Peg-IFN α -2a ("PEG-IFN", 180 μ g per week) and RBV (1000 or 1200mg per day) for a period of 28 days. Upon completion of the 28 day study, patients began receiving subsequent treatment with Peg-IFN α -2a and RBV at the clinical care of their physicians. In the case of Peg-IFN-2a/RBV treatment, additional HCV RNA evaluations were performed at the discretion of the attending physician. The above evaluations included evaluation at time points after 4 th, 8 th and 14 th weeks of treatment after study.

The results show that the VX-950/Peg-IFN/RBV combination is well tolerated and without serious adverse events in the 28-day study. The profile of adverse events observed is consistent with the profile common to treatment with Peg-IFN/RBV combinations. All patients demonstrated a response to the study regimen, suggesting that VX-950 has a rapid and significant antiviral effect. Specifically, the HCV RNA in the plasma of 2 patients reached undetectable levels (< 10IU/mL, Roche) within 8 days after the start of dosingAssay), whereas HCV RNA was undetectable in all patients at the end of the 28-day study dosing period. HCV RNA levels were undetectable in 11 patients at 12 weeks of follow-up treatment (with Peg-IFN/RBV) after completion of the study dose for 28 days. All patients continued to receive Peg-IFN/RBV treatment and were followed up for response according to standard practice. 7 patients received a total of 48 weeks of treatment and achieved a Sustained Virological Response (SVR). A patient who received Peg-IFN/RBV for 18 weeks only (22 weeks total treatment) subsequently discontinued treatment, but reachedThe SVR is reached. Two patients experienced viral rebound at 12 and 24 weeks of treatment, and these two patients received no follow-up. Overall, 8 out of 10 patients had effective results, reaching SVR. The profile of side effects observed during post-study dosing was consistent with the expected profile of Peg-IFN/RBV treatment.

Eight patients were observed to reach SVR, including 1 patient who completed only 22 weeks of treatment, suggesting that a VX-950 based regimen may increase SVR rates compared to current therapy.

Comparing current therapies

For chronic HCV patients with genotype 1, current therapy typically consists of 48-week treatment with only pegylated interferon alpha-2 a/2b (Peg-IFN-2a) and RBV, of which only about 50% of patients with genotype 1 HCV achieve SVR and patients typically show poor tolerance to treatment.

Example 9: study at phase 1b

VX-950 has rapid and powerful antiviral activity as a single agent or in combination with Peg-IFN-2a and is well tolerated for 14 days. The study was designed to provide information on HCV kinetics 14 days after treatment with VX-950 and Peg-IFN-2 a.

The study randomized twenty first-treatment patients with chronic genotype 1 hepatitis c infection into three groups (table 1). At the completion of the 14-day study, 19 of 20 patients selected Peg-IFN-2a/RBV, starting within 5 days after the 14-day dosing period; while another patient decided to use a combination of Peg-IRN-2a and RBV. After completion of the study-prescribed follow-up at weeks 1 and 12, a follow-up (clinicvisit) was performed at the discretion of the investigator. Nineteen patients received follow-up within 24 weeks after completion of study dosing. After discussion with the attending physician, ten patients (4 with VX-950, 6 with VX-950/Peg-IFN-2a) stopped Peg-IFN-2a/RBV treatment at 24 weeks. The schedule of the patient is shown in table 1 below.

Table 1: patient scheduling

On the last day of follow-up after the study (12 weeks after the last follow-up in the study), HCV RNA levels were not detectable in patients initially randomized in both the VX-950 and VX-950/Peg-IFN-2a groups alone and continued to be treated with Peg-IFN-2 a/RBV. The data are provided in table 2 below.

Table 2: study of non-detectable HCV RNA in groups during post-treatment

^aCOBAS Taqman HCV RNA assay. Roche Molecular Diagnostics

^bTaqman HCV RNA assay (15IU/mL) and 5 IU/mL): after the study

As shown in Table 3 below, of the 10 patients who discontinued post-study Peg-IFN-2a/RBV treatment after 24 weeks total treatment, 2 of the 4 patients who initially received VX-950 alone demonstrated undetectable HCV RNA levels in plasma at 12 weeks follow-up after Peg-IFN-2a discontinuation; of the 6 patients who initially received VX-950/Peg-IFN-2a, 5 of the patients who after 12 weeks of Peg-IFN-2a withdrawal demonstrated undetectable HCV RNA levels in plasma.

Table 3: HCV RNA undetectable in groups after Peg-IFN-2a/RBV withdrawal

Situation(s)

	Number of undetectable persons (N) treated with peg-IGN-2a/RBV HCV RNA after 24 weeks of study	Number of patients (N)/number of patients (N) who discontinued peg-IFN-2a/RBV at week 24	HCV RNA undetectable by people (N)/number of people (N) in 12 weeks follow-up after peg-IFN-2a/RBV withdrawal
				VX-950(N-7)	7*	4/7	2/4
VX-950/Peg-IFN-2a(N-8)	8	6/8	5/6

	Number of undetectable persons (N) treated with peg-IGN-2a/RBV HCV RNA after 24 weeks of study	Number of patients (N)/number of patients (N) who discontinued peg-IFN-2a/RBV at week 24	HCV RNA undetectable by people (N)/number of people (N) in 12 weeks follow-up after peg-IFN-2a/RBV withdrawal
				Peg-IFN-2a(N-4)	3	0/4	N/A

One patient selected Peg-IFN-2 a/RBV.

In the follow-up after 24 weeks of the study, all patients initially randomized in the VX-950 group and continuing treatment with Peg-IFN-2a/RBV remained undetectable for HCV RNA. Viral load data for follow-up (Peg-IFN-2a/RBV) after early (12 weeks) treatment was consistent with the model, suggesting that the time required to reach SVR correlates with the kinetics of early viral clearance. Of 15 patients who received 14 days VX-950 and optionally combined with Peg-INF and then re-treated with Peg-INF/RBV for 22 or 46 weeks, 10 reached SVR.

At week 12, HCV RNA was undetectable in all 8 patients who received the initial combination of VX-950 and PEG-IFN, and 5 of 7 patients who received VX-950 alone. At week 24, HCV RNA was undetectable in all 15 patients who received VX-950. 10 patients (8 with 6 of PEG-IFN and VX-950 and 7 with 4 of VX-950 alone) chose to discontinue PEG-IFN/RBV at week 24, while 5 patients continued to be treated with PEG-IFN/RBV for a total of 48 weeks. All groups were followed for 24 weeks. Of the patients who received VX-950 (alone or in combination with PEG-IFN) for at least 14 days and then started PEG-IFN containing RBV, 7 out of 10 patients treated for 24 weeks and 3 out of 5 patients treated for 48 weeks reached SVR.

All documents cited herein are incorporated herein by reference.

Other embodiments

While a number of embodiments and examples of the present invention are described herein, it will be apparent that these embodiments and examples can be modified to provide additional embodiments and examples utilizing the pharmaceutical formulations and drug regimens of the present invention. It is, therefore, to be understood that the scope of the invention is defined by the appended claims rather than by the specific embodiments shown in the examples above.

Sequence listing

<110> Futex drugs Co., Ltd

<120> dosage forms comprising VX-950 and dosing regimens thereof

<130>125805/00487

<160>1

<170>PantentIn version 3.3

<210>1

<211>10

<212>PRT

<213> Artificial

<220>

<223> synthetically prepared peptide

<220>

<221>MOD_RES

<222>(5)..(5)

<223> alpha-aminobutyric acid

<400>1

Glu Asp Val Val Xaa Cys Ser Met Ser Tyr

1 5 10

Claims (54)

1. A therapeutic regimen comprising administering VX-950 in an amount of about 100mg to about 1500mg, wherein the amount of VX-950 is administered once, twice, or three times daily.

2. The therapeutic regimen of claim 1, wherein the amount of VX-950 is about 300mg to about 1500 mg.

3. The therapeutic regimen of claim 1, wherein the amount of VX-950 is about 300mg to about 1250 mg.

4. The therapeutic regimen of claim 1, wherein the amount of VX-950 is about 450 mg.

5. The therapeutic regimen of claim 1, wherein the amount of VX-950 is about 750 mg.

6. The therapeutic regimen of claim 1, wherein the amount of VX-950 is about 1250 mg.

7. A method for treating or preventing a hepatitis c infection in a patient comprising administering to a patient in need thereof about 100mg to about 1500mg of VX-950.

8. The method of claim 7, wherein the amount of VX-950 is about 300mg to about 1500 mg.

9. The method of claim 7, wherein the amount of VX-950 is about 300mg to about 1250 mg.

10. The method of claim 7, wherein the amount of VX-950 is about 450 mg.

11. The method of claim 7, wherein the amount of VX-950 is about 750 mg.

12. The method of claim 7, wherein the amount of VX-950 is about 1250 mg.

13. The method of any one of claims 7-12, wherein the amount of VX-950 is administered once daily.

14. The method of any one of claims 7-12, wherein the amount of VX-950 is administered twice daily.

15. The method of any one of claims 7-12, wherein the amount of VX-950 is administered three times daily.

16. The method of any one of claims 7-12, further comprising administering to the patient an immunomodulator, an antiviral drug, an additional inhibitor of HCV NS3/4A protease, an inhibitor of a target other than NS3/4A protease in the HCV life cycle, an inhibitor of internal ribosome entry, a broad-spectrum viral inhibitor, an additional inhibitor of cytochrome P-450, an inhibitor of viral entry into cells, or a combination thereof.

17. The method of claim 16, wherein the immunomodulator is alpha, beta or gamma interferon or thymosin; the antiviral drug is ribavirin, amantadine or telbivudine; while inhibitors of additional targets in the HCV life cycle are inhibitors of helicase, polymerase or metalloprotease of HCV.

18. A method for treating a patient infected with a hepatitis c virus, comprising administering to a patient in need thereof about 750mg VX-950 three times daily, once every 8 hours.

19. A method for treating a patient infected with hepatitis C virus comprising administering to a patient in need thereof an amount effective to reduce hepatitis C virus RNA in plasma by at least about 2log₁₀VX-950 in an amount of (a).

20. The method of claim 19, wherein the amount of VX-950 is effective to reduce hepatitis c viral RNA by at least about 4log in plasma₁₀。

21. The method of claim 19, wherein the amount of VX-950 is effective to reduce the hepatitis c viral RNA to undetectable levels in the plasma.

22. A method for treating a patient infected with a hepatitis c virus, comprising administering to a patient in need thereof VX-950 in an amount effective to achieve a sustained virological response.

23. The method of claim 22, wherein said patient is infected with hepatitis c virus genotype 1.

24. A method for treating liver injury, hepatitis, steatosis, fatty liver, NAFLD, NASH, alcoholic steatosis, or reye's syndrome in a patient, comprising administering to a patient in need thereof VX-950 in an amount of about 1350mg, about 2250mg, or about 2500mg per day.

25. The method of claim 24, wherein about 450mg or about 750mg of VX-950 is administered every 8 hours per day, or about 1250mg of VX-950 is administered every 12 hours per day.

26. A method for protecting the liver of a patient comprising administering to a patient in need thereof about 1350mg, about 2250mg, or about 2500mg of VX-950 per day.

27. The method of claim 26, wherein about 450mg or about 750mg of VX-950 is administered every 8 hours per day, or about 1250mg of VX-950 is administered every 12 hours per day.

28. A method for reducing ALT levels in a patient comprising administering to a patient in need thereof a therapeutically effective amount of VX-950.

29. The method of claim 28, wherein about 1350mg, about 2250mg, or about 2500mg of VX-950 is administered to the patient per day.

30. The method of claim 29, wherein about 450mg or about 750mg of VX-950 is administered every 8 hours per day, or about 1250mg of VX-950 is administered every 12 hours per day.

31. The method of any one of claims 28-30, wherein the patient is infected with HCV.

32. The method of any one of claims 28-30, wherein the patient is not infected with HCV.

33. A method for providing VX-950 to a patient in need thereof, comprising administering to the patient a dosage form comprising VX-950, wherein the dosage form provides the patient with a mean plasma concentration of VX-950 of at least about 750ng/mL after administration.

34. The method of claim 33, wherein the mean plasma concentration of VX-950 is about 750ng/mL to about 1250ng/mL after the administration.

35. The method of claim 34, wherein the mean plasma concentration of VX-950 is about 1000ng/mL after the administration.

36. The method of any of claims 33-35, wherein the mean plasma concentration of VX-950 is obtained or attained within 3 hours after administration.

37. The method of claim 36, wherein the mean plasma concentration of VX-950 is obtained or attained within 2 hours after administration.

38. The method of claim 37, wherein the mean plasma concentration of VX-950 is obtained or attained within 1 hour after the administration.

39. The method of any one of claims 33-38, further comprising maintaining a trough plasma level of VX-950 of about 750ng/mL to about 1500ng/mL over a 24-hour period.

40. A method of treating a patient infected with HCV, comprising administering to a patient in need thereof a dosage form comprising VX-950 that is effective over 24 hours, wherein the dosage form is administered to maintain a trough plasma level of VX-950 of about 750ng/mL to about 1500ng/mL over 24 hours.

41. The method of claim 40, wherein the dosage form is administered to maintain a trough plasma level of VX-950 of about 1000ng/mL to about 1500ng/mL over a 24 hour period.

42. The method of any one of claims 40-41, wherein the dosage form comprises about 750mg VX-950.

43. The method of claim 40, wherein the dosage form is administered three times per day.

44. The method of any one of claims 33-43, wherein the mean plasma concentration or trough level of VX-950 is maintained for about 12 weeks.

45. The method of any one of claims 7-44 further comprising the step of administering an interferon or ribavirin.

46. The method of claim 45, wherein the interferon is a pegylated interferon.

47. The method of claim 45 or 46, wherein the interferon is administered in an amount of about 180 μ g/mL.

48. A pharmaceutical formulation comprising VX-950, povidone-alkane, and sodium lauryl sulfate.

49. The pharmaceutical formulation of claim 48, wherein the polyvinylpyrrolidone is povidone K29/32.

50. A treatment regimen comprising administering VX-950 in an initial phase that lasts for a period of less than about 12 weeks.

51. The therapeutic regimen of claim 50, further comprising administering the pegylated interferon over a second phase, wherein the second phase occurs after the initial phase and lasts for less than 36 weeks.

52. The therapeutic regimen of claim 50, further comprising administering a pegylated interferon in combination with VX-950 in an initial phase.

53. The therapeutic regimen of claim 50, further comprising administering ribavirin in association with VX-950 and pegylated interferon during an initial phase.

54. The therapeutic regimen of claim 50, further comprising administering ribavirin in association with pegylated interferon in a second phase.