OA17352A - Compositions and methods for treating hepatitis C virus. - Google Patents

Abstract

Disclosed herein are a composition and unit dosage form for the treatment of hepatitis C virus (HCV) infection comprising GS-7977 and at least one pharmaceutically acceptable excipient, as well as methods for making said composition and unit dosage form. Also disclosed herein is a method of treating a subject, preferably a human, infected with hepatitis C virus, said method comprising administering to the subject for a time period an effective amount of GS-7977 and an effective amount of ribavirin. In one aspect, the method comprises administering to the subject an interferon-free treatment regimen comprising an effective amount of GS-7977 and an effective amount of ribavirin. In a particular aspect, the method is sufficient to produce an undetectable amount of HCV RNA in the subject for at least 12 weeks after the end of the time period.

Description

Disclosed herein are a composition and unit dosage form for the treatment of hepatitis C virus (HCV) infection comprising GS-7977 and at least one pharmaceutically acceptable excipient, as well as methods for making the said composition and unit dosage form. Also disclosed herein is a method of treating a subject, preferably a human, infected with hepatitis C virus, said method comprising administering to the subject for a time period an effective amount of GS-7977 and an effective amount of ribavirin. In one aspect, the method comprises administering to the subject an interferon-free treatment regimen comprising an effective amount of GS-7977 and an effective amount of ribavirin. In a particular aspect, the method is sufficient to produce an undetectable amount of HCV RNA in the subject for at least 12 weeks after the end of the time period.

Background

Hepatitis C virus (“HCV”) infection is a major health problem that leads to chronic liver disease, such as cirrhosis and hepatocellular carcinoma, in a substantial number of infected individuals, estimated by the World Health Organization to be about 3% of the world’s population. (World Health Organization, Hepatitis C (2002).) According to the U.S. Centers for Disease Control and Prévention, HCV is the most common blood-bome infection in the United States, with an estimated 3.2 million people (1.8%) chronically infected in the United States alone. (U.S. Centers for Disease Control and Prévention, Viral Hepatitis Surveillance - United States, 2010; U.S. Centers for Disease Control and Prévention, Morbidity and Mortality Weekly Report 70(17): 537-539 (May 6, 2011).) An estimated 150-180 million individuals are chronically infected with HCV worldwide, with 3 to 4 million people infected each year. (World Health Organization, Hepatitis C, Fact Sheet No. 164 (July 2012); Ghany et al., Hepatology (2009) 49(4): 1335-1374.) Once infected, about 20% of people clear the virus, but the rest can harbor HCV for the rest of their lives. Ten to twenty percent of chronically infected individuals eventually develop liver-destroying cirrhosis or cancer. (Naggie et ak, J. Antimicrob. Chemother. (2010) 65: 2063-2069.) The viral disease is transmitted parenterally by contaminated blood and blood products, contaminated needles, or sexually and vertically from infected mothérs or carrier mothers to their offspring.

The HCV virion is an enveloped positive-strand RNA virus with a single oligoribonucleotide genomic sequence of about 9600 bases which encodes a polyprotein of about 3,010 amino acids. The protein products of the HCV gene consist of the structural proteins C, El, and E2, and the non-structural proteins NS2, NS3, NS4A and NS4B, and NS5A and NS5B. The nonstructural (“NS”) proteins are believed to provide the catalytic machinery for viral réplication. The NS3 protease releases NS5B, the RNA10 dépendent RNA polymerase, from the polyprotein chain. HCV NS5B polymerase is required for the synthesis of a double-stranded RNA from a single-stranded viral RNA that serves as a template in the réplication cycle of HCV. Therefore, NS5B polymerase is considered to be an essential component in the HCV réplication complex. (K. Ishi, et al, Hepatology (1999) 29: 1227-1235; V. Lohmann, et al., Virology (1998) 249: 108-118.)

Inhibition of HCV NS5B polymerase prevents formation of the double-stranded HCV RNA and therefore constitutes an attractive approach to the development of HCV-specific antiviral thérapies.

A number of potential molecular targets for drug development of direct acting antivirals as anti-HCV therapeutics hâve now been identified including, but not limited to, the NS2-NS3 autoprotease, the N3 protease, the N3 helicase, and the NS5B polymerase. The RNA-dependent RNA polymerase is essential for réplication of the single-stranded, positive sense, RNA genome, and this enzyme has elicited significant . interest among médicinal chemists. Another auxiliary protein of HCV is referred to as NS5A. The NS5A nonstructural protein is a phosphoprotein, with no apparent enzymatic activity; however it acts as a multifunctional regulator of cellular pathways, including host cell growth, immunity and innate immunity, and virus réplication. (Appel et al., J.

Virol. (2005) 79: 3187-3194; Evans et al., Proc. Natl. Acad. Sci. USA (2004) 101: 1303813043; Gale et al., Nature (2005) 436: 939-945; Gale et al., Virology (1997) 230: 217227; Ghosh et al., J. Gen. Virol. (1999) 80(Pt 5): 1179-1183; Neddermann et al., J. Virol.

(1999) 73: 9984-9991; Polyak et al., Hepatology (1999) 29: 1262-1271; Shimakami et al.,

J. Virol. (2004) 78: 2738-2748; Shirota et al., J. Biol. Chem. (2002) 277: 11149-11155; and Tan et al., Proc. Natl. Acad. Sci. U. S. A. (1999) 96: 5533-5538.) NS5A is associated with host cell membranes through its N-terminal amphipathic hélix, where it is a part of the réplication complex. (Elazar et al., J. Virol. (2004) 78: 11393-11400 and Penin et al., J. Biol. Chem. (2004) 279: 40835-40843.) Recent studies suggest that NS5A is organized into three domains: the first 213 amino acids in the N-terminal domain constitutes domain I and contains a zinc binding motif suggesting that the protein is a zinc metalloprotein and domains II and III are in the C-terminal région of the protein. (Tellinghuisen et al., J. Biol. Chem. (2004) 279:48576-48587 and Tellinghuisen et al., Nature (2005) 435: 374-379.) NS5A exists in two phosphorylated forms: a basal form of 56 kD and a hyperphosphorylated form of 58 kD. The protein is phosphorylated at spécifie sites, primarily on serine residue within domains II and 111, by host cell kinases. (Ide et al., Gene (1997) 201: 151-158; Kaneko et al., Biochem. Biophys. Res. Commun. (1994) 205: 320-326; Katze et al., Virology (2000) 278: 501-513; Reed et al., J. Biol. Chem. (1999) 274: 28011-28018; Reed et al., J. Virol. (1997) 71: 7187-7197; and Tanji et al., J. Virol. (1995) 69: 3980-3986.)

The initially-approved standard of care (“SOC”) for the treatment of chronic HCV infection is a combination therapy with pegylated interferon alfa-2a or pegylated interferon alfa-2b (collectively “peginterferon” or “PEG”) used alone or in combination with ribavirin (“RBV”). The primary goal of treatment for chronic hepatitis C is a sustained virologie response (“SVR”), which refers to an undetectable level of sérum HCV RNA maintained.for a period of time post-treatment. Host factors including âge, ... body weight, race, and advanced fibrosis influence the outcome of treatment (Dienstag and McHutchison Gastroenterology (2006)130: 231-264 and Missiha et al., Gastroenterology (2008) 134: 1699-1714), but are poor predictors of response. In contrast, viral factors like the génotype and the on-treatment pattern of viral response can be used to détermine the likelihood of treatment success and guide treatment duration individually, and they hâve proven to be very useful in clinical practice. (Ge et al., Nature (2009)461:399-401.)

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In spite of an encouraging response in some patients to SOC treatment, the overall response to peginterferon/ribavirin combination therapy among patients infected with Hepatitis C virus is only about 50%. SVR rates are <50% for patients infected with génotype 1 HCV treated with a prolonged duration (48-72 weeks) of peginterferon/ribavirin therapy. (Naggie et al., J. Antimicrob. Chemother. (2010) 65: 2063-2069.) Accordingly, there is a need to provide a therapy resulting in improved SVR compared to the outcome of treatment with peginterferon alone or in combination with ribavirin. There is also a need to provide a therapy that reduces the time in which patients show evidence of complété viral suppression (négative HCV status) following the initiation of treatment.

Peginterferon alfa-2a (PEG-IFN-à-2a or peginterferon a-2a), marketed under the trademark PEGASYS®, is an antiviral administered by subcutaneous injection indicated for, among other things, treatment of chronic hepatitis C (CHC) when administered alone or in combination with ribavirin. PEGASYS® is indicated for the treatment of CHC in patients with compensated liver disease not previously treated with interferon alpha, in patients with histological evidence of cirrhosis and compensated liver disease, and in adults with CHC/HIV co-infection. Combination therapy using PEG-IFNa-2a and ribavirin is recommended unless the patient has contraindication to or signifïcant intolérance to ribavirin.

Peginterferon alfa-2b (PEG-IFN-a-2b or peginterferon a-2b”), marketed under the trademark PEGINTRON®, is also administered by subcutaneous injection and is indicated for use alone or in combination with ribavirin to treat CHC in patients with compensated liver disease. Like PEG-IFN-a-2a, PEG-IFN-a-2b has undesirable side effects.

Ribavirin (RBV), marketed under the trademark COPEGUS®, is a nucleoside analogue indicated for the treatment of CHC virus infection in combination with peginterferon in patients 5 years of âge and older with compensated liver disease not previously treated with peginterferon, and in adult CHC patients co-infected with HIV. Ribavirin alone is not approved for the treatment of CHC. (COPEGUS® FDA-approved label, revised 08/2011.) Clinical trials hâve shown that ribavirin alone can normalize

17352“ alanine aminotransferase (“ALT”) levels transiently during the course of treatment in some patients with CHC infections. However, these studies hâve reported that ribavirin alone did not reduce HCV RNA levels during or after therapy arid did not produce any sustained virologie response. (Di Bisceglie et al., Ann. Intem. Med. (1995) 123(12): 897903; Dusheiko et al., J. Hepatology (1996) 25: 591-598; Bodenheimer, Jr., et al., Hepatology (1997) 26(2}: 473-477.) One clinical study reported observing a decrease in HCV RNA from treatment with ribavirin monotherapy (1.0 to 1.2 g daily for 24 weeks); however, the observed HCV RNA decrease was transient and no patient receiving ribavirin monotherapy cleared HCV RNA. (Pawlotsky et al., Gastroenterology (2004) 126:703-714.)

Treatment of CHC using peginterferon alone or in combination with ribavirin has several disadvantages. First and foremost, this therapy is not effective for many patients. For instance, certain Phase 3 clinical trials using the combination of peginterferon and ribavirin reported SVR rates of 54 to 63%, but additional studies show that the SVR rates may be much lower in certain populations. (Feurstadt et al., Hepatology (2010) 51(4): 1137-1143.) Second, use of peginterferon and ribavirin is associated with certain adverse events. For instance, the boxed waming on the PEGASYS® label states that use of peginterferon may cause or aggravate fatal or life-threatening neuropsychiatrie, autoimmune, ischémie, and infectious disorders. (PEGASYS® (peginterferon alfa-2a) FDA-approved label, revised 09/2011.) Additionally, the boxed waming on the COPEGUS® label states that ribavirin adverse effects may include hemolytic anémia and that significant tératogénie and embryocidal effects hâve been demonstrated in ail animal species exposed to ribavirin. (COPEGUS® (ribavirin) FDA-approved label, revised 08/2011.) Finally, the peginterferon/ribavirin treatment protocol is quite expensive. Given these disadvantages, there has been a recognized need to develop new anti-FICV drug substances and treatment regimens.

The FDA recently approved two additional drug products for the treatment of génotype 1 CHC, boceprevir and telaprevir, both of which are HCV NS3/4 protease inhibitors. Boceprevir, marketed under the trademark V1CTRELIS®, is indicated for the treatment of génotype 1 CHC infection, in combination with interferon and ribavirin, in adult patients (>18 years of âge) with compensated liver disease, including cirrhosis, who are previously untreated or who hâve failed previous interferon and ribavirin therapy. Telaprevir, marketed under the trademark INCIVEK®, is indicated, in combination with interferon and ribavirin, for the treatment of génotype 1 CHC in adult patients with compensated liver disease, including cirrhosis, who are treatment-naïve or who hâve been previously treated with interferon-based treatment, inclüding prior nul! responders, partial responders, and relapsers. Both boceprevir and telaprevir are approved for administration in combination with peginterferon and ribavirin only; neither is approved for monotherapy or for administration with ribavirin alone. (INCIVEK® (telaprevir) FDAapproved label, revised 06/2012; VICTRELIS® (boceprevir) FDA-approved label, revised 07/2012.)

The introduction of both boceprevir and telaprevir has increased the therapeutic options àvaiiable to HCV-infected patients; however, both treatment regimens hâve certain disadvantages. A principle disadvantage is that the boceprevir and telaprevir regimens still require the use of peginterferon. Additional disadvantages are summarized below.

Boceprevir (used in combination with peginterferon a-2a and ribavirin) has a complicated dosing regimen, e.g., 800 mg (4 x 200 mg) three times daily (every 7 to 9 hours) with food. Moreover, late-stage clinical studies show that boceprevir used in combination with peginterferon and ribavirin results in a 66% SVR rate. (Manns et al., Liver Int’l (2012) 27-31.) Additionally, the boceprevir regimen must be administered for 48 weeks, which means that the treatment costs are quite expensive. Finally, use of boceprevir in combination with peginterferon and ribavirin is presently limited to those subjects infected with HCV génotype 1.

The telaprevir regimen (used in combination with peginterferon and ribavirin) requires a dosing regimen of 750 mg (2 x 375 mg) three times daily (7-9 hours apart) with food. An SVR rate of 79% was reported for patients receiving telaprevir in combination with peginterferon and ribavirin for 12 weeks. (Jacobson et al., New Engl. J. Med. (2011) 364: 2405-2416.) However, reports reveal that about half of the treated patients developed a skin rash or itching, and a small number of patients developed the severe •r

Stevens-Johnson Syndrome, a life-threatening skin condition, in which case the regimen must be terminated. Finally, use of telaprevir in combination with peginterferon and ribavirin is presently limited to those subjects infected with HCV génotype l. Although the treatment period is reduced for telaprevir as compared to that for boceprevir, the treatment costs for the two regimens are about the same.

Despite the additional options offered by the boceprevir and telaprevir regimens, these alternative treatments still hâve disadvantages. Further, génotype l patients who fail therapy with boceprevir and/or telaprevir in combination with peginterferon and ribavirin may develop undesirable NS3 protease inhibitor résistance. (E.g., Pawlotsky, Hepatology (2011) 53(5): 1742-1751.) There is a need for improved treatment regimens that are more effective, safe, tolerable, shorter in duration, and which are associated with reduced rates of viral breakthrough and/or viral résistance. In particular, there is a need for interferon-free treatment regimens that are effective for treating CHC but resuit in reduced side-effects compared to treatment regimens involving interferon or peginterferon. There is also a need for interferon-free treatment regimens for patients suffering from CHC infection who are interferon-ineligible or interferon-intolerant.

GS-7977 (also called sofosbuvir and formerly called PSI-7977) is a nucléotide analog prodrug currently in Phase 2/Phase 3 trials for treatment of chronic HCV infection.

Several Phase 2 clinical trials hâve been conducted to evaluate the efficacy, safety and tolerability of GS-7977 400 mg administered for 8 or 12 weeks with or without ribavirin and optionally peginterferon in subjects with GT1, GT2 or GT3 HCV. The results of these trials, alongwith the results if in vitro studies, revealed several potential and hereto unknown advantages of HCV treatment regimens utilizing GS-7977 in combination with ribavirin. These results provide a basis for the disclosed and claimed method and composition for treating HCV infection.

Summary

Disclosed herein are a composition and unit dosage form for the treatment of hepatitis C virus (HCV) infection comprising GS-7977 and at least one pharmaceutically acceptable excipient, as well as methods for making said composition and unit dosage form.

Also disclosed herein is a method of treating a subject, preferably a human, infected with hepatitis C virus, said method comprising administering to the subject for a time period an effective amount of GS-7977 and an effective amount of ribavirin. In one aspect, the method comprises administering to the subject an interferon-free treatment regimen comprising an effective amount of GS-7977 and an effective amount of ribavirin. In a particular aspect, the method is sufficient to produce an undetectable amount ofHCV RNA in the subject for at least 12 weeks after the end of the time period.

Brief Description of the Drawings

Figure 1. Plot of Mean HCV RNA (logio RJ/mL) versus time during treatment and for up to 12 weeks after the end of treatment (“EOT”) for HCV GT2/GT3 treatment-naïve patients receiving a combination of GS-7977 (400 mg QD) and RBV (1000/1200 mg BID based on weight) for 12 weeks (ELECTRON Group 1).

Figure 2. Fold-change in EC50 for HCV replicons containing lb, la, 2a, 2b, 3a, 4a, and 5a NS5B harboring the S282T mutation (compared to the corresponding wild-type) treated with GS-7977 or ribavirin.

· < ... fMW-w.··

Figure 3. Percentage of wild-type at S282 position in HCV replicons before and after treatment with GS-7977, ribavirin, and a combination of GS-7977 and ribavirin in long-term passaging study (15-30 days).

Detailed Description

Définitions

The phrase a or an entity as used herein refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound. As such, the tenus a” (or an), one or more, and at least one can be used interchangeably herein.

The term about (also represented by has its plain and ordinary meaning of approximately except as related to an amount of GS-7977, an amount of ribavirin, or an amount of HCV RNA. As related to an amount of GS-7977, an amount of ribavirin, or an amount of HCV RNA, the qualifier about reflectsthe standard experimental enor.

The terms optional or optionally as used herein means that a subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not.

The term subject as used herein means a mammal. Preferably the subject is a human.

The term effective amount as used herein means an amount sufficient to reduce symptoms of the HCV infection in a subject.

The term undetectable amount refers to an amount of HCV RNA, as determined by the assay methodology described herein, that is less than the limit of détection (LOD”) of about 15 lU/mL.

A sustained virologie response (SVR) for a patient treated according to one of the treatment regimens described herein is defïned as a patient who complétés the HCV treatment regimen and who has an undetectable amount of HCV RNA (i.e., < about 15 RJ/mL) for a period of time post-treatment as measured iij accordance with the assay methodology described herein. SVR-N is the abbreviation for sustained virologie response N weeks after completion of one of the HCV treatment regimens disclosed herein. For example, SVR-4 is the abbreviation for sustained virologie response 4 weeks after completion of one of the HCV treatment regimens disclosed herein.

The term préparation or dosage form is intended to include both solid and liquid formulations of the active compound and one skilled in the art will appreciate that an active ingrédient can exist in different préparations depending on the desired dose and pharmacokinetic parameters.

The term “unit dosage form” refers to a physically discrète unit containing a predetermined quantity of the active compound. Preferred unit dosage forms are those

A t' ( Τ’”’ containing a daily dose or unit daily sub-dose, or an appropriate fraction thereof, of GS7977.

The ternis “pharmaceutically acceptable excipient” and pharmaceutical excipient as used herein refer to a compound that is used to préparé a pharmaceutical composition, and is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipients that are acceptable for veterinary use as well as human pharmaceutical use.

RVR is the abbreviation for rapid virologie response and refers to an undetectable level of HCV RNA in the blood at week 4 of treatment. The occurrence of RVR has been reported to be prédictive of ultimate SVR for a full treatment course of 48 weeks with peginterferon/ribavirin combination treatment in HCV GT-I patients. (Poordad et al., Clin. Infect. Dis. (2008) 46: 78-84.)

QD means that the dose is administered once a day.

BID means that the dose is administered twice a day.

TID means that the dose is administered three times a day. QID means that the dose is administered four times a day.

The highest activities of alanine aminotransferase (ALT) are found in hépatocytes and striated (skeletal and cardiac) muscle cells. Increased sérum ALT activity can accompany hepatocellular injury or necrosis of striated muscle. With cell injury or death, ALT escapes from the cytosol. In addition, release of ALT from the cytosol can occur secondary to cellular necrosis or as a resuit of cellular injury with membrane damage. Détermination of ALT activity is a relatively sensitive indicator of hepatic damage. Mechanisms of increased activity of ALT in sérum include enzyme release from damaged cells or induction of enzyme activity, such as increased enzyme synthesis from drug administration. (Zeuzem, et al., Aliment Pharmacol Ther. 2006 Oct 15; 24(8) 11331149).

The interleukin 28B (IL28B) gene encodes a cytokine distantly related to type I interferons and the IL-10 family. The IL28B gene, interleukin 28A (IL28A), and interleukin 29 (IL29) are three closely related cytokine genes that form a cytokine gene cluster on a chromosomal région mapped to 19ql 3. Expression of the cytokines encoded by the three genes can be induced by viral infection. Ail three cytokines hâve been shown to interact with a heterodimeric class Π cytokine receptor that consists of interleukin 10 receptor, beta (DL10RB), and interleukin 28 receptor, alpha (IL28RA). (National Center for Biotechnology Information, Entrez Gene Entry for 1L28B, Gene ID: 282617, updated on 23-Oct-2010.)

Body mass index (“BMI”) is a measurement based on a person's weight and height and is used to estimate a healthy body weight based on a person's height, assuming an average body composition. The units of BMI are kg/m .

LOD is the abbreviation for limit of détection. As used herein with regard to HCV RNA measurements, in one aspect LOD is from about 1 IU/mL to about 60 IU/mL, more preferably from about 5 IU/mL to about 30 IU/mL, and even more preferably from about 10 RJ/mL ίο about 20 IU/mL. In a particulariy preferred embodiment, the LOD is about 15 IU/mL.

GT is the abbreviation for génotype.

ΙΌ is the abbreviation for international unit, which is a measure of the amount of a substance based on biological activity or effect.

There are several recognized HCV Génotypes (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11), which can be further categorized by different sub-types: 1 (la, lb, and le), 2 (2a, 2b, 2c), 3 (3a and 3b), 4 (4a, 4b, 4c, 4d, and 4e), 5 (5a), 6 (6a), 7 (7a and 7b), 8 (8a and 8b), 9 (9a), 10 (10a), and 11 (lia). Génotype 1 is the prédominant form found in North and South America, Europe, Asia, Australia, and New Zealand. Génotypes 2 and 3 are also widely distributed throughout North America, Europe, Australia, East Asia and some...........

portions of Africa. In some portions of Africa, Génotype 4 prédominâtes, while in others (such as South Africa) génotype 5 prédominâtes. The method disclosed herein is contemplated to be independently effective for the treatment of each of the HCV génotypes, and in particular each genotype-sub-type.

The term “interferon-free” as used herein refers to a treatment regimen that does not involve the administration of interferon or pegylated interferon to the subject.

GS-7977, (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4dihydropyrimidin^;-l(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2«K

-17352 ~ yl)methoxy)(phenoxy)phosphoryl)amino)propanoate, available from Gilead Sciences,

Inc., is described and claimed in U.S. Patent No. 7,964,580. (See also US 2010/0016251,

US 2010/0298257, US 2011/0251152 and US 2012/0107278.) GS-7977 has the structure:

GS-7977 can be crystalline or amorphous. Examples of preparing crystalline and amorphous forms of GS-7977 are disclosed in US 2010/0298257 (US 12/783,680) and US 2011/0251152 (US 13/076,552), both of which are incorporated by reference.

Polymorphie Forms 1-6 of GS-7977 disclosed in US 2010/0298257 and/or US 2011/0251152 hâve the following characteristic X-ray powder diffraction (XRPD) pattern 20-values measured according to the XRPD methods disclosed therein:

(1) 20-reflections (°) at about: 5.2, 7.5, 9.6, 16.7,18.3, and 22.2 (Form 1);

(2) 20-reflections (°) at about: 5.0, 7.3, 9.4, and 18.1 (Form 1);

(3) 20-reflections (°) at about: 4.9, 6.9, 9.8, 19.8, 20.6, 24.7, and 26.1 (Form 2);

(4) 20-reflections (°) at about: 6.9, 9.8, 19.7,20.6, and 24.6 (Form 3);

(5) 20-reflections (°) at about: 5.0, 6.8, 19.9, 20.6, 20.9, and 24.9 (Form 4);

(6) 20-reflections (°) at about: 5.2, 6.6, 7.1, 15.7,19.1, and 25.0 (Form 5); and (7) 20-reflections (°) at about: 6.1, 8.2, 10.4, 12.7, 17.2, 17.7, 18.0, 18.8, 19.4,

19.8,20.1,20.8, 21.8, and 23.3 (Form 6).

Polymorphie Forms 1 and 6 are altematively characterized by the following characteristic XRPD pattern 20-values measured according to the methods disclosed in US 2010/0298257 (US 12/783,680) and US 2011/0251152 (US 13/076,552):

(1) 20-reflections (°) at about: 5.0 and 7.3 (Form 1); and (2) 20-reflections (°) at about: 6.1 and 12.7 (Form 6).

In one aspect, the disclosed composition comprises polymorphie Form 6 of GS7977. It has been found that Form 6 has a melt onset of approximately 121°C and is not hygroscopic, with less than 0.2% moisture sorption at room température and 90% RH. Form 6 is chemically stable when stored under opened conditions at 40°C/75% RH for 30 days.

In one aspect, GS-7977 is substantially free from its corresponding phosphorous5 based diastereomer (S)-isopropyl 2-(((R)-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4dihydropyrimidin-1 (2H)-yI)-4-fluoro-î-hydroxy-4-methyïtetrahydrofùran -2yl)methoxy)(phenoxy)phosphoryl)amino)propanoate. In one embodiment, GS-7977 is at least 95% free from its corresponding phosphorous-based diastereomer. In another embodiment, GS-7977 is at least 97% free from its corresponding phosphorous-based diastereomer. In another embodiment, GS-7977 is at least 99% free from its corresponding phosphorous-based diastereomer. In a further embodiment, GS-7977 is at least 99.9% free from its corresponding phosphorous-based diastereomer.

Ribavirin, l-P-D-ribofuranosyl-lH-l,2,4-triazole-3-carboxamide, is described in the Merck Index (I2th Edition), monograph no. 8365. (See also U.S. Patent No.

4,530,901.)

As used herein, treatment or treating is an approach for obtaining bénéficiai or desired clinical results. Bénéficiai or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether détectable or undetectable. Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment. Treatment is an intervention.performed with the intention of preventing the development or altering the pathology of a disorder. The term treatment of an HCV infection, as used herein, also includes treatment or prophylaxis of a disease or a condition associated with or mediated by HCV infection, or the clinical symptoms thereof.

Compositions and Unit Dosage Forms

A first embodiment is directed to a composition for the treatment of hepatitis C virus (HCV) comprising a) GS-7977, and b) a pharmaceutically acceptable excipient.

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In a first aspect of the first embodiment, the composition for the treatment of HCV comprises from about 25% to about 35% w/w of GS-7977. In another aspect, the composition comprises from about 30% to about 35% w/w of GS-7977. In another aspect, the composition comprises from about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, or about 35% w/w of GS-7977. In oné subembodiment, the composition comprises about 30% w/w of GS-7977. In another subembodiment, the composition comprises about 33% w/w of GS7977. In another subembodiment, the composition comprises about 33.33% w/w of GS7977.

In a second aspect of the first embodiment, the composition comprises crystalline GS-7977. In one subembodiment, the composition comprises crystalline GS-7977 having XRPD 26-reflections (°) at about: (1) 5.2, 7.5, 9.6,16.7, 18.3, and 22.2; (2) 5.0, 7.3, 9.4, and 18.1; (3)4.9, 6.9, 9.8, 19.8, 20.6, 24.7, and 26.1; (4) 6.9, 9.8, 19.7, 20.6, and 24.6; (5) 5.0, 6.8, 19.9, 20.6, 20.9, and 24.9; (6) 5.2, 6.6, 7.1, 15.7, 19.1, and 25.0; or (7) 6.1, 8.2, 10.4, 12.7, 17.2, 17.7, 18.0, 18.8, 19.4, 19.8, 20.1, 20.8, 21.8, and 23.3. In another subembodiment, the composition comprises crystalline GS-7977 having XRPD 20reflections (°) at about: (1) 5.0 and 7.3; or (2) 6.1 and 12.7. In one preferred subembodiment, the composition comprises crystalline GS-7977 having XRPD 20reflections (°) at about: (1) 5.2, 7.5, 9.6, 16.7, 18.3, and 22.2; or (2) XRPD 20-reflections (°) at about: 5.0, 7.3, 9.4, and 18.1. In another preferred subembodiment, the composition comprises crystalline GS-7977 having XRPD 20-reflections (°) at about 6.1, 8.2, 10.4, 12.7,17.2, 17.7, 18.0, 18.8, 19.4, 19.8, 20.1, 20.8, 21.8, and 23.3. In another preferred subembodimenζ the composition comprises crystalline GS-7977 having XRPD 20reflections (°) at about: 5.0 and 7.3. In a further preferred subembodiment, the composition comprises crystalline GS-7977 having XRPD 20-reflections (°) at about 6.1 amd 12.7.

In a third aspect of the first embodiment, the pharmaceutically acceptable excipient comprises at least one of a diluent, a disintegrant, a glidant, and a lubricant.

In one subembodiment, the diluent is selected from the group consisting of calcium carbonate, dicaicium phosphate, dry starch, calcium sulfate, cellulose, compressible sugars, confectioner's sugar, dextrates, dextrin, dextrose, dibasic calcium phosphate dihydrate, glyceryl palmitostearate, hydrogenated vegetable oil (type I), inositol, kaolin, lactose, magnésium carbonate, magnésium oxide, maltodextrin, mannitol, microcrystalline cellulose, polymethacrylates, potassium chloride, powdered cellulose, powdered sugar, pregelatinized starch, sodium chloride, sorbitol, starch, sucrose, sugar spheres, talc, tribasic calcium phosphate, and combinations thereof. In a preferred subembodiment, the diluent is selected from the group consisting of dicalcium phosphate, cellulose, compressible sugars, dibasic calcium phosphate dehydrate, lactose, mannitol, microcrystalline cellulose, starch, tribasic calcium phosphate, and combinations thereof. In another preferred subembodiment, the diluent is selected from the group consisting of mannitol, microcrystalline cellulose, and combinations thereof.

In another subembodiment, the disintegrant is selected from the group consisting of agar, alginic acid, bentonite, carboxymethylcellulose calcium, carboxymethylcellulose sodium, carboxymethylcellulose, cellulose, a cation exchange resin, cellulose, gums, citrus pulp, colloïdal silicon dioxide, com starch, croscarmellose sodium (e.g., Ac-DiSol®), crospovidone, guar gum, hydrous aluminum silicate, an ion exchange resin (e.g., polyacrin potassium), magnésium aluminum silicate, methyl cellulose, microcrystalline cellulose, modified cellulose gum, modified com starch, montmorillonite clay, natural sponge, polyacrilin potassium, potato starch, powdered cellulose, povidone, pregelatinized starch, sodium alginate, sodium bicarbonate in admixture with an acidulant such as tartaric acid or citric acid, sodium starch glycolate, starch, silicates (e.g., Veegum® HV), and combinations thereof. In a preferred subembodiment, the disintegrant is selected from the group consisting of croscarmellose sodium (e.g., Ac-DiSol®), crospovidone, microcrystalline cellulose, modified com starch, povidone, pregelatinized starch, sodium starch glycolate, and combinations thereof. In another preferred subembodiment, the disintegrant is croscarmellose sodium (e.g., Ac-Di-Sol®).

In another subembodiment, the glidant is selected from the group consisting of colloïdal silicon dioxide, talc, starch, starch dérivatives, and combinations thereof. In a preferred subembodiment, the glidant comprises colloïdal silicon dioxide.

In another subembodiment, the lubricant is selected from the group consisting of calcium stéarate, glyceryl monostearate, glyceryl palmitostearate, hydrogenated castor oil, hydrogenated vegetable oil, light minerai oil, magnésium stéarate, minerai oil, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, sodium stearyl fumarate, stearic acid, talc, zinc stéarate, and combinations thereof. In a preferred subembodiment, the lubricant is selected from the group consisting of calcium stéarate, magnésium stéarate, polyethylene glycol, sodium stearyl fumarate, stearic acid, talc, and combinations thereof. In another preferred subembodimenq the lubricant is magnésium stéarate.

In another subembodiment, the pharmaceutically acceptable excipient comprises:

a) about 55% w/w to about 65% w/w of a diluent; b) about 2.5 % w/w to about 7.5% w/w of a désintégrant; c) about 0.25% w/w to about 0.75% w/w of a glidant; and d) about 1.25% w/w to about 1.75% w/w of a lubricant. In a preferred subembodiment, the pharmaceutically acceptable excipient comprises a) about 60% w/w a diluent; b) about 5 %w/w of a désintégrant; c) about 0.5% w/w a glidant; and d) about 1.5% w/w a lubricant. In another preferred subembodiment, the pharmaceutically acceptable excipient comprises a) about 60% w/w a diluent comprising mannitol and/or microcrystalline cellulose; b) about 5 %w/w of croscarmellose sodium; c) about 0.5% w/w of colloïdal silicon dioxide; and d) about 1.5% w/w of magnésium stéarate. In another preferred subembodiment, the pharmaceutically acceptable excipient comprises a) about 30% w/w of mannitol and about 30% w/w of microcrystalline cellulose; b) about 5 % w/w of croscarmellose sodium; c) about 0.5% w/w of colloïdal silicon dioxide; and d) about 1.5% w/w of magnésium stéarate.

In a fourth aspect of the first embodiment, the composition further comprises a coating agent. In one subembodiment, the coating agent is formed from an aqueous félm coat composition, wherein the aqueous film coat composition comprises a film-forming polymer, water and/or an alcohol as a vehicle, and optionally one or more adjuvants such as are known in the film-coating art. In another subembodiment, the coating agent is selected from the group consisting of hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, cellulose acetate phthalate, sodium ethyl cellulose sulfate, carboxymethyl cellulose,

-L- I ~ · t f polyvinylpyrolidone, zein, and an acrylic polymer (e.g., methacrylic acid/methacrylic acid ester copôlymers such as methacrylic acid/methylmethacrylate copolymers, etc.), and a polyvinyl alcohol. In another subembodiment, the coating agent comprises a polyvinyl alcohol.

In a fifth aspect of the first embodiment, the composition comprises about 25% w/w to about 35% w/w of crystalline GS-7977; aboui-30% w/w of mannitoland about 30% w/w of microcrystalline cellulose; about 5 %w/w of croscarmellose sodium; about 0.5% w/w of colloïdal silicon dioxide; and about l .5% w/w of magnésium stéarate. In one subembodiment, the composition comprises about 30% w/w to about 35% w/w of crystalline GS-7977; about 30% w/w of mannitol and about 30% w/w of microcrystalline cellulose; about 5 %w/w of croscarmellose sodium; about 0.5% w/w of colloïdal silicon dioxide; and about 1.5% w/w of magnésium stéarate. In another subembodiment, the composition comprises about 30% w/w of crystalline GS-7977; about 30% w/w of mannitol and about 30% w/w of microcrystalline cellulose; about 5 %w/w of croscarmellose sodium; about 0.5% w/w of colloïdal silicon dioxide; and about 1.5% w/w of magnésium stéarate. In one subembodiment, the composition comprises about 30% w/w of crystalline GS-7977 having XRPD 20-reflections (°) at about 6.1, 8.2, 10.4, 12.7, 17.2, 17.7, 18.0, 18.8, 19.4, 19.8, 20.1,20.8, 21.8, and 23.3; about 30% w/w of mannitol and about 30% w/w of microcrystalline cellulose; about 5 %w/w of croscarmellose sodium; about 0.5% w/w of colloïdal silicon dioxide; and about 1.5% w/w of magnésium stéarate. In another subembodiment, the composition comprises about 30% w/w of crystalline GS-7977 having XRPD 20-reflections (°) at about 6.1 and 12.7; about 30% w/w of mannitol and about 30% w/w of microcrystalline cellulose; about 5 %w/w of croscarmellose sodium; about 0.5% w/w of colloïdal silicon dioxide; and about 1.5% w/w of magnésium stéarate. In another subembodiment, the composition comprises about 33% w/w of crystalline GS-7977; about 30% w/w of mannitol and about 30% w/w of microcrystalline cellulose; about 5 %w/w of croscarmellose sodium; about 0.5% w/w of colloïdal silicon dioxide; and about 1.5% w/w of magnésium stéarate. In another subembodiment, the composition comprises about 33% w/w of crystalline GS-7977 having XRPD 20-reflections (°)at about 6.1, 8.2, 10.4, 12.7, 17.2, 17.7, 18.0, 18.8,19.4, ,-r· » 17352

19.8, 20.1, 20.8, 21.8, and 23.3; about 30% w/w of mannitol and about 30% w/w of microcrystalline cellulose; about 5 %w/w of croscarmellose sodium; about 0.5% w/w of colloïdal siliçon dioxide; and about 1.5% w/w of magnésium stéarate. In another subembodiment, the composition comprises about 33% w/w of crystalline GS-7977 having XRPD 20-reflections (°) at about 6.1 and 12.7; about 30% w/w of mannitol and about 30% w/w of microcrystaHine cellulose; about 5 %w/w of croscarmellose sodium; about 0.5% w/w of colloïdal silicon dioxide; and about 1.5% w/w of magnésium stéarate. In another subembodiment, the composition further comprises a coating agent.

A second embodiment is directed to a unit dosage form for the treatment of hepatitis C virus (HCV), said composition comprising a) about 400 mg of GS-7977, and

b) a pharmaceutically acceptable excipient.

In a first aspect of the second embodiment, the unit dosage form comprises crystalline GS-7977. In one subembodiment, the composition comprises crystalline GS7977 having XRPD 20-reflections (°) at about: (J) 5.2, 7.5, 9.6, 16.7, 18.3, and 22.2; (2) 5.0,7.3, 9.4, and 18.1; (3) 4.9, 6.9, 9.8, 19.8, 20.6, 24.7, and 26.1; (4) 6.9, 9.8, 19.7, 20.6, and 24.6; (5) 5.0, 6.8, 19.9, 20.6, 20.9, and 24.9; (6) 5.2, 6.6, 7.1, 15.7, 19.1, and 25.0; or (7) 6.1, 8.2,10.4, 12.7, 17.2, 17.7, 18.0, 18.8, 19.4, 19.8,20.1,20.8,21.8, and 23.3. In another subembodiment, the composition comprises crystalline GS-7977 having XRPD 20-reflections (°) at about: (I) 5.0 and 7.3; or (2) 6.1 and 12.7. In one preferred subembodiment, the unit dosage form comprises crystalline GS-7977 having XRPD 20reflections (°) at about: (1) 5.2, 7.5, 9.6, 16.7, 18.3, and 22.2; or (2) XRPD 20-reflections (°) at about: 5.0, 7.3, 9.4, and 18.1. In another preferred subembodiment, the unit dosage form comprises crystalline GS-7977 having XRPD 20-reflections (°) at about 6.1, 8.2, 10.4,12.7, 17.2, 17.7, 18.0, 18.8, 19.4, 19.8, 20.1, 20.8,21.8, and 23.3. In another preferred subembodiment, the composition comprises crystalline GS-7977 having XRPD 20-reflections (°) at about: 5.0 and 7.3. In a further preferred subembodiment, the composition comprises crystalline GS-7977 having XRPD 20-reflections (°) at about 6.1 amd 12.7.

In a second aspect of the second embodiment, the pharmaceutically acceptable excipient comprises at least one of a diluent, a disintegrant, a glidant, and a lubricant.

In a one subembodiment, diluent is selected from the group consisting of calcium carbonate, dicalcium phosphate, dry starch, calcium sulfate, cellulose, compressible sugars, confectioner's sugar, dextrates, dextrin, dextrose, dibasic calcium phosphate dihydrate, glyceryl palmitostearate, hydrogenated vegetable oil (type I), inositol, kaolin, lactose, magnésium carbonate, magnésium oxide, maltodextrin, mannitol, microcrystalline cellulose, pofymethacrylates,· potassium chloride, powdered cellulose, powdered sugar, pregelatinized starch, sodium chloride, sorbitol, starch, sucrose, sugar spheres, talc, tribasic calcium phosphate, and combinations thereof. In a preferred subembodiment, the diluent is selected from the group consisting of dicalcium phosphate, cellulose, compressible sugars, dibasic calcium phosphate dehydrate, lactose, mannitol, microcrystalline cellulose, starch, tribasic calcium phosphate, and combinations thereof. In another preferred subembodiment, the diluent is selected from the group consisting of mannitol, microcrystalline cellulose, and combinations thereof.

In another subembodiment, the désintégrant is selected from the group consisting of agar, alginic acid, bentonite, carboxymethylcellulose calcium, carboxymethylcellulose sodium, carboxymethylcellulose, cellulose, a cation exchange resin, cellulose, gums, citrus pulp, colloïdal silicon dioxide, com starch, croscarmellose sodium (e.g., Ac-DiSol®), crospovidone, guar gum, hydrous aluminum silicate, an ion exchange resin (e.g., polyacrin potassium), magnésium aluminum silicate, methyl cellulose, microcrystalline cellulose, modified cellulose gum, modified com starch, montmorillonite clay, natural sponge, polyacrilin potassium, potato starch, powdered cellulose, povidone, pregelatinized starch, sodium alginate, sodium bicarbonate in admixture with an acidulant such as tartaric acid or citric acid, sodium starch glycolate, starch, silicates (e.g., Veegum® HV), and combinations thereof. In a preferred subembodiment, the disintegrant is selected from the group consisting of croscarmellose sodium (e.g., Ac-DiSol), crospovidone, microcrystalline cellulose, modified com starch, povidone, pregelatinized starch, sodium starch glycolate, and combinations thereof. In another preferred subembodiment, the disintegrant is croscarmellose sodium (e.g., Ac-Di-Sol).

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In another subembodiment, the glidant is selected from the group consisting of colloïdal silicon dioxide, talc, starch, starch dérivatives, and combinations thereof. In a preferred subembodiment, the glidant comprises colloïdal silicon dioxide.

In another subembodiment, the lubricant is selected from the group consisting of calcium stéarate, glyceryl monostearate, glyceryl palmitostearate, hydrogenated castor oil, 'hydrogenated vegetable oil, light minerai oil, magnésium stéarate, minerai oil, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, sodium stearyl fumarate, stearic acid, talc, zinc stéarate, and combinations thereof. In a preferred subembodiment, the lubricant is selected from the group consisting of calcium stéarate, magnésium stéarate, polyethylene glycol, sodium stearyl fumarate, stearic acid, talc, and combinations thereof. In another preferred subembodiment, the lubricant is magnésium stéarate.

In another subembodiment, the pharmaceutically acceptable excipient comprises: a) about 660 mg to about 780 mg of a diluent; b) about 30 mg to about 90 mg of a disintegrant; c) about 3 mg to about 9 mg of a glidant; and d) about 15 mg to about 21 mg 15 of a lubricant. In a preferred subembodiment, the pharmaceutically acceptable excipient comprises a) about 710 mg to about 720 mg of a diluent; b) about 60 mg of a disintegrant;

c) about 6 mg of a glidant; and d) about 18 mg of a lubricant. In another preferred subembodiment, the pharmaceutically acceptable excipient comprises a) about 716 mg of a diluent; b) about 60 mg of a disintegrant; c) about 6 mg of a glidant; and d) about 18 mg 20 of a lubricant. In another preferred subembodiment, the pharmaceutically acceptable excipient comprises a) about 710 mg to about 720 mg of a diluent comprising mannitol and/or microcrystalline cellulose; b) about 60 mg of croscarmellose sodium; c) about 6 mg of colloïdal silicon dioxide; and d) about 18 mg of magnésium stéarate. In another preferred subembodiment, the pharmaceutically acceptable excipient comprises a) about 25 716 mg of a diluent comprising mannitol and/or microcrystalline cellulose; b) about 60 mg of croscarmellose sodium; c) about 6 mg of colloïdal silicon dioxide; and d) about 18 mg of magnésium stéarate. In another preferred subembodiment, the pharmaceutically acceptable excipient comprises a) about 360 mg of mannitol and about 356 mg of microcrystalline cellulose; b) about 60 mg of croscarmellose sodium; c) about 6 mg of 30 colloïdal silicon dioxide; and d) about 18 mg of magnésium stéarate.

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In a third aspect of the second embodiment, the unit dosage form further comprises a coating agent. In one subembodiment, the coating agent further comprises a taste-masking agent. In one subembodiment, the coating agent is formed from an aqueous film coat composition, wherein the aqueous film coat composition comprises a 5 film-forming polymer, water and/or an alcohol as a vehicle, and optionally one or more -- adjuvants-sueh«as-are known in the film-coating art. In another subembodimenî, the — coating agent is selected from among hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, cellulose acetate phthalate, sodium ethyl cellulose sulfate, carboxymethyl cellulose, polyvinylpyrolidone, zein, and an acrylic polymer (e.g., methacrylic acid/methacrylic acid ester copolymers such as methacrylic acid/methylmethacrylate copolymers, etc.), and a polyvinyl alcohol. In another subembodiment, the coating agent comprises a polyvinyl alcohol. In another subembodiment, the unit dosage comprises about 24 mg to about 60 mg of a coating agent. In another subembodiment, the unit dosage comprises about 36 mg to about 48 mg of a coating agent. In another subembodiment, the unit dosage comprises about 36 mg of a coating agent. In another subembodiment, the unit dosage comprises about 36 mg of a coating agent that further comprises a taste-masking agent.

In a fourth aspect of the second embodiment, the unit dosage form comprises about 400 mgof crystalline GS-7977; about 360 mg of mannitol and about 356 mg of 20 microcrystalline cellulose; about 60 mg of croscarmellose sodium; about 6 mg of colloïdal silicon dioxide; and about 18 mg of magnésium stéarate. In one subembodiment, the unit dosage form comprises about 400 mg of crystalline GS-7977 having XRPD 20-reflections (°) at about 6.1, 8.2, 10.4, 12.7, 17.2, 17.7, T 8.0, 18.8, 19.4, 19.8, 20.1, 20.8, 21.8, and 23.3; about 360 mg of mannitol and about 356 mg of microcrystalline cellulose; about 60 mg of croscarmellose sodium; about 6 mg of colloïdal silicon dioxide; and about 18 mg of magnésium stéarate. In another subembodiment, the unit dosage form comprises about 400 mg of ciystalline GS-7977 having XRPD 20-reflections (°) at about 6.1 and 12.7; about 360 mg of mannitol and about 356 mg of microcrystalline cellulose; about 60 mg of croscarmellose sodium; about 30 6 mg of colloïdal silicon dioxide; and about 18 mg of magnésium stéarate.

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In a fifth aspect of the second embodiment, the unit dosage form comprises a capsule or a tablet. In one subembodiment, the unit dosage form comprises a tablet. In another subembodiment, the unit dosage form comprises a tablet and further comprises a coating agent.

With respect to the coating agent, film-forming polymers are typically provided in either aqueous or organic sofvent-based solutions or aqueous dispersions. However, the polymers may be provided in dry form, alone or in a powdery mixture with other components (e.g., a plasticizer and/or colorant), which is made into a solution or dispersion by the user by admixing with the aqueous vehicle.

It will be appreciated that the aqueous film coat composition further comprises water as a vehicle for the other components, to facilitate their delivery to the surface of the unit dosage form. The vehicle may optionally further comprise one or more water soluble solvents, e.g., an alcohol and/or a ketone. Examples of an alcohol include but are not limited to methanol, isopropanol, propanol, etc. A non-limiting example for the ketone is acetone. The skilled artisan can select appropriate vehicle components to provide good interaction between the film-forming polymer and the vehicle to ensure good film properties. In general, polymer-vehicle interaction is designed to yield maximum polymer chain extension to produce films having the greatest cohesive strength and thus mechanical properties. The components are also selected to provide good déposition of the film-forming polymer onto the surface of the unit dosage form, such that a cohérent and adhèrent film is achieved.

Suitable aqueous film coating compositions include those commercially available from Colorcon, Inc. of West Point, Pa., under the trade name OPADRY and OPADRY II (non-limiting examples includes Opadry II Purple and Opadiy Π Yellow).

A third embodiment is directed to a method of treating a subject infected with hepatitis C virus comprising administering to the subject a composition comprising a) about 25-35% w/w of GS-7977, and b) a pharmaceutically acceptable excipient.

In a first aspect of the third embodiment, the composition comprising a) about 2535% w/w of GS-7977, and b) a pharmaceutically acceptable excipient is administered to the subject in combination with ribavirin.

In a second aspect of the third embodiment, the subject is a human.

A fourth embodiment is directed to a method of treating a subject infected with hepatitis C virus comprising administering to the subject a unit dosage form comprising a) about 400 mg of GS-7977, and b) a pharmaceutically acceptable excipient.

In a first aspect of the fourth embodiment, the a unit dosage form comprising a) about 400 ing of GS-7977, and b) a pharmaceutically acceptable excipient is administered .... to the subject in combination with ribavirin.

In a second aspect of the fourth embodiment, the subject is a human.

Tablet Préparation

The choice of particular types and amounts of excipients, and tabletting technique employed dépends on the further properties of GS-7977 and the excipients, e.g., compressibility, flowability, particle size, compatibility, and density. In this regard, reference is made Remington: The Science and Practice of Pharmacy 2006, 21 st édition, Lippincott Williams & Wilkins; see also Handbook of Pharmaceutical Excipients 1994, edited by A. Wade and P. J. Weller, The Pharmaceutical Press, 2nd Edition, London. A skilled formulation scientist may modify the formulations within the teachings of the spécification to provide numerous formulations for a particular route of administration without rendering compositions containing GS-7977 unstable or compromising their therapeutic activity.

Tablets may be prepared according to methods known in the art, including dry granulation (e.g., roller compaction), wet granulation (e.g., fluid bed granulation and high shear granulation), and direct compression, and the type of excipients used will vary accordingly. It has been found that dry granulation is particularly suitable for providing high strength, low breakage tablets comprising relatively high concentrations of crystalline GS-7977 (e.g., about 33%), on a scale suitable for commercial production. Suitable dry granulated tablets comprise granules comprising GS-7977 and one or more of a diluent, a disintegrant, a glidant, and a lubricant, wherein the granules are mixed with one or more of a diluent, a disintegrant, a glidant, and a lubricant to form a granulation mixture that is compressed to form tablets.

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A fïfth embodiment is directed to a process for preparing a tablet composition comprising about 400 mg of GS-7977, said process comprising blending an intragranular composition and an extragranular composition to obtain a blended composition; compressing the blended composition to obtain a tablet composition; and optionally coating the tablet composition.

... ... ..... in a first aspect ofthe fifth embodiment, the intragranular composition comprises GS-7977, a first intragranular diluent, optionally a second intragranular diluent, an intragranular désintégrant, an intragranular glidant, and an intragranular fabricant; and the extragranular composition comprises a first extragranular diluent, optionally a second extragranular diluent, an extragranular disintegrant, an extragranular glidant, and an extragranular fabricant, wherein the first intragranular diluent, the second intragranular diluent, the first extragranular diluent, and the second extragranular diluent are the same or different, the intragranular disintegrant and the extragranular disintegrant are the same or different, the intragranular glidant and the extragranular glidant are the same or different, and the intragranular fabricant and the extragranular fabricant are the same or different.

In a second aspect of the fifth embodiment, the intragranular composition comprises GS-7977, a first intragranular diluent, an intragranular disintegrant, an intragranular glidant, and an intragranular fabricant; and the extragranular composition comprises a first extragranular diluent, a second extragranular diluent, an extragranular disintegrant, an extragranular glidant, and an extragranular fabricant,, wherein the first intragranular diluent, the first extragranular diluent, and the second extragranular diluent are the same or different, the intragranular disintegrant and the extragranular disintegrant are the same or different, the intragranular glidant and the extragranular glidant are the same or different, and the intragranular fabricant and the extragranular fabricant are the same or different.

In a third aspect of the fifth embodiment, the intragranular composition comprises GS-79.77, a first intragranular diluent, a second intragranular diluent, an intragranular disintegrant, an intragranular glidant, and an intragranular fabricant; and the extragranular composition comprises a first extragranular diluent, an extragranular disintegrant, an extragranular glidant, and an extragranular lubricant, wherein the first intragranular diluent, the second intragranular diluent, and the first extragranular diluent are the same or different, the intragranular disintegrant and the extragranular disintegrant are the same or different, the intragranular glidant and the extragranular glidant are the same or different, and the intragranular lubricant and the extragranular lubricant are the same or different.-........... · < ........................... ... .......

A fourth aspect of the fïfth embodiment comprises at least one of the following steps:

(!) Sifting/Blending: GS-7977 and pharmaceutically acceptable excipients are sifted and/or blended during the formulation process. In one non-limiting example, first, GS-7977 and intragranular excipients (first diluent, optional second diluent, glidant, disintegrant; except for the intragranular lubricant) are sifted through a 20-mesh screen, added to a blender, and blended for a first blending time period to produce an initial blend. In one aspect, the first blending time period ranges from about 5 to about 30 minutes. Separatety, an intragranular lubricant is passed through a 20-mesh screen, mixed with a portion of the initial blend, added to the blender, and blended for a second blending time period. In one aspect, the second blending time period is from about l minute to about 10 minutes. In another aspect, the second blending time period is from about 1 minute to about 5 minutes. In another aspect, the second blending time period is from about 5 minutes to about 10 minutes. Second, extragranular excipients (first diluent, optional second diluent, glidant, disintegrant) (except for the extragranular lubricant) are sifted through a 20-mesh screen and used in the final blending. It is contemplated that the blending time periods may increase as the scale of the formulation process increases.

(2) Dry Granulation:

(A) Roller Compaction: GS-7977 and pharmaceutically acceptable excipients are passed through a roller compacter to product compacts. Compacts are then milled (below) te achieve granules. In one non-limiting example, a blend comprising GS-7977, intragranular excipients, and lubricant, is passed through a roller compacter until granulation is achieved. The non-limiting example has the

I following parameters: granulator speed ranges from about 50 to about 90 rpm, more specifically about 70 rpm; compactor speed ranges from about 4 to about 6 rpm, more specifically about 5 rpm; and pressure ranges from about 65 to about 100 barr, more specifically about 75 to about 100 bar.

(B) Milling (préparation of milled/sifted granule): GS-7977 and ' pharmaceuticaly acceptable excipients are mi lied and/or sifted. In one nonlimiting example, after GS-7977 and intragranular excipients hâve passed through the roller compactor, the material is passed/forced through a 20-mesh screen using a Comill or Fitz Mill, and then sifted with a 60 mesh screen. In this non-limiting example, material which remains on the 60 mesh screen is considered to be an acceptable granule, but material which passes through the 60 mesh screen is considered fines and is re-circulated through the roller compactor. This process is repeated until the percentage of fines is less than 20%. In one non-limiting example, the mill speed ranges from about 50 to about 90 rpm, more specifically about 70 rpm, (3) Final Blending: Granules comprising GS-7977 and intragranular excipients that hâve been milled/sifted are blended with extragranular excipients in a final blending. In one non-limiting example, the milled/sifted granules comprising GS-7977 and intragranular exceipients are added to a blender (e.g., a double-cone blender, a bin blender, or a V-shell blender) along with extragranular excipients (first diluent and/or second diluent, glidant, and disintegrant) and blended for about 10 to about 30 minutes. The extragranular lubricant is passed through a 20-mesh screen and added to the blend. The blend/mixture is blended for about 5 minutes. It is contemplated that the blending time periods may increase as the scale of the formulation process increases.

(4) Compressing: The final blend is compressed into tablets using a tablet press (e.g., a Globe Pharma Mini Press).

(5) Optionally, tablets are film-coated with a film-coating agent.

In a fifth aspect of the fifth embodiment, GS-7977 is blended with intragranular excipients comprising microcrystalline cellulose, mannitol, croscarmellose sodium and colloïdal silicon dioxide in a blender. The mixture is milled and blended with a portion

of magnésium stéarate, then dry granulated using a roller compacter and mill. The resulting granules are then blended with extragranular excipients comprising microcrystalline cellulose, croscarmellose sodium, and colloïdal silicon dioxide. An additional portion of magnésium stéarate is added and the resulting composition is mixed to yield a powder blend comprising 33.33% w/w GS-7977. The powder blend is compressed into tablet cores to yield tablets comprising about 400 mg of GS-7977. The tablet cores are film-coated, and the resulting film-coated tablets are then packaged.

The embodiments described herein may be modified by one of ordinary skill without straying from the expressed intent using materials and methods described in Remington: The Science and Practice ofPharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19th édition, Easton, Pennsylvania; see also Handbook of Pharmaceutical Excipients 1994, edited by A. Wade and P. J. Weller, The Pharmaceutical Press, 2nd Edition, London. One of ordinary skill may modify the formulations within the teachings of the spécification to provide numerous formulations without rendering compositions containing GS-7977 unstable or compromising its therapeutic activity. The following non-limiting examples provide further guidance related to additional aspects of the disclosed methods and compositions.

Methods of Treatment

A sixth embodîment is directed to a method for treating a subject infected with hepatitis C virus comprising administering to the subject for a time period an effective amount of GS-7977 and an effective amount of ribavirin.

In a first aspect of the sixth embodiment, the time period is selected from among from about 2 weeks to about 12 weeks, from about 3 weeks to about 12 weeks, from about 4 weeks to about 12 weeks, from about 5 weeks to about 12 weeks, from about 6 weeks to about 12 weeks, from about 7 weeks to about 12 weeks, from about 8 weeks to about 12 weeks, from about 9 weeks to about 12 weeks, from about 10 weeks to about 12 weeks, from about 11 weeks to about 12 weeks, and about 12 weeks. In one subembodiment the time period is 12 weeks. In another subembodiment the time period is 8 weeks.

In a second aspect of the sixth embodiment, the effective amount of GS-7977 is a daily dose selected from about 100 mg to about 800 mg, from about 200 mg to about 800 mg, from about 400 mg to about 800 mg, from about 600 mg to about 800 mg, from about 100 mgto about 600 mg, from about 100 mg to about 400 mg, from about 100 mg to about 200 mg, from about 200 mg to about 600 mg, from about 200 mg to about 400 mg, from about 400 mg to about 600 mg, and about 400 mg: In one subembodiment, the daily dose of GS-7977 is administered to the subject QD, BID, TID, or QID. In another subembodiment, a daily dose of about 400 mg of GS-7977 is administered to the subject QD, BID, TID, or QID. In another subembodiment, a daily dose of about 400 mg of GS7977 is administered to the subject QD.

In a third aspect of the sixth embodiment, an effective amount of GS-7977 is administered to the subject in combination with an effective amount of ribavirin, wherein the administration is concurrent or alternative.

In a fourth aspect of the sixth embodiment, the effective amount of ribavirin is a daily dose selected from about 600 mg to about 1400 mg, and from about 800 mg to about 1200 mg. In one subembodiment, the effective amount of ribavirin is a daily dose of about 1000 mg to about 1200 mg. In another subembodiment, the effective amount of ribavirin is a daily dose of about 1000 mg to about 1200 mg based on the subject’s body weight. In another subembodiment, the effective amount of ribavirin is a daily dose of about 800 mg. In another subembodiment, the daily dose of ribavirin is administered to the subject QD, BID, TID, or QID. In a further subembodiment, the daily dose of ribavirin is administered to the subject BID. —

In a fifth aspect of the sixth embodiment, a daily dose of about 400 mg of GS7977 is administered tothe subject in combination with a daily dose of about 800 mg to about 1200 mg of ribavirin. In one subembodiment, a daily dose of about 400 mg of GS7977 is administered to the subject in combination with a daily dose of about 800 mg of ribavirin. In another subembodiment, a daily dose of about 400 mg of GS-7977 is administered to the subject in combination with a daily dose of about 1000 mg to about 1200 mg of ribavirin.

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In a sixth aspect of the sixth embodiment, the subject is infected with HCV génotype 1,2,3,4,5 or 6, or any combination thereof. In one subembodiment, the subject is infected with HCV génotype I, 2, or 3, or any combination thereof.

In a seventh aspect of the sixth embodiment, the subject has an undetectable amount of HCV RNA for at least 12 weeks after the end of the time period. In one

..... subembodiment, the subject has an undetectable amount of HCV RNA for at least 24 weeks after the end of the time period. In another subembodiment, the subject has an undetectable amount of HCV RNA for at least 36 weeks after the end of the time period. In a further subembodiment, the subject has an undetectable amount of HCV RNA for at least 48 weeks after the end of the time period.

In an eighth aspect of the sixth embodiment, the subject is a human.

In a ninth aspect of the sixth embodiment, an effective amount of GS-7977 and an effective amount of ribavirin are administered to the subject according to an interferonfree treatment regimen. In one subembodiment, the interferon-free treatment regimen consists of administering an effective amount of GS-7977 and an effective amount of ribavirin to the subject for the time period.

In a tenth aspect of the sixth embodiment, the effective amount of GS-7977 comprises a composition comprising GS-7977 and at least one pharmaceutically acceptable excipient as disclosed herein.

In an eleventh aspect of the sixth embodiment, the effective amount of GS-7977 comprises a unit dosage form comprising GS-7977 and at least one pharmaceutically j acceptable excipient as disclosed herein.

J A seventh embodiment is directed to a method of treating a subject infected with | hepatitis C virus, said method comprising administering to the subject for a time period an effective amount of GS-7977 and an effective amount of ribavirin sufficient to produce an undetectable amount of HCV RNA in the subject for at least 12 weeks after the end of the time period.

In a first aspect of the seventh embodiment, the time period is selected from among from about 2 weeks to about 12 weeks, from about 3 weeks to about 12 weeks, from about 4 weeks to about 12 weeks, from about 5 weeks to about 12 weeks, from i

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I about 6 weeks to about 12 weeks, from about 7 weeks to about 12 weeks, from about 8 weeks to about 12 weeks, from about 9 weeks to about 12 weeks, from about 10 weeks to about 12 weeks, from about 11 weeks to about 12 weeks, and about 12 weeks. In one subembodiment the time period is 12 weeks. In another subembodiment the time period is 8 weeks.

In a second aspect ôf the seventh embodiment, the effective amount of GS-7977 is a daily dose selected from about 100 mg to about 800 mg, from about 200 mg to about 800 mg, from about 400 mg to about 800 mg, from about 600 mg to about 800 mg, from about 100 mg to about 600 mg, from about 100 mg to about 400 mg, from about 100 mg to about 200 mg, from about 200 mg to about 600 mg, from about 200 mg to about 400 mg, from about 400 mg to about 600 mg, and about 400 mg. In one subembodiment, the daily dose of GS-7977 is administered to the subject QD, BID, TID, or QID. In another subembodiment, a daily dose of about 400 mg of GS-7977 is administered to the subject QD, BID, TID, or QID. In another subembodiment, a daily dose of about 400 mg of GS7977 is administered to the subject QD.

In a third aspect of the seventh embodiment, an effective amount of GS-7977 is administered to the subject in combination with an effective amount of ribavirin, wherein the administration is concurrent or alternative.

In a fourth aspect of the seventh embodiment, the effective amount of ribavirin is a daily dose selected from about 600 mg to about 1400 mg, and from about 800 mg to about 1200 mg. In one subembodiment, the effective amount of ribavirin is a daily dose of about 1000 mg to about 1200 mg. In another subembodiment, the effective amount of ribavirin is a daily dose of about 1000 mg to about 1200 mg based on the subject’s body weight. In another subembodiment, the effective amount of ribavirin is a daily dose of about 800 mg. In another subembodiment, the daily dose of ribavirin is administered to the subject QD, BID, TID, or QID. In a further subembodiment, the daily dose of ribavirin is administered to the subject BID.

In a fifth aspect of the seventh embodiment, a daily dose of about 400 mg of GS7977 is administered to the subject in combination with a daily dose of about 800 mg to about 1200 mg of ribavirin. In one subembodiment, a daily dose of about 400 mg of GS

7977 is admînistered to the subject in combination with a daily dose of about 800 mg of ribavirin. In another subembodiment, a daily dose of about 400 mg of GS-7977 is admînistered to the subject in combination with a daily dose of about 1000 mg to about 1200 mg of ribavirin.

In a sixth aspect of the seventh embodiment, the subject is infected with HCV génotype 1, 2, 5,4, 5 or 6, or any combination thereof. In one subembodiment, the subject is infected with HCV génotype 1,2, 3, or any combination thereof.

In a seventh aspect of the seventh embodiment, the subject has an undetectable amount of HCV RNA for at least 24 weeks after the end of the time period. In one subembodiment, the subject has an undetectable amount of HCV RNA for at least 36 weeks after the end of the time period. In another subembodiment, the subject has an undetectable amount of HCV RNA for at least 48 weeks after the end of the time period.

In an eighth aspect of the seventh embodiment, the subject is a human.

In a ninth aspect of the seventh embodiment, an effective amount of GS-7977 and an effective amount of ribavirin are admînistered to the subject according to an interferon-free treatment regimen. In one subembodiment, the interferon-free treatment regimen consists of administering an effective amount of GS-7977 and an effective amount of ribavirin to the subject for the time period.

In a tenth aspect of the seventh embodiment, the effective amount of GS-7977 comprises a composition comprising GS-7977 and at least one pharmaceutically acceptable excipient as disclosed herein.

In an eleventh aspect of the seventh embodiment, the effective amount of GS7977 comprises a unit dosage form comprising GS-7977 and at least one pharmaceutically acceptable excipient as disclosed herein.

An eighth embodiment is directed to a method of treating a human infected with hepatitis C virus, said method comprising administering to the human for a time period an effective amount of GS-7977 and an effective amount of ribavirin suffirent to produce an undetectable amount of HCV RNA in the human for at least 12 weeks after the end of the time period.

In a first aspect of the eighth embodiment, the time period is selected from among from about 2 weeks to about 12 weeks, from about 3 weeks to about 12 weeks, from about 4 weeks to about 12 weeks, from about 5 weeks to about 12 weeks, from about 6 weeks to about 12 weeks, from about 7 weeks to about 12 weeks, from about 8 weeks to about 12 weeks, from about 9 weeks to about 12 weeks, from about 10 weeks to about 12 weeks, from about 11 weeks to about 12 weeks, and about 12 weeks. In one subembodiment the time period is 12 weeks. In another subembodiment the time period is 8 weeks.

In a second aspect of the eighth embodiment, the effective amount of GS-7977 is a daily dose selected from about 100 mg to about 800 mg, from about 200 mg to about 800 mg, from about 400 mg to about 800 mg, from about 600 mg to about 800 mg, from about 100 mg to about 600 mg, from about 100 mg to about 400 mg, from about 100 mg to about 200 mg, from about 200 mg to about 600 mg, from about 200 mg to about 400 mg, from about 400 mg to about 600 mg, and about 400 mg. In one subembodiment, the daily dose of GS-7977 is administered to the human QD, BID, TID, or QID. In another subembodiment, a daily dose of about 400 mg of GS-7977 is administered to the human QD, BID, TID, or QID. In another subembodiment, a daily dose of about 400 mg of GS7977 is administered to the human QD.

In a third aspect of the eighth embodiment, an effective amount of GS-7977 is administered to the subject in combination with an effective amount of ribavirin, wherein the administration is concurrent or alternative.

In a fourth aspect of the eighth embodiment, the effective amount of ribavirin is a daily dose selected from about 600 mg to about 1400 mg, and from about 800 mg to about 1200 mg. In one subembodiment, the effective amount of ribavirin is a daily dose of about 1000 mg to about 1200 mg. In another subembodiment, the effective amount of ribavirin is a daily dose of about 1000 mg to about 1200 mg based on the human’s body weight. In another subembodiment, the effective amount of ribavirin is a daily dose of about 800 mg. In another subembodiment, the daily dose of ribavirin is administered to the human QD, BID, TID, or QID. In a further subembodiment, the daily dose of ribavirin is administered to the human BID.

In a fifth aspect of the eighth embodiment, a daily dose of about 400 mg of GS7977 is administered to the human in combination with a daily dose of about 800 mg to about 1200 mg ot ribavirin. In one subembodiment, a daily dose of about 400 mg of GS7977 is administered to the human iri combination with a daily dose of about 800 mg of ribavirin. In another subembodiment, a daily dose of about 400 mg of GS-7977 is administered to the human in combination with a daily dose of about 1000 mg to about 1200 mg of ribavirin.

In a sixth aspect of the eighth embodiment, the human is infected with HCV génotype 1,2, 3,4, 5, or 6, or any combination thereof. In one subembodiment, the subject is infected with HCV génotype 1, 2, or 3, or any combination thereof.

In a seventh aspect of the eighth embodiment, the human has an undetectable amount of HCV RNA for at least 24 weeks after the end of the time period. In one subembodiment,, the human has an undetectable amount of HCV RNA for at least 36 weeks after the end of the time period. In another subembodiment, the human has an undetectable amount of HCV RNA for at least 48 weeks after the end of the time period.

In an eighth aspect of the eighth embodiment, an effective amount of GS-7977 and an effective amount of ribavirin are administered to the human according to an interferon-ffee treatment regimen. In one subembodiment, the interferon-free treatment regimen consists of administering an effective amount of GS-7977 and an effective amount of ribavirin to the subject for the time period.

In a ninth aspect of the eighth embodiment, the effective amount of GS-7977 comprises a composition comprising GS-7977 and at least one pharmaceutically acceptable excipient as disclosed herein.

In a tenth aspect of the eighth embodiment, the effective amount of GS-7977 comprises a unit dosage form comprising GS-19T1 and at least one pharmaceutically acceptable excipient as disclosed herein.

A ninth embodiment is directed to a method of treating a human infected with hepatitis C virus, said method comprising administering to the human for a time period an effective amount of GS-7977 and an effective amount of ribavirin sufficient to produce an

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t amount of HCV RNA in the human that is less than about 15 IU/mL for at least 12 weeks after the end of the time period.

In a first aspect of the ninth embodiment, the time period is selected from among from about 2 weeks to about 12 weeks, from about 3 weeks to about 12 weeks, from about 4 weeks to about 12 weeks, from about 5 weeks to about 12 weeks, from about 6 weeks to about 12 weeks, from about 7 weeks to about 12 weeks, from about 8 weeks to about 12 weeks, from about 9 weeks to about 12 weeks, from about 10 weeks to about 12 weeks, from about î 1 weeks to about 12 weeks, and about 12 weeks. In one subembodiment the time period is about 12 weeks. In another subembodiment the time period is about 8 weeks.

In a second aspect of the ninth embodiment, the effective amount of GS-7977 is a daily dose selected from about 100 mg to about 800 mg, from about 200 mg to about 800 mg, from about 400 mg to about 800 mg, from about 600 mg to about 800 mg, from about 100 mg to about 600 mg, from about 100 mg to about 400 mg, from about 100 mg to about 200 mg, from about 200 mg to about 600 mg, from about 200 mg to about 400 mg, from about 400 mg to about 600 mg, and about 400 mg. In one subembodiment, the daily dose of GS-7977 is administered to the human QD, BED, TED, or QID. In another subembodiment, a daily dose of about 400 mg of GS-7977 is administered to the human QD, BED, TDD, or QID. In another subembodiment, a daily dose of about 400 mg of GS7977 is administered to the human QD.

In a third aspect of the ninth embodiment, an effective amount of GS-7977 is administered to the human in combination with an effective amount of ribavirin wherein the administration is concurrent or alternative.

In a fourth aspect of the ninth embodiment, the effective amount of ribavirin is a daily dose selected from about 600 mg to about 1400 mg, and from about 800 mg to about 1200 mg. In one subembodiment, the effective amount of ribavirin is a daily dose of about 1000 mg to about 1200 mg. In another subembodiment, the effective amount of ribavirin is a daily dose of about 1000 mg to about 1200 mg based on the human’s body weight. In another subembodiment, the effective amount of ribavirin is a daily dose of about 800 mg. In another subembodiment, the daily dose of ribavirin is administered to

* the human QD, BID, TID, or QID. In a further subembodiment, the daily dose of ribavirin is administered to the human ΒΠ).

In a fifth aspect of the ninth embodiment, a daily dose of about 400 mg of GS7977 is administered to the human in combination with a daily dose of about 800 mg to about 1200 mg of ribavirin. In one subembodiment, a daily dose of about 400 mg of GS7977 is administered to the-human in combination with a daily dose of about 800 mg of ribavirin. In another subembodiment, a daily dose of about 400 mg of GS-7977 is administered to the human in combination with a daily dose of about 1000 mg to about 1200 mg of ribavirin.

In a sixth aspect of the ninth embodiment, the human is infected with HCV génotype 1, 2, 3, 4, 5, or 6, or any combination thereof. In one subembodiment, the human is infected with HCV génotype 1, 2, or 3, or any combination thereof.

In a seventh aspect of the ninth embodiment, the human has an amount of HCV RNA less than about 15 IU/mL for at least 24 weeks after the end of the time period. In one subembodiment, the human has an amount of HCV RNA less than about 15 IU/mL for at least 36 weeks after the end of the time period. In another subembodiment, the human has an amount of HCV RNA less than about 15 IU/mL for at least 48 weeks after the end of the time period.

In an eighth aspect of the ninth embodiment, an effective amount of GS-7977 and an effective amount of ribavirin are administered to the human according to an interferonfree treatment regimen. In one subembodiment, the interferon-free treatment régiment consists of administering an effective amount of GS-7977 and an effective amount of ribavirin to the subject for the time period.

In a ninth aspect of the ninth embodiment, the effective amount of GS-7977 comprises a composition comprising GS-7977 and at least one pharmaceutically acceptable excipient as disclosed herein.

In a tenth aspect of the ninth embodiment, the effective amount of GS-7977 comprises a unit dosage form comprising GS-7977 and at least one pharmaceutically acceptable excipient as disclosed herein.

A tenth embodiment is directed to a method of treating a human infected with hepatitis C virus, said method consisting of administering to the human for a time period about 400 mg of GS-7977 and about 800 mg to about 1200 mg of ribavirin.

In a first aspect of the tenth embodiment, the time period is selected from among from about 2 weeks to about 12 weeks, from about 3 weeks to about 12 weeks, from about 4 weeks to about 12 weeks, from about >5 weeks to about 12 weeks, from about 6 weeks to about 12 weeks, from about 7 weeks to about 12 weeks, from about 8 weeks to about 12 weeks, from about 9 weeks to about 12 weeks, from about 10 weeks to about 12 weeks, from about 11 weeks to about 12 weeks, and about 12 weeks. In one subembodiment the time period is 12 weeks. In another subembodiment the time period is 8 weeks.

In a second aspect of the tenth embodiment, about 400 mg of GS-7977 is administered to the human daily. In One subembodiment, a daily dose of about 400 mg of GS-7977 is administered to the human QD, BID, TID, or QID. In another subembodiment, a daily dose of about 400 mg of GS-7977 is administered to the human QD.

In a third aspect of the tenth embodiment, about 400 mg of GS-7977 is administered to the human in combination with about 800 mg to about 1200 mg of ribavirin, wherein the administration is concurrent or alternative.

In a fourth aspect of the tenth embodiment, about 1000 mg to about 1200 mg of ribavirin is administered to the human daily. In one subembodiment, a daily dose of about 1000 mg to about 1200 mg of ribavirin is administered to the human QD, BID, TID, or QID. In another subembodiment, a daily dose of about 1000 mg to about 1200 mg of ribavirin is administered to the human BID. In a further subembodiment, a daily dose of 1000 mg or 1200 mg of ribavirin is administered to the subject based on body weight.

In a fifth aspect of the tenth embodiment, about 800 mg of ribavirin is administered to the human daily. In one subembodiment, a daily dose of about 800 mg of ribavirin is administered to the human QD, BID, TD or QID. In another subembodiment, a daily dose of about 800 mg of ribavirin is administered to the human BED.

In a sixth aspect of the tenth embodiment, the human is infected with HCV génotype 1,2, 3,4, 5 or 6, or any combination thereof. In one subembodiment, the human is infected with HCV génotype 1,2, or 3, or any combination thereof.

In a seventh aspect of the tenth embodiment, the human has an undetectable amount of HCV RNA for at least 12 weeks after the end of the time period. In one subembodiment, the human has an undetectable amount of HCV RNA for at least 24 weeks after the end of the time period. In another subembodiment, the human has an undetectable amount of HCV RNA for at least 36 weeks after the end of the time period. In a further subembodiment, the human has an undetectable amount of HCV RNA for at least 48 weeks after the end of the time period.

In an eighth aspect of the tenth embodiment, the about 400 mg of GS-7977 comprises a composition comprising GS-7977 and at least one pharmaceutically acceptable excipient as disclosed herein.

In a ninth aspect of the tenth embodiment, the about 400 mg of GS-7977 comprises a unit dosage form comprising GS-7977 and at least one pharmaceutically acceptable excipient as disclosed herein.

An eleventh embodiment is directed to a composition useful for the treatment of hepatitis C virus infection in a subject, said composition comprising an effective amount of GS-7977 and an effective amount of ribavirin.

In a first aspect of the eleventh embodiment, the composition does not comprise peginterferon.

In a second aspect of the eleventh embodiment, the effective amount of GS-7977 comprises from about 100 mg to about 800 mg, from about 200 mg to about 800 mg, from about 400 mg to about 800 mg, from about 600 mg to about 800 mg, from about 100 mg to about 600 mg, from about 100 mg to about 400 mg, from about 100 mg to about 200 mg, from about 200 mg to about 600 mg, from about 200 mg to about 400 mg, from about 400 mg to about 600 mg, and about 400 mg of GS-7977 administered to the subject daily. In one subembodiment, the composition comprises about 400 mg of GS7977 administered to the subject QD.

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In a third aspect of the eleventh embodiment, the effective amount of ribavirin comprises from about 600 mg to about 1400 mg, or from about 800 mg to about 1200 mg administered to the subject daily. In one subembodiment, the effective amount of ribavirin is about 1000 mg to about 1200 mg administered to the subject daily. In another subembodiment, the effective amount of ribavirin is about 1000 mg to about 1200 mg administered to the subject daily based on the subject’s body weight. In another subembodiment, the effective amount of ribavirin about 800 mg administered to the subject daily. In another subembodiment, the composition comprises an effective amount ribavirin administered to the subject QD, BID, TID, or QID. In a further subembodiment, the composition comprises an effective amount of ribavirin administered to the subject BID.

In a fourth aspect of the eleventh embodiment, the composition comprises about 400 mg of GS-7977 administered to the subject QD and about 800 mg to about 1200 mg of ribavirin administered to the subject BID. In one subembodiment, the composition comprises about 400 mg of GS-7977 administered to the subject QD and about 800 mg of ribavirin administered to the subject BID. In another subembodiment, the composition comprises about 400 mg of GS-7977 administered to the subject QD and about 100 mg to about 1200 mg of ribavirin administered to the subject BID

In a fifth aspect of the eleventh embodiment, the composition is capable of providing an undetectable amount of HCV RNA for at least 12 weeks after the end of a time period following treatment of a subject infected with hepatitis C virus for the time period. In one subembodiment, the composition is capable of providing an undetectable amount of HCV RNA for at least 24 weeks after the end of a time period following treatment of a subject infected with hepatitis C virus for the time period. In another subembodiment, the composition is capable of providing an undetectable amount of HCV RNA for at least 36 weeks after the end of a time period following treatment of a subject infected with hepatitis C virus for the time period. In a further subembodiment, the composition is capable of providing an undetectable amount of HCV RNA for at least 48 weeks after the end of a time period following treatment of a subject infected with hepatitis C virus for the time period.

A — t

In a sixth aspect of the eleventh embodiment, the composition is capable of providing less than about 15 IU/mL of HCV RNA for at least 12 weeks after the end of a time period following treatment of a subject infected with hepatitis C virus for the time period. In one subembodiment, the composition is capable of providing less than about 15 IU/mL of HCV RNA for at least 24 weeks after the end of a time period following rreatment of a subject infected withhepatitis C virus for the time period. In another subembodiment, the composition is capable of providing less than about 15 IU/mL of HCV RNA for at least 36 weeks after the end of a time period following treatment of a subject infected with hepatitis C virus for the time period. In a further subembodiment, the composition is capable of providing less than about 15 IU/mL of HCV RNA for at least 48 weeks after the end of a time period following treatment of a subject infected with hepatitis C virus for the time period.

In a seventh aspect of the eleventh embodiment, the effective amount of GS-7977 comprises a unit dosage form comprising GS-7977 and at least one pharmaceutically acceptable excipient as disclosed herein administered to the subject. In one subembodiment, the unit dosage form comprising GS-7977 and at least one pharmaceutically acceptable excipient as disclosed herein is administered to the subject QD.

A twelfth embodiment is directed to use of an effective amount of GS-7977 and an effective amount of ribavirin to treat hepatitis C virus infection in a subject in need thereof.

In a first aspect of the twelfth embodiment, the usçjcomprises administering an effective amount of GS-7977 and an effective amount of ribavirin to the subject for a time period selected from among from about 2 weeks to about 12 weeks, fforn about 3 weeks to about 12 weeks, from about 4 weeks to about 12 weeks, from about 5 weeks to about 12 weeks, from about 6 weeks to about 12 weeks, from about 7 weeks to about 12 weeks, from about 8 weeks to about 12 weeks, from about 9 weeks to about 12 weeks, from about 10 weeks to about 12 weeks, from about 11 weeks to about 12 weeks, and about 12 weeks. In one subembodiment the time period is 12 weeks. In another subembodiment the time period is 8 weeks.

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In a second aspect of the twelfth embodiment, the effective amount of GS-7977 is a daily dose selected from about 100 mg to about 800 mg, from àbout 200 mg to about 800 mg, from about 400 mg to about 800 mg, from about 600 mg to about 800 mg, from about 100 mg to about 600 mg, from about 100 mg to about 400 mg, from about 100 mg to about 200 mg, from about 200 mg to about 600 mg, from about 200 mg to about 400 mg, from about 400 mg to about 600 mg, and about 400 mg. In one subembodiment, the daily dose of GS-7977 is administered to the subject QD, BID, TID, or QID. In another subembodiment, a daily dose of about 400 mg of GS-7977 is administered to the subject QD, BED, TID, or QID. In another subembodiment, a daily dose of about 400 mg of GS7977 is administered to the subject QD.

In a third aspect of the twelfth embodiment, an effective amount of GS-7977 is used in combination with an effective amount of ribavirin, wherein the administration of GS-7977 and ribavirin is concurrent or alternative.

In a fourth aspect of the twelfth embodiment, the effective amount of ribavirin is a daily dose selected from about 600 mg to about 1400 mg, and from about 800 mg to about 1200 mg. In one subembodiment, the effective amount of ribavirin is a daily dose of about 1000 mg to about 1200 mg. In another subembodiment, the effective amount of ribavirin is a daily dose of about 1000 mg to about 1200 mg based on the subject’s body weight. In another subembodiment, the effective amount of ribavirin is a daily dose of about 800 mg. In another subembodiment, the daily dose of ribavirin is administered to the subject QD, BID, TID, or QID. In a further subembodiment, the daily dose of ribavirin is administered to the subject BID,

In a fifth aspect of the twelfth embodiment, the effective amount of GS-7977 is about 400 mg QD and the effective amount of ribavirin is about 800 mg to about 1200 mg BID. In one subembodiment, the effective amount of GS-7977 is about 400 mg QD and the effective amount of ribavirin is about 800 mg BID. In another subembodiment, the effective amount of GS-7977 is about 400 mg QD and the effective amount of ribavirin is about 1000 mg to about 1200 mg BID.

fF’F’*

In a sixth aspect of the twelfth embodiment, the subject is infected with HCV génotype 1,2,3,4, 5 or 6, or any combination thereof. In one subembodiment, the subject is infected with HCV génotype 1,2, or 3, or any combination thereof.

In a seventh aspect of the twelfth embodiment, the subject has an undetectable amount of HCV RNA for at least 12 weeks after the end of the time period. In one subembodiment, the subject has an undetectable amount of HCV RNA for ai least 24 weeks after the end of the time period. In another subembodiment, the subject has an undetectable amount of HCV RNA for at least 36 weeks after the end of the time period. In a further subembodiment, the subject has an undetectable amount of HCV RNA for at least 48 weeks after the end of the time period.

In an eighth aspect of the twelfth embodiment, the subject has an amount of HCV RNA less than about 15 IU/mL for at least 12 weeks after the end of the time period. In on subembodiment, the subject has an amount of HCV RNA less than about 15 IU/mL for at least 24 weeks after the end of the time period. In one subembodiment, the subject 15 has an amount of HCV RNA less than about 15 ΙΌ/mL for at least 36 weeks after the end of the time period. In another subembodiment, the subject has an amount of HCV RNA less than about 15 IU/mL for at least 48 weeks after the end of the time period.

In a ninth aspect of the twelfth embodiment, the subject is a human.

In a tenth aspect of the twelfth embodiment, an effective amount of GS-7977 and an effective amount of ribavirin are used according to an interferon-free treatment regimen. In one subembodiment, the interferon-free treatment regimen consi sts of administering an effective amount of GS-7977 and an effective amount of ribavirin to the subject for a time period.

In an eleventh aspect of the twelfth embodiment, the effective amount of GS-7977 comprises a composition comprising GS-7977 and at least one pharmaceutically acceptable excipient as disclosed herein.

In a twelfth aspect of the twelfth embodiment, the effective amount of GS-7977 comprises a unit dosage form comprising GS-7977 and at least one pharmaceutically acceptable excipient as disclosed herein.

A

According to the FDA-approved label dated August 22, 2011, which is hereby incorporated by reference, the recommended dose of COPEGUS® (ribavirin) tablets when used in combination with peginterferon dépends on body weight and the HCV génotype to be treated, as shown in the following table.

HCV Génotype	PEGASYS® Dose*	COPEGUS® Dose	Duration
Génotypes 1,4	180pg	<75 kg — 1000 mg >75 kg = 1200 mg	48 weeks 48 weeks
Génotypes 2, 3	180 gg	800 mg	24 weeks
Génotypes 2 and 3 showed no increased response to treatment beyond 24 weeks. *See PEGASYS® Package Insert for further details on PEGASYS® dosing and administration. The FDAapproved label for PEGASYS® dated September 29, 2011 is incorporated by reference.

The daily dose of COPEGUS® indicated for use in combination with peginterferon is 800 mg to 1200 mg administered orally in two divided doses (BID). The dose should be individualized to the subject depending on baseline disease characteristics (e.g., génotype), response to therapy, and tolerability of the regimen. Based on the foregoing, as well as the exàmples described below, an effective amount ribavirin when used in combination with an effective amount of GS-7977 is contemplated to include 800 mg and 1000 mg to 1200 mg, including daily doses ôf 1000 mg or 1200 mg depending on body weight.

Based on the data reported herein, an effective amount of GS-7977 is 400 mg QD, which can also be administered B ED, TID, or QID. It is also contemplated that an effective amount of GS-7977 can include 100 mg to 400 mg and ail integer values in between.

When administered as .a combination, GS-7977 is administered to the subject in association with ribavirin. That is, the GS-7977 dose is administered during the same time period that the subject receives doses of ribavirin. Concurrent or alternative administration is consîdered, which means that while the GS-7977 and ribavirin are administered during the same time period, the spécifie order of administration on a daily basis can be: GS-7977 followed by ribavirin, GS-7977 and ribavirin together, or ribavirin followed by GS-7977. GS-7977 may be administered orally in capsule or tablet form, or any other suitable unit dosage form, in association with the oral (capsule or tablet form) administration of ribavirin. Of course, other types of administration of both médicaments, as they become available, are contemplated, such as by nasal spray, by a buccal or sublingual administration dosage form, transdermally, by suppository,- by sustained release dosage form, etc. Any form of administration will work so long as the proper dosages are delivered without destroying the active ingrédient and/or without impeding the effective amount of GS-7977 and/or an effective amount of ribavirin delivered to the subject.

Examples

GS-7977 Formulation Compositions Using Roller Compaction Process

A sériés of formulations containing polymorphie Form 1 GS-7977 with different quantitative compositions of excipients were prepared and screened using the roller compaction process to evaluate the impact of various diluents and compression aids on granulation powder properties and on tablet disintegration and dissolution times. Considérations were also given to moisture sorption properties of the tablets due to the sensitivity of Form 1 ôf GS-7977 to moisture.

Ail formulations were compressed into tablets at both high and low hardness levels. Formulation and tablet performance were determined by tablet disintegration time, content uniformity, and dissolution, as presented in Table IA.

Table IA. Formulation Compositions for GS-7977 Form 1 Tablets Using a Roller

Compaction Process

	Formulation Composition (% w/w)
Ingrédient	A	B1	B2	C	G1	G2	H
Intragranular
GS-7977	25.0	33.3	33.3	33.3	33.3	33.3	33.3
Microcrystalline Cellulose	25.0	33.3	33.3

.

Mannitol	-	-	-	33.3	30.6	33.3	30.6
Croscannellose Sodium					2.00	3.0	2.00
Colloïdal Silicon Dioxide	0.5	0.25	0.3	0.25	0.25	0.3	0.25
Magnésium Stéarate		0.5	0.5	0.5	0.5	0.5	0.5
Extragranular
Microcrystalline Cellulose	49.0	31.9	31.8		15.3	21.0
Mannitol	-	-	-	31.9	15.3	5.8	-
Croscarmellose Sodium					2.0	2.0	2.00
Dicalcium Phosphate							30.6
Colloïdal Silicon Dioxide		0.25	0.25	0.25	0.25	0.3	0.25
Magnésium Stéarate	0.5	0.5	0.5	0.5	0.5	0.5	0.5
Total Tablet Weight (mg>	400	300	300	300	300	300	300
Hardness (kp) Low/High	8.1	16.3	7.4	17.2	10.2	5.8	NA	5.4	12.1	8.2	5.1	9.9
Disintegration time (min:sec)	0:17	0:33	0:13	3:16	0:48	45:00		0:14	6:27	1:43	1:23	8:06
Dissolution @ 45 min (%LS)	98	102	94	91	82	87	NA	101	95	96	60	64

The results in Table IA show that use of microcrystalline cellulose as the sole diluent (Formulations A, Bl, B2) produced tablets with acceptable hardness, disintegration and dissolution, even without incorporating a disintegrant. In contrast, incorporation of mannitol as the sole diluent (Formulation C) without a disintegrant resulted in lower compressibility and a longer disintegration time resulting in slower dissolution. When used in combination with microcrystalline cellulose, mannitol levels as high as 75% of total filler amount (Formulation G) produced an acceptable tablet as long as a disintegrant was added to the formulation. However, lowering mannitol levels produced a harder and more robust tablet. Dicalcium phosphate used in combination with mannitol (Formulation H) failed to produce an acceptable tablet with respect to dissolution and hardness. The data in Table IA support the use of formulations i

Γ Τι· containing microcrystalline cellulose and mannitol/microcrystalline cellulose, in particular, as diluents.

Formulations B2 and G2 in Table IA prepared per a roller compaction/granulation process were further evaluated. The prototype tablet batches were packaged 30 tablets per bottle and placed on stability in 40°C/75% RH conditions, with each bottle containing a molecular sieve (Tri-Sorb®) dessicant. The data shown in Table IB show a decrease in moisture level as the amount of mannitol is increased (concomitant with réduction in microcrystalline cellulose).

Table IB. Stability Data for GS-7977 Form 1 Tablets Using a Roller Compaction

Process

			HPLC Assay	Dissolution (% Dissolved)*
Formulation	Stability Condition	Time (mo.)	%LS	Unknowa % img/deg at RRT 0.67	15 min	30 min	45 min	60 min	Water Content (%)
B2	25 °C/60% RH	0	98.5		76 (4)	80 (3)	82 (3)	85 (3)	4.3
3	98.4		73 (2)	77 (3)	79 (3)	81 (3)	3.2
6	96.2	<0.04	76 (2)	80 (2)	82 (2)	85 (2)	3.2
40 °CZ75% RH	3	97.0		71 (5)	74 (5)	77 (4)	79 (4)	3.3
6	96.8	0.04	79 (8)	84 (8)	87 (8)	89 (7)	3.5
G2	25 °C/60% RH	0	99.9		84 (6)	93 (3)	96 (3)	98 (3)	1.7
3	98.3	<0.04	72 (8)	92 (2)	96 (2)	97 (3)	1.4
6	97.9	<0.04	82 (4)	93 (2)	95 (3)	97 . (2)	1.3
40°C/75% RH	3	98.0	<0.04	77 (4)	84 (4)	88 (3)	89 (3)	1.5
6	99.0	<0.04	80 (5)	90 (5)	93 (4)	94 (4)	1.2
40 °CZ75% RH (no dessicant)	3	97.9	0.08	79 (4)	91 (3)	96 (2)	97 (2)	1.9
6	97.8	0.18	72 (8)	85 (2)	91 (2)	93 (3)	1.9

•Dissolution method: USP Apparatus Π (paddles) with 900 mL, pH 6.8 (50 mM sodium phosphate), 0.5% SLS, 75 rpm, 37 C

GS-7977 400 mg Tablets

Formulations (Tablets A and B) comprising GS-7977 polymorphie Form l were prepared by dry granulation. The formulations contained GS-7977 (polymorphie Form l) (33.33%), mannitol (30.00%), microcrystalline cellulose (29.67%), croscarmellose sodium (5.00%), colloïdal silicon dioxide (0.50%), and magnésium stéarate (1.50%), as described in Table 2.

Table 2. GS-7977 Polymorphie Form 1 400 mg Tablet Compositions

	% w/w of 400 mg tablet
	Tablet A	Tablet B
Intragranular Components
GS-7977 (Form 1)	33.33	33.33
Mannitol	30.0	30.0
Croscarmellose Sodium	3.0	3.0
FD&C Red 40 Aluminum Lake	-	0.27
FD&C Blue 2 Aluminum Lake	-	0.10
Colloïdal Silicon Dioxide	0.25	0.25
Magnésium Stéarate	0.50	0.5
Extragranular Components
Microcrystalline Cellulose	29.67	29.12
Croscarmellose Sodium	2.00	2.00
FD&C Red 40 Aluminum Lake	-	0.13
FD&C Blue 2 Aluminum Lake	-	0.05
Colloïdal Silicon Dioxide	0.25	0.25
Magnésium Stéarate	1.00	1.00
Total	100.00	100.00
Coating Agent	3.00	-

Tablets containing about 400 mg of GS-7977 (Form l) per tablet and an Opadry II purple film coating (Tablet A in Table 2) were prepared as follows:

(1) A composition comprising GS-7977 (Form 1) and the intragranular excipients (mannitol, croscarmellose sodium, and colloïdal silicon dioxide) was sifted through a 20mesh screen and added to a blender (V-shell blender) and blended for about 10-15 minutes to obtain an initial blend. Separately, the intragranular magnésium stéarate was passed through a 20-mesh screen and mixed with a portion of the initial blend, added to the blender, and blended for about 5 minutes to obtain an intragranular blend.

(2) Separately, the extragranular excipients microcrystalline cellulose, croscarmellose sodium, and colloïdal silicon dioxide were sifted through a 20-mesh screen for use in the final blending (step (4), below).

(3) The intragranular blend comprising GS-7977, mannitol, croscarmellose sodium, colloïdal silicon dioxide, and magnésium stéarate was passed through a roller compacter equipped with a 20-mesh (0.84 mm) milling screen on the granulator and both 20- and 60-mesh (0.25 mm) screens on the separator until granulation was achieved. The roller compacter parameters were: (i) granulator speed ranges from about 50 to about 90 rpm, more specifically about 70 rpm, compacter speed ranges from about 4 to about 6 rpm, more specifically about 5 rpm, and pressure ranges from about 65 to about 100 barr, more specifically about 75 to about 100 bar. Ribbons were produced using fiat straightgrooved rollers. Upon passing through the roller compacter, the material was passed/forced through a 20-mesh screen and then sifted with a 60 mesh screen. Granules were sorted into three categories (coarse, acceptable, and fine) using the separator portion of the dry granulator. ‘Coarse’ granules retained on the 20-mesh (0.84 mm) screen on the separator were passed through a Comil with a 0.055-inch (1.4 mm) round screen. Granules that remained on the 60 mesh screen were considered to be ‘acceptable’ granules. The milled/sifted granule material was passed te the final blending step. Material that passed through the 60 mesh screen was considered ‘fine’ and was recirculated through the roller compacter. This process was repeated until a minimum amount (e.g., less than 20%) of fines remained.

t *» »w y»* < j<

(4) The milled/sifted granules from step (3) and the sifted extragranular excipients (microcrystalline cellulose, mannitol, croscarmellose sodium and silicon dioxide) from step (2) were added to a blender (V-shell blender) and blended for about 15 minutes. Separately, magnésium stéarate was passed through a 20-mesh screen. The magnésium stéarate was added to the blender and blended for about 5 minutes to obtain a final powder blend comprising 33.33% w/w GS-7977. Blend uniformity samples were taken prior to removing the blend from the blender.

(5) The final blend was compressed into tablets using a tablet press (e.g., a Globe Pharma Mini Press) to obtain 1200 mg uncoated tablets comprising about 400 mg of GS7977. As needed, a 15% w/w aqueous suspension for film-coating comprising polyvinyl alcohol (Opadry II Purple) was prepared and applied to achieve a target weight gain of 3% (range: 2-4%). The coating suspension was spràyed at 300 g/min/4 guns (range: 200400 g/min/4 guns) at a target pan speed of 5 rpm (range: 4-8 rpm) and an exhaust température of 46 ±5 °C. The GS-7977 tablets were packaged with 30 tablets and 1 gram of desiccant per bottle.

Uncoated tablets comprising 400 mg of GS-7977 (Form 1) were prepared in a similar manner using blue and red lake in the blend (Tablet B in Table 2).

Another formulation (Tablet C) was prepared contaîning GS-7977 polymorphie Form 6 (33.33%), mannitol (30.00%), microcrystalline cellulose-(29.67%), croscarmellose sodium (5.00%), colloïdal silicon dioxide (0.50%), and magnésium stéarate (1.50%), as described in Table 3. While a low moisture grade of microcrystalline cellulose (PH 112) was used in the Form 1 formulation to improve the stability of Form 1 GS-7977, the microcrystalline cellulose grade was changed to PH 102 for the Tablet C formulation due to the non-hygroscopic nature of Form 6. In addition, incorporation of a large proportion of the excipients into the intragranular composition decreased the potential for powder ségrégation and the variability in the blend, and improved the tablet content uniformity for the Tablet C formulation.

Table 3. GS-7977 Polymorphie Form 6 400 mg Tablet Composition

	Tablet C
	%.w/w	mg/tablet
Intragranular Components
GS-7977 (Form 6)	33.33	400.0
Mannitol	30.00	360.0
Microcrystalline Cellulose	24.67	296.0
Croscarmellose Sodium	2.50	30.0
Colloïdal Silicon Dioxide	0.45	5.4
Magnésium Stéarate	0.75	9.0
Extragranular Components
Microcrystalline Cellulose	5.00	60.0
Croscarmellose Sodium	2.50	30.0
Colloïdal Silicon Dioxide	0.05	0.6
Magnésium Stéarate	0.75	9.0
Total	100.00	1200
Coating Agent	3.0	36.0

The Tablet C formulation was prepared by blending the intragranular components listed in Table 3, other than magnésium stéarate (i.e., GS-7977, microcrystalline cellulose, mannitol, croscarmellose sodium and colloïdal silicon dioxide) in a blender. The mixture was milled, blended with the intragranular magnésium stéarate and dry granulated using a roller compaction process train and mill. The resulting ribbons were milled through a milling screen and then blended with the extragranular excipients (microcrystalline cellulose, croscarmellose sodium, colloïdal silicon dioxiade, magnésium stéarate) to yield a powder blend comprising 33.33% w/w GS-7977. The powder blend was compressed to a target tablet weight of 1200 mg, with each tablet comprising about 400 mg of GS-7977. An aqueous suspension for the film-coating process was prepared and applied to achieve a target weight gain of 3%.

Λ • 1

Moisture content was tested for Tablets A-C and tablet stability (30 tablets/bottle with a 1 gram Tri-sorb® dessicant in a 60 cc HDPE bottle) was tested for Tablets B and C, the results of which are presented in Table 4.

Table 4. Moisture and Stability Data for GS-7977 400 mg Tablets

	Tablet A	Tablet B	Tablet C
	Moisture (% w/w)	HPLC Assay	Moisture (% w/w)	HPLC Assay	HPLC Assay
	% GS-7977	% Impurity	% GS-7977	% Impurity	% GS-7977
Initial	1.8	100	0.05	1.5	99.5	0.05	101.7
40°C/75% RH
1 month	1.4	102.4	0.04	1.5	101.7	0.05	101.1
2 months	1.5	101.5	0.04	1.7	101.1	0.04	100.9

The results in Table 4 show that the exemplary tablet compositions described herein exhibit stability to both moisture and dégradation.

The dissolution profile (75 RPM, Apparatus II (Paddle), Phosphate buffer pH 6.8

900 mL) of tablets having the Tablet B formulation was tested initially and after storage at 40°C and 75% relative humidity. The results are presented in Table 5.

Table 5. Dissolution Data for GS-7977 (Form 1) 400 mg Tablet B Composition

	Mean Dissolution (± RSDa)
	15 min	30 min	45 min	60 min
Initial	97 + 1	102 ±2	103 ± 1	102 + 1
40°C/75% RH
1 month	87 ±3	99 ±2	101 ±2	100 + 3
2 month	96 ± 1	102+1	102+1	102 + 1

^aRSD = Relative Standard Déviation

In Vitro Antiviral Synergy for the Combination of GS-977 and Ribavirin

The antiviral effect of GS-7977 in combination with ribavirin was evaluated using the HCV génotype la replicon. (Robinson et al., Antimicrob. Agents Chemother. (2010) 54(8): 3099-3106.) The cells were grown in cell culture medium containing Dulbecco’s Modified Eagle Medium (DMEM) with Gibco® GlutaMAX supplemented with 10% HyClone FPS, 100 units/mL penicillin, 100 pg/mL streptomycin, and 0.1 mM nonessential amino acids. Replicon cells were maintained· in 0.5 mg/mL Geneticin®. The cells were passaged every 3-4 days before reaching confluency. Ail compounds were supplied in 100% DMSO and compound serial dilutions were performed in 100% DMSO. To each well of a 384-well plate was added 90 pL of cell culture medium (without Geneticin®) containing 2000 suspended HCV replicon cells and 0.4 pL of compound solution. The DMSO concentration of the final assay wells was 0.44%. The plates were incubated for 3 days at 37°C with 5% CO₂ and 85% humidity.

For the CC₅o assay, the media in the 384-well plate was aspirated and the wells were washed four times with 100 pL 1 X PBS each. A volume of 50 pL of a solution containing 400 nM calcein AM in 1 X PBS was added to each well and the plate was incubated for 30 minutes at room température before the fluorescence signal (excitation 490 nm, émission 520 nm) was measured.

EC₅₀ assays were performed in the same wells as CC50 assays. The calcein-PBS solution was aspirated and a volume of 20 pL of Dual-Glo® luciferase buffer was added to each well. The plate was incubated for 10 minutes at room température and a volume of 20 pL of a solution containing a 1:100 mixture of Dual-Glo® Stop & Glo® substrate and Dual-Glo® Stoj) & Glo® bufifer.was added to each well. The plate was incubated at room température for 10 minutes before the luminescence signal was measured.

The combination study experimental data were analyzed for two-compound synergy using the MacSynergy II program developed by Prichard and Shipman. (Prichard et al., MacSyngergy™ II, Version 1.0, University of Michigan, Ann Arbor (1993).) Two-compound synergy définitions are provided in Table 6:

Table 6. Two-Compound Synergy Définitions

Synergy/Antagonism Volume (nM2%)

Interaction

•r- ·· -» ι ’tiw

Auiiml» w.'.ei i ··»* * ··*·· !>»»*—««??*·*>

>100	Strong Synergy
>50 and <100	Moderate Synergy
> 25 and < 50	Minor Synergy -
< 25 and > -25	Additive
< -25 and > -50	Minor Antagonism
<-50and>-100	Moderate Antagonsim
<-100	Strong Antagonsim

GS-7977 in combination with ribavirin showed a synergy volume of

35.3 ± 3.2 nM²% indicating a synergistic interaction. A cytotoxicity study analyzing the combined effect of GS-7977 and ribavirin showed cell viability greater than 85% at the highest combined drug concentrations (320 nM GS-7977,1600 nM ribavirin, 14.0 ± 4.4 % inhibition on cell growth). (See also Hebner et al., 63^rd Annual Meeting of the American Association for the Study of Liver Diseases, Poster 1875, Nov. 12, 2012.) These findings support the potentiel of GS-7977 administered in combination with ribavirin to achieve enhanced viral suppression compared to GS-7977 or ribavirin monotherapy.

In Vitro Susceptibility of S282T Mutants to GS-7977, Ribavirin, and the Combination of GS-7977 andRibavirin

In vitro studies hâve shown that S282T is the primary mutation selected by GS7977 in HCV génotype la, lb and 2a replicon cells. (Lam et ah, J. Virology (2011) 85(23): 12334-12342; Lam et al., Antimicrob. Agents Chemother. (2012) 56(6): 33593368.) S282T mutations in NS5B were created by site-directed mutagenesis in la-H77, lb con-1, and 2a JFH1 sub-genomic replicons. lb con-l-based chimeric replicons containing 2b, 3a, 4a, 5a, or 6a NS5B were also engineered to harbor the S282T mutation. (See Wong et al., Virology (2012) 429:57-62.) Réplication capacities and drug susceptibilities of S282T to GS-7977 and ribavirin were determined in transient replicon assays. The susceptibilities of S282T and wild-type (WT) NS5B to GS-7977 and ribavirin were further studied by passaging the mixture of 50% S282T and 50% WT in

S

GT2a in the presence of GS-7977 and ribavirin individually and in combination. Relative percentages of mutant and WT were assessed by deep sequencing.

Introduction of the NS5B S282T mutation into lb, la, 2a, 2b, 3a, 4a, and 5a HCV replicons resulted in reduced susceptibility to GS-7977 for ail seven génotypes, producing 5 a 2- to 16-fold increase in ECso values compared to the wild-type from the corresponding génotypes. Surprisingly, the S282T replicons weFe 3- to 10-fold more sensitive to treatment with ribavirin than their corresponding wild-type for these seven génotypes. EC50 values were not calculated for génotype 6a S282T mutants due to low signal-tonoise ratios; the génotype 6a mutant did not replicate sufficiently to obtain drug susceptibility data. The results of these studies are presented in Table 7, below, and in Figure 2.

Table 7. Antiviral Activity of GS-7977 and Ribavirin Against S282T Mutants in

Génotype 1-6 Replicons

Génotype	GS-7977	Ribavirin
EC50 nM1	Fold Change1*	ECso nMa	Foldb Change
WT	S282T	WT	S282T
lb	21.5	189.2	8.8	6.6	1.6	0.2
la	25.1	253.1	10.1	21.0	5.0	0.2
2a	146.8	346.1	2.4	8.3	0.6	0.1
2bc	13.3	215.6	16.2	2.6	0.6	0.2
3ac	33.9	117.1	3.5	6.7	1.0	0.2
4ac	35.8	217.5	6.1	6.2	0.6	0.1
5ac	9.91	142.2	14.35	1.9	0.6	0.3
6ac	39.8	n/ad	-	5.3	n/ad	-

‘EC₅o indicates average of 2 or more independent experiments.

Told change from corresponding wild-type.

These chimeric replicons carry NS5B from génotypes 2b, 3a, 4a; however, the NS5A sequence in ali of these chimeric replicons is derived from génotype lb.

^JECso was not determined due to low signal-to-noise ratio.

A long-term passaging study in GT 2a replicons revealed that GS-7977 alone displayed greater inhibition of WT than S282T, resulting in a population that was 92% mutant S282T over fifteen days. Ribavirin alone suppressed S282T more than WT, resulting in a population that was 96% WT after fifteen days. The combination of GS5 7977 and ribavirin also preferentially inhibited S282T over WT, resulting in a population that was 91% WT following thirty days of treatment. The results of the passaging study are presented in Figure 3. (See also Han et al., 63^rd Annual Meeting of the American Association for the Study of Liver Diseases, Poster 1078, Nov. 11, 2012.)

Thus, while the S282T replicon has been shown to confer reduced susceptibility to GS-7977 in vitro, the mutant replicon has demonstrated increased susceptibility to ribavirin over the wile-type, suggesting that treatment of CHC with the combination of GS-7977 and ribavirin may resuit in reduced viral breakthroughs and incidence of résistance compared to monotherapy with GS-7977 alone. The hypersensitivity of S282T mutants to ribavirin may provide an additional advantage to combination treatment comprising GS-7977 and ribavirin, in tenus of preventing or delaying the emergency of S282T mutants.

Quantification of HCV RNA in Human Clinical Studies

Quantitative HCV RNA testing for clinical trials was performed using the Roche

COBAS® AmpliPrep/COBAS® HCV TaqMan® assay using a standardized, automatic RNA extraction System and standardized controls and calibrators. The established LOD of the assay was 15 IU/mL (defined by a 95% hit rate with WHO Standards). HCV RNA levels were measured using sérum samples.

US 2010/0226885 (US 12/376,180), which is incorporated byreference, also discloses a method for measuring whether a patient has achieved an HCV négative status using RT-PCR to measure HCV RNA levels.

Treatment Regimens — P7977-0221 and PROTON Clinical Studies

A Phase 2a, 3-cohort placebo-controlled study (P7977-0221) evaluated treatment with GS-7977 (100 mg, 200 mg or 400 mg QD) in combination with peginterferon and

ribavirin in treatment-naïve GT1 HCV subjects for 4 weeks, followed by up to an additîonal 44 weeks of treatment with SOC peginterferon and ribavirin. High RVR (8894%) was observed for ail three GS-7977 treatment groups. Following discontinuation of GS-7977, the durability of antiviral response (SVR-12 and SVR-24) was greatest in the 400 mg treatment group (86.7% and 80.0%, respectively). SVR-12 and SVR-24 rates were 72.2% and 833%, Fespectively, for patients receiving a 200 mg GS-7977 treatment regimen, and the majority of GS-7977-treated patients who failed to achieve SVR received a 100 mg QD dose of GS-7977.

The Phase 2b PROTON study evaluated treatment with a combination of GS7977, peginterferon, and ribavirin at daily dosage Ievels of200 mg and 400 mg of GS7977 for 12 weeks, followed by up to an additîonal 36 weeks of treatment with SOC peginterferon and ribavirin. A greater number of subjects experienced viral breakthrough after cessation of the GS-7977 200 mg dosage level while still receiving peginterferon/ribavirin treatment compared to no viral breakthroughs after cessation of the GS-7977 400 mg dosage level while still receiving peginterferon/ribavirin treatment.

The preceding studies indicate enhanced efficacy for a GS-7977 400 mg daily dose level compared to a 200 mg daily dose level.

Treatment Regimens - ELECTRON Clinical Study

The ongoing Phase 2a ELECTRON clinical study evaluated GS-7977 400 mg QD for 8 or 12 weeks in combination with or without ribavirin and/or peginterferon in subjects with GT1, GT2 or GT3 HCV infection. Preliminary data demonstrates 100% SVR-12 for treatment-naïve GT2 or GT3 HCV patients treated with a combination of GS-7977 and ribavirin, regardless of the presence of peginterferon, as well as 84% SVR12 for treatment-naïve GT1 HCV patients receiving combination treatment with GS-7977 and ribavirin. In comparison, only 60% of treatment-naïve GT2/GT3 HCV patients receiving GS-7977 monotherapy achieved SVR-12.

Part 1 of the ELECTRON trial evaluated 12-week regimens of GS-7977 400 mg QD in combination with ribavirin (RBV) only (1000/12000 mg by weight BID) and, in separate arms, with abbreviated durations of peginterferon for 4, 8, or 12 weeks in treatment-naïve patients with HCV GT2 or GT3:

Group 1 : GS-7977 (400 mg QD) with RBV (1000/1200 mg BID) for 12 weeks (no peginterferon) (GT2/GT3 treatment-naïve); and

Groups 2, 3,4: GS-7977 (400 mg QD) with RBV (1000/1200 mg BID) for 12 weeks and PEG (180 pg weekly) weeks 1-4 only / PEG (1-80 pg weekly) weeks 1-8 only / PEG (180 pg weekly) weeks 1-12 (GT2/GT3 treatment-naïve).

In Part 2 of the ELECTRON trial, an additional 30 patients were enrolled in exploratory regimens of GS-7977 monotherapy and abbreviated durations of total therapy with the combination of GS-7977, RBV and PEG:

Group 5: GS-7977 (400 mg QD) monotherapy for 12 weeks (GT2/GT3 treatmentnaïve);

Group 6: GS-7977 (400 mg QD) with PEG (180 pg weekly) and RBV (1000/1200 mg BID) for 8 weeks (GT2/GT3 treatment-naïve); and

Group 7: GS-7977 (400 mg QD) with RBV (1000/1200 mg BID) for 12 weeks (GT1 null responders).

In Part 3 of the ELECTRON trial, two additional peginterferon-free regimens were explored in treatment-naïve patients with HCV GT1 and treatment-experienced patients with HCV GT2 or HCV GT3 :

Group 8: GS-7977 (400 mg QD) with RBV (1000/1200 mg BID) for 12 weeks (GT1 treatment-naïve); and

Group 9: GS-7977 (400 mg QD) with RBV (1000/1200 mg BID) for 12 weeks (GT2/GT3 treatment-experienced).

In Part 4 of the ELECTRON trial, two further peginterferon-free regimens were added:

Group 10: GS-7977 (400 mg QD) with RBV (1000/1200 mg BID) for 8 weeks (GT2/GT3 treatment-naïve); and

Group 11 : GS-7977 (400 mg QD) with RBV (800 mg BID) for 12 weeks (GT2/GT3 treatment-naïve).

't t

f ♦· ’ 1 · Λ» *·»♦ · «r · F'

Null responders were defined as patients with <2 logio IU/mL décliné from baseline HCV RNA after at least 12 weeks of treatment with peginterferon and ribavirin.

Treatment-experienced patients were defined as those who had any of the following responses after at least 12 weeks of treatment with peginterferon and ribavirin: 5 (1) < 2 logio IU/mL. décliné from baseline HCV RNA, (2) > logio IU/mL réduction in

HCV RNA, but HCV RNA > limit of quantitation (“LOQ”) at end of treatment, and (3) HCV RNA < LOQ at end of treatment but subséquent HCV RNA > LOQ (relapsers).

The preliminary results of the ELECTRON trial are presented below.

The patient population and demographics for ELECTRON Groups 1 -9 are summarized in Tables 8A and 8B, below.

Table 8A. ELECTRON Patient Demographics (Groups 1-5)

	GS-7977 RBV NO PEG	GS-7977 RBV 4 Wks PEG	GS-7977 RBV 8 Wks PEG	GS-7977 RBV 12 Wks PEG	GS-7977 NO RBV NO PEG
	GT2/GT3 Tx-Naive
	(Group J)	(Groups 2, 3, 4)	(Group 5)
Number (N)	10	9	10	11	10
Male (n, %)	8(80)	5(56)	5(50)	9(82)	4(40)
Race (Caucasian, %)	7(70)	4(44)	8(80)	8(73)	7(70)
Age (Mean, range)	47 (35-53)	47 (29-66)	49 (29-66)	46 (22-57)	43 (22-57)
BMI (Mean, range) (kg/m2)	28 (23.7-35.7)	26 (21.3-32.2)	25 (18.1-32.5)	24 (20.8-28.4)	26 (18.2-39.4)
HCV RNA (Mean, SD) (logio IU/mL)	6.7 (0.42)	6.6 (0.52)	6.4 (0.57)	6.3 (0.76)	5.7 (0.89)
HCV RNA (Médian, range)	6.7 (6.6-7.3)	6.6 (5.8-7.3)	6.4 (5.1-7.0)	6.4 (5.2-7.1)	5.7 (4.6-7.3)
HCV GT-2:GT-3	4:6	3:6	4:6	4:7	3:7
IL28B CC/CT/TT	5/4/1	4/4/1	4/4/2	4/5/2	2/6/2
IL28B CC (n, %)	5(50)	4 (44)	4(40)	4(36)	2(20)

---1

Table 8B. ELECTRON Patient Demographics (Groups 6-9)

	GS-7977 RBV PEG 8 Wks	GS-7977 RBV NO PEG 12 Wks	GS-7977 RBV NO PEG 12 Wks	GS-7977 RBV NO PEG 12 Wks
	GT2/GT3 Tx-Naive	GT1 Null	GT1 Tx-Naîve	GT2/GT3 Tx-Ëxperienced
	(Group 6)	(Group 7)	(Group 8)	(Group 9)
Number (N)	10	10	25	25
Male (n, %)	50	70	60	76
Race (Caucasian, %)	70	90	80	68
BMI (Mean,.range)	24.8 (21-34.9)	28.1 (19.5-35.7)	25.6 (19.3-37.6)	26.8 (19.2-40.0)
HCV RNA (Mean, SD) (logjo lU/mL)	6.1 (4.3-7.3)	6.8 (5.6-7.5)	6.1 (4.4-7.2)	6.5 (4.8-7.7)
GT la(%)	n/a	90	88	n/a
GT 3 (%)	100	n/a	n/a	76
IL28B CC/CT/TT	3/6/1	2/5/3	11/12/2	11/12/2
IL28B CC (n, %)	3 (30)	2(20)	11 (44)	11 (44)

A summary of the patient results for treatment-naïve HCV GT2/GT3 Groups l-5 as related to the percentage of patients having an amount of HCV RNA below the limits 5 of détection (LOD) is provided in Table 9.

Table 9. ELECTRON Groups 1-5 Patient Results

Time (Wks)	GS-7977 RBV NO PEG	GS-7977 RBV 4 wks PEG	GS-7977 RBV 8 wks PEG’	GS-7977 RBV 12 weeks PEG’	GS-7977 NO RBV NO PEG
(Group 1)	(Groups 2, 3,4)	(Group 5)
n/N	%<LOD	n/N	%<LOD	n/N	%<LOD	n/N	%<LOD	n/N	%<LOD
0	0/10	0	0/9	0	0/10	0	0/ïl	0	10/0	0
4	10/10	100	9/9	100	10/10	100	11/11	100	10/10	100
8	10/10	100	9/9	100	10/10	100	11/11	100	10/10	100
12	10/10	100	9/9	100	10/10	100	11/11	100	10/10	100
SVR-4	10/10	100	9/9	100	10/10	100	11/11	100	6/10	60
SVR-8	10/10	100	9/9	100	10/10	100	11/11	100	6/10	60
SVR-12	10/10	100	9/9	100	10/10	100	11/11	100	6/10	60
SVR-24	10/10	100	9/9	100	10/10	100	11/11	100	6/10	60

From Table 9 it can be seen that ali treatment-naïve HCV GT2 and GT3 patients treated with GS-7977 and RBV for 12 weeks (Groups 1 -4) had no détectable amount of

HCV RNA during the entire treatment period (with or without PEG). Ail such patients treated with a combination of GS-7977 and RBV (with or without PEG) had no détectable amount of HCV RNA at 12 weeks and at 24 weeks after the termination of treatment.

Table 9 also reveals that ail HCV GT2/GT3 treatment-naïve patients receiving 12 10 weeks of GS-7977 (400 mg QD) monotherapy (Group 5) had no détectable amount of

HCV RNA during the entire treatment period. However, only 60% of the patients receiving GS-7977 monotherapy achieved SVR-12 and SVR-24.

Comparing Group 1 (GS-7977 + RBV) with Group 5 (GS-7977 monotherapy), the combination of GS-7977 and ribavirin appears to provide a synergistic increase in SVR-4, 15 SVR-8, SVR-12 and SVR-24 rates, as ribavirin alone has been reported to hâve little to no effect on HCV RNA levels.

Table 10 provides the mean HCV RNA values (logio IU/mL) for treatment-naive HCV GT2/GT3 patients (N=10) for time of treatment (12 weeks) up to 12 weeks after •r treatment (W24) for patients receiving a combination of 400 mg QD of GS-7977 and 1000/1200 mgBID (based on weight) of RBV (Group 1). Table 10 also provides the mean HCV RNA values (logio RJ/mL) for treatment-naïve HCV GT2/GT3 patients (N=10) for the time of treatment (12 weeks) for patients receiving a 12-week regimen of

400 mg QD of GS-7977 only (Group 5). The terms DI (6 hr) and DI (12 hr) refer to the recorded measurements made 6 hrs and 12 hrs, respectively, on day 1 following day 1 dosing. The data presented in Table 10 is also illustrated in Figure 1.

Table 10. ELECTRON Groups 1 and 5 HCV RNA values (logjoIU/mL)

Time	HCV RNA (logio IU/mL)
GS-7977 RBV (Group 1)	GS-7977 NO RBV (Group 5)
T=0a	6.79	6.08
DI	6.67	5.74
DI (6 hr)b	6.65	5.63
DI (12hr)c	5.86	4.98
D2	4.50	3.75
D3	3.41	2.62
W1	2.16	1.56
W2	1.36	1.22
W3	1.18	1.15
W4	1.15	1.15
W5	1.15	1.15
W6	1.15	1.15
W7	1.15	1.15
W8	1.15	1.15
W9	1.15	1.15
W10	1.15	1.15
Wll	1.15	1.15
W12	1.15	1.15
W14	1.15	1.66
W16	1.15	2.95
W20	1.15	3.12

·»— -r - ·

W24	1.15	3.17
“Initial Screening Values for patients. ’Day 1 results 6 hrs after dosing. cDay 1 results 12 hrs after dosing.

The data in Table 10 and Figure 1 clearly show that treatment of HCV GT2/GT3 treatment-naïve patients with a combination of GS-7977 and RBV (in the amounts noted above) results in mean HCV RNA levels below the limit of détection during weeks 4-12 of the treatment period, as well as SVR-12. This data also shows that the mean HCV RNA value is below the limit of détection during weeks 3-12 of the treatment period for patients receiving GS-7977 monotherapy. However, Table 10 and Figure 1 also illustrate that patients who received a combination of GS-7977 and ribavirin for 12 weeks (Group 1) maintained lower mean HCV RNA levels for the 12 weeks following cessation of treatment compared to patients who received monotherapy with GS-7977 (Group 5).

These results demonstrate that the combination of GS-7977 and ribavirin is advantageous in that patients can be treated for HCV without receiving peginterferon treatment and achieve a high rate of SVR-12.

A summary of the preliminary patient results for ail nine fully reportcd cohorts of the ELECTRON trial as related to the percentage of patients having an amount of HCV RNA below the limits of détection (LOD) is summarized in Table 11.

Table 11. ELECTRON Groups 1-9 Patient Results

Time (Wks)	Génotype 2/3 Treatment Naïve	Génotype 1 Null Responders	Génotype 1	Génotype 2/3 Treatment Experienced
GS-7977 RBV NOPEG 12 wks (Group 1) (N =10) n (%)	GS-7977 RBV PEG 12 wks (Groups 2,3,4) (N = 30) n (%)	GS-7977 NO RBV NOPEG 12 weeks (Group 5) (N =10) n (%)	GS-7977 RBV PEG 8 weeks (Group 6) (N =10) n(%)	GS-7977 RBV NO PEG 12 weeks (Group 7) (N =10) n (%)	GS-7977 RBV NO PEG 12 weeks (Group 8) (N = 25) n (%)	GS-7977 RBV NO PEG 12 weeks (Group 9) (N = 25) n (%)
0	0	0	0	0	0	0	0
1	2(20)	8(27)	5(50)	6(60)	1(10)	8(32)	8(32)
2	8(80)	23(77)	8(80)	10(100)	7(70)	17(68)	21 (84)
3	9(90)	25 (83)	10(100)	10(100)	10(100)	22(88)	25 (100)
4	10(100)	30 (100)	10(100)	10(100)	10(100)	25(100)	25(100)
8	10(100)	30(100)	10(100)	N/A	10(100)	25 (100)	25 (100)
12	10(100)	30 (100)	10(100)	10(100)	10(100)	25(100)	25 (100)
SVR-4	10(100)	30(100)	6(60)	10(100)	1 (10)	22 (88)	19(76)
SVR-12	10(100)	30(100)	6(60)	10(100)	I (10)	21 (84) ...	17(68)

The data in Table 11 demonstrate an SVR.-12 rate of 100% for treatment-naïve patients with HCV GT2/GT3 (Groups 1-4, 6) when treated with a combination of GS5 7977 (400 mg QD) and RBV, regardless of the presence of peginterferon. The data in

Table 11 also demonstrates an S.VR-12 rate of 84% for patients with HCV GT1 (Group 8) treated with a combination of GS-7977 and RBV in the absence of peginterferon. In contrast, monotherapy with GS-7977 (Group 5) for GT2/GT3 treatment-naïve patients produced an SVR-12 rate of 60%.

Ail patients enrolled in Group 10 (8 weeks of GS-7977 + ribavirin combination therapy in treatment-naïve GT2/GT3 HCV subjects) achieved rapid virological response, and there were no discontinuations or on-treatment breakthroughs.

Treating a subject infected with HCV by administering an effective amount of GS-7977, either alone or in combination with an effective amount of RBV, means that the 15 side-effects normally associated with peginterferon may be avoided. Table 12 présents adverse events reported in at least 15% of the subjects in any treatment group for

ELECTRON Groups 1-9.

Table 12. ELECTRON Groups 1-9 Adverse Events Reported in at Least 15% of

Subjects in Any Treatment Group

Adverse Event	GS-7977 RBV NOPEG 12 wks N = 70 (Groups 1, 7, 8,9)	GS-7977 PEG RBV 12 wks N = 30 (Groups 2. 3,4)	GS-7977 NO RBV NOPEG 12 wks N = 10 (Group 5)	GS-7977 RBV PEG 8 wks N = 10 (Group 6)
>1 AE: n (%)	69 (99)	30(100)	10(100)	10(100)
Blood and Lymphatic System Disorders	10(14)	10(33)	0	3(30)
Anémia	3(4)	5(17)	0	3(30)
Gastrointestinal Disorders	32 (46)	17 (57)	8(80)	7(70)
Nausea	18(26)	9(30)	3(30)	2(20)
Diarrhoea	10(14)	4(13)	0	3(30)
Abdominl Pain	Kl)	1 O)	0	2(20)
Flatulence	KD	0	0	2(20)
General Disorders and Administration Site Conditions	43(61)	22 (73)	8(80)	10(100)
Fatigue	27 (39)	11 (37)	3(30)	7(70)
Irritability	8(11)	5(17)	1(10)	2(20)
Pyrexia	KD	4(13)	0	5(50)
Pain	KD	2(7)	0	2(20)
Chills	0	2(7)	0	2(20)
Injection Site Erythema	0	1(3)	0	2(20)
Axillary Pain	0	0	2(20)	0
Infections and Infestations	33 (47)	12(40)	5(50)	6(60)
Upper Respiratory Tract Infection	11(16)	3(10)	2(20)	1(10)
Metabolism and Nutrition Disorders	5(7)	11 (37)	0	50 (50)
Decreased Appetite	4(6)	5(17)	0	50 (50)
Musculoskeletal and Connective Tissue Disorders	23 (33)	19(63)	2(20)	7(70)

Myalgia	10(14)	9(30)	KIO)	4(40)
Back Pain	3(4)	4(13)	1 (10)	2(20)
Arthralgia	4(6)	5(17)	0	1(10)
Nervous System Disorders	40 (57)	26 (87)	9(90)	7(70)
Headache	28 (40)	24 (80)	8(80)	6(60)
Dizziness	7(10)	9 (30)	2(20)	1(10)
Dizziness Postural	0	0	0	2(20)
Psychiatrie Disorders	26 (37)	23(77)	6(60)	5(50)
Insomnia	15(21)	16(53)	6(60)	1(10)
Respiratory, Thoracic and Médiastinal Disorders	18(26)	15(50)	3(30)	5(50)
Oropharyngeal Pain	5(7)	3(10)	2(20)	1(10)
Dyspnoea	2(3)	5,(17)	0	1(10)
Skin and Subeutaneous Tissue Disorders	31 (44)	25 (83)	3(30)	8(80)
Rash	16(23)	9(30)	1(10)	5(50)
Pruritus	4(6)	8(27)	0	2(20)
Dry Skin	7(10)	5(17)	0	2(20)
Alopecia	0	5(17)	0	1(10)

The data in Table 12 reveal that lower incidence rates (%) were reported for a number of types of adverse events for treatment regimens involving the combination of GS-7977 and ribavirin (Groups 1, 7, 8, 9) compared to treatment regimens also involving peginterferon (Groups 2, 3, 4). For example, reduced rates of the following adverse events were reported for the interferon-free treatment regimens combining GS-7977 and ribavirin: blood and lymphatic System disorders (including anémia); pain and chills; metabolism and nutrition disorders (including decreased appetite); musculoskeletal and connective tissue disorders (including myalgia, back pain and arthralgia); nervous System disorders (including headache and dizziness); psychiatrie disorders (including insomnia); respiratory, thoracic and médiastinal disorders (including dyspnoea); and skin and subeutaneous tissue disorders (including pruritis, dry skin and alopecia).

-x

Î7352

The data in Table 13, below, reveals reduced frequencies of Grade 3 and Grade 4 hématologie abnormalities for interferon-free Groups 1,5, 7, 8 and 9 compared to Groups 2, 3,4 and 6 receiving treatment regimens including peginterferon:

Table 13. ELECTRON Groups 1-9 Reported Grade 3/4 Hématologie Abnormalities

Laboratory Abnormalities	GS-7977 RBV NOPEG 12wks (Group 1) (N = 10) n (%)	.GS-7977 RBV PEG 12 wks (Groups 2,3,4) (N = 30) n(%)	GS-7977 NO RBV NOPEG 12 weeks (Group 5) (N= 10) n (%)	GS-7977 RBV PEG 8 weeks (Group 6) (N =10) n (%)	GS-7977 RBV NOPEG 12 weeks (Group 7) (N = 10) n (%)	GS-7977 RBV NOPEG 12 weeks (Group 8) (N = 25) n (%)	GS-7977 RBV NOPEG 12 weeks (Group 9) (N = 25) n (%)
Alanine aminotransferase
Grade 3	0	1(3)	0	0	0	1(4)	0
Hemoglobin
Grade 3	0	1(3)	0	1(10)	1 (10)	0	0
Lymphocytes
Grade 3	0	3(10)	0	0	0	0	0
Grade 4	0	0	1(10)	0	0	1(4)	0
Neutropenia
Grade 3	0	5(17)	0	2(20)	0	0	0
Grade 4	0	5(17)	0	0	0	0	0
White blood cells
Grade 3	0	6(20)	0	0	0	0	0
INR
Grade 3	1(10)	0	0	0	1 (10)	0	0

Additional results, not shown here, show a rapid normalization of ALT levels in ail patients in ELECTRON Groups 1-5 during the treatment period (12 weeks), and to the extent of available data, for periods after the end of the treatment period.

GS-7977 Résistance in Human Clinical Studies

To date, no virologie breakthrough has been observed during treatment with GS7977, suggesting a high barrier to résistance. Across the P7977-0221, PROTON, ELECTRON (Groups 1-9) and ATOMIC Phase 2 human clinical studies of treatment

regimens involving GS-7977 alone or in combination with ribavirin and/or peginterferon, 53 out of 621 patients hâve experienced viral relapse after cessation of GS-7977containing treatment. Population sequencing of the viral relapse samples showed that S2S2T was detected in only one ofthe 53 patients, who was GT2b and relapsed 4 weeks after completion of 12 weeks of GS-7977 monotherapy. Deep sequencing revealed 99% S2c2T in this GT2b patient at relapse. Population and clonal phenotypic analysis de nonstrated that the GT2b S282T-containing sample was 8- to 13-fold less susceptible to GS-7977 compared to corresponding baseline virus. For the other 52 patients experiencing relapse, deep sequencing at baseline and relapse showed no S282T, and no spécifie NS5B mutation at other residues was identified by population or deep sequencing as being associated with GS-7977 résistance. (See also Svarovskaia et al., 63^rd Annual Meeting of the American Association for the Study of Liver Diseases, Poster 753, Nov. 11,2012.)

The foregoing illustrâtes that GS-7977 has a high résistance barrier. Notably, the S282T mutation has not been observed in any patient receiving a treatment regimen combining GS-7977 and ribavirin.

Concordance ofSVR-4 with SVR-12 and SVR-24 for Treatment Regimens Combining GS7977 with Ribavirin and Optionally Peginterferon

Florian et al. hâve reported that SVR-12 and SVR-24 were concordant across a large population database of HCV clinical trials including trials involving peginterferon/ribavirin combination treatment and treatment regimens combining peginterferon, ribavirin and telaprevir or boceprevir, with SVR-12 having a positive prédictive value of 98% for SVR-24. (Florian et al., AASLD 2011, Abstract LB-28; see also Martinot-Peignoux et al., Hepatology (2010) 51(4): 1122-1126.)

HCV data from treatment-naive GT1, GT2 and GT3 patients in the PROTON, ELECTRON and ATOMIC Phase 2 studies who received at least 12 weeks of treatment with GS-7977, either alone or in combination with ribavirin and optionally peginterferon, were evaluated. Only patients treated for at least 12 weeks with 400 mg GS-7977 who had SVR-4 and SVR-12 or SVR-4 and SVR-24 data were included in the analysis (259 of

596 patients). The analysis found 99-100% concordance between SVR-4 and both SVR12 and SVR-24 across ail regimens for patients who achieved SVR-4 and for whom posttreatment week 12 data were available. These results show that SVR-4 is highly concordant with SVR-12 and SVR-24 for GT1, GT3 and GT3 HCV patients treated with 400 mg GS-7977 and ribavirin, and optionally with peginterferon. (Lawitz et al., GS7977 Phase 2 Trials: Concordance of SVR4 with SVR 12 and SVR24 in HCV Génotypes 1-3, EASL (April 18-22, 2012).)

The foregoing suggests that the SVR data presented herein may hâve prédictive value for longer-term SVR rates încluding SVR-24, SVR-36 and SVR-48.

The compositions and unit dosage forms comprising GS-7977 disclosed herein provide good stability to moisture and dégradation, as well as désirable dissolution and désintégration profiles. They may be used to treat HCV infection optionally in combination with ribavirin, peginterferon or any other antiviral agent.

Additionally, the foregoing data illustrate that GS-7977 administered in combination with ribavirin (with or without peginterferon) elicited a rapid décliné in HCV RNA and end of treatment response (EOTR) in patients with HCV GT1, GT2 and GT3. No viral breakthrough has been observed during the course of treatment with GS7977, încluding when combined with ribavirin and optionally peginterferon. SVR-12 was 100% for HCV GT2 and GT3 treatment-naïve patients who received a combination of GS-7977 and ribavirin for 12 weeks and 84% for HCV GT1 treatment-naïve patients who received a combination of GS-7977 and ribavirin for 12 weeks, compared to 60% SVR12 for HCV GT2 and GT3 treatment-naïve patients who received GS-7977 alone. Given that ribavirin, alone, has been shown to hâve little to no effect on HCV RNA levels in human clinical trials, the foregoing clinical and in vitro data demonstrates that the combination of GS-7977 and ribavirin produces a synergistic réduction in HCV RNA levels.

Further, treatment arms in the ELECTRON trial receiving GS-7977 in combination with ribavirin, compared to treatment arms also receiving peginterferon, reported reduced incidences of side effects, suggesting that interferon-ffee treatment with a combination of GS-7977 and ribavirin may offer advantages over treatment regimens involving peginterferon.

Even further, in vitro results showing that HCV replicons with the S282T mutation, which show reduced susceptibility to GS-7977, display increased susceptibility to ribavirin suggest that the combination of GS-7977 and ribavirin may provide a treatment regimen resulting in reduced rates of résistance compared to monotherapy with GS-7977. Thus far, the S282T mutation has not been observed in a patient receiving GS7977 and ribavirin combination therapy, compared to the observation of the mutation in one patient receiving GS-7977 monotherapy.

The ability to provide effective therapy without peginterferon according to the methods described herein has the potential to significantly improve therapeutic options for individuals living with HCV infection.

The foregoing description of the présent invention provides illustration and description, but is not intended to be exhaustive or to limit the invention to the précisé 15 one disclosed. Modifications and variations are possible in light of the above teachings or may be acquired from practice of the invention.

Claims (25)

What is claimed is:
1. (1) 5.0 and 7.3; or (2) 6.1 and 12.7.

   43. The unit dosage form according to claim 24, wherein the unit dosage form comprises a capsule or a tablet.

   44. A process for preparing a tablet composition comprising about 400 mg of GS7977 comprising:

   blending an intragranular composition and an extragranular composition to obtain a blended composition; . „ , ., .

   compressing the blended composition to obtain a tablet composition; and optionally coating the tablet composition;

   wherein the intragranular composition comprises GS-7977, a first intragranular diluent, optionally a second intragranular diluent, an intragranular disintegrant, an intragranular glidant, and an intragranular lubricant; and •••t fl ·+·“-* the extragranular composition comprises a first extragranular diluent, optionally a second extragranular diluent, an extragranular glidant, an extragranular disintegrant, and an extragranular lubricant.

   45. A tablet composition comprising about 400 mg of GS-7977 made according

   (1) 5.0,7.3, 9.4, and 18.1; or (2) 6.1,8.2, 10.4, 12.7,17.2, 17.7,18.0, 18.8, 19.4, 19.8, 20.1, 20.8, 21.8, and

   (1) 5.0, 7.3, 9.4, and 18.1; or (2) 6.1, 8.2, 10.4, 12.7, 17.2, 17.7, 18.0, 18.8, 19.4, 19.8, 20.1, 20.8,21.8, and

   (1) 5.2, 7.5, 9.6, 16.7, 18.3, and 22.2;

   (1) 5.0 and 7.3; or (2) 6.1 and 12.7.

   (1) 5.0, 7.3, 9.4, and 18.1; or (2) 6.1, 8.2, 10.4, 12.7, 17.2, 17.7, 18.0, 18.8, 19.4, 19.8, 20.1, 20.8, 21.8, and

   (1) 5.0 and 7.3; or !

   , >· u · .-«B·· F ¹ t «►«t-nwiii I «IB »au| »- nWrt «T WT «-»· -W ·«· (2) 6.1 and 12.7.

   (1) 5.0,7.3, 9.4, and 18.1; or (2) 6.1,8.2, 10.4, 12.7, 17.2, 17.7, 18.0, 18.8, 19.4, 19.8, 20.1,20.8,21.8, and

   (1) 5.2, 7.5, 9.6, 16.7, 18.3, and 22.2;

   1. A pharmaceutical composition comprising:

   a) about 25% to about 35% w/w of GS-7977; and

   b) at least one pharmaceutically acceptable excipient.
2. (2) 5.0,7.3, 9.4, and 18.1 ;

   (2) 5.0,7.3, 9.4, and 18.1;

   2. The composition according to claim 1, wherein the composition comprises about 30% to about 35% w/w of GS-7977.
3. (3) 4.9,6.9, 9.8, 19.8,20.6,24.7, and 26.1 ;

   (3) 4.9, 6.9, 9.8, 19.8,20.6, 24.7, and 26.1;

   3. The composition according to claim 1, wherein the composition comprises about 30% w/w of GS-7977.
4. (4) 6.9, 9.8, 19.7,20.6, and 24.6;

   (4) 6.9, 9.8, 19.7, 20.6, and 24.6;

   4. The composition according to claim 1, wherein the composition comprises about 33% w/w of GS-7977.
5. 5 h Use of the composition according to claim 1 to treat hepatitis C virus infection in a human in need thereof.

   52. The use according to claim 51, wherein the composition according to claim 1

   5 to the process of claim 44.

   46. A method of treating a human infected with hepatitis C virus comprising administering to the human the composition according to claim 1.

   47. The method according to claim 46, wherein the composition according to claim 1 is administered to the human in combination with ribavirin.

   (5) 5.0, 6.8, 19.9, 20.6, 20.9, and 24.9;

   (5) 5.0, 6.8, 19.9, 20.6, 20.9, and 24.9;

   5. The composition according to claim 1, wherein the composition comprises crystalline GS-7977.
6. (6) 5.2, 6.6, 7.1, 15.7, 19.1, and 25.0; or (7) 6.1,8.2, 10.4, 12.7, 17.2, 17.7, 18.0, 18.8, 19.4, 19.8, 20.1, 20.8, 21.8, and

   (6) 5.2, 6.6, 7.1, 15.7, 19.1, and 25.0; or (7) 6.1, 8.2, 10.4, 12.7, 17.2, 17.7, 18.0, 18.8, 19.4, 19.8, 20.1, 20.8, 21.8, and

   6. The composition according to claim 5, wherein the crystalline GS-7977 has XRPD 20-reflections (°) at about:
7. 7. The composition according to claim 6, wherein the crystalline GS-7977 has XRPD 20-reflections (°) at about:
8. 8. The composition according to claim 5, wherein the crystalline GS-7977 has XRPD 20-reflections (°) at about:
9. 9. The composition according to claim 1, wherein the at least one pharmaceutically acceptable excipient comprises at least one of a diluent, a disintegrant, a glidant, and a lubricant.
10. 10 48. A method of treating a human infected with hepatitis C virus comprising administering to the human the unit dosage form according to claim 24.

    49. The method according to claim 48, wherein the unit dosage form according to claim 24 is administered to the human in combination with ribavirin.

    50. The method according to claim 49, wherein the unit dosage form according to 15 claim 24 is administered to the human in combination with ribavirin as part of an interferon-free treatment regimen.

    10. The composition according to claim 9, wherein the at least one pharmaceutically acceptable excipient comprises a diluent selected front the group consisting of dicalcium phosphate, cellulose, compressible sugars, dibasic calcium phosphate dehydrate, lactose, mannitol, microcrystalline cellulose, starch, tribasic calcium phosphate, and combinations thereof.
11. 11. The composition according to claim 10, wherein the diluent is selected from the group consisting of mannitol, microcrystalline cellulose, and combinations thereof.
12. 12. The composition according to claim 9, wherein the at least one pharmaceutically acceptable excipient comprises a disintegrant selected from the group consisting of croscarmellose sodium, crospovidone, microcrystalline cellulose, modified com starch, povidone, pregelatinized starch, sodium starch glycolate, and combinations thereof.
13. 13. The composition according to claim 12, wherein the disintegrant is croscarmellose sodium.
14. 14. The composition according to claim 9, wherein the at least one pharmaceutically acceptable excipient comprises a glidant selected from the group consisting of colloïdal silicon dioxide, talc, starch, starch denvatîves, and combinations thereof.
15. 15. The composition according to claim 14, wherein the glidant is colloïdal silicon dioxide.
16. 16. The composition according to claim 9, wherein the at least one pharmaceutically acceptable excipient comprises a lubricant selected from the group consisting of calcium stéarate, magnésium stéarate, polyethylene glycol, sodium stearyl fumarate, stearic acid, talc, and combinations thereof.
17. 17. The composition according to claim 16, wherein the lubricant is magnésium stéarate.
18. 18. The composition according to claim 1 further comprising a coating agent.
19. 19. The composition according to claim 1, wherein the at least one pharmaceutically acceptable excipient comprises:

    a) about 55% w/w to about 65% w/w of a diluent;

    b) about 2.5 % w/w to about 7.5% w/w of a disintegrant;

    c) about 0.25% w/w to about 0.75% w/w ofa gîidant; and

    d) about 1.25% w/w to about 1.75% w/w of a lubricant.
20. 20 is administered to the human in combination with ribavirin.

    53. Use of the unit dosage form according to claim 24 to treat hepatitis C virus infection in a human in need thereof.

    t

    54. The use according to claim 53, wherein the unit dosage form according to claim 24 is administered to the human in combination with ribavirin.

    20. The composition according to claim 1, wherein the at least one pharmaceütically acceptable excipient comprises:

    a) about 30% w/w of mannitol and about 30% w/w of microcrystalline cellulose;

    b) about 5 %w/w of croscarmellose sodium;

    c) about 0.5% w/w of colloïdal silicon dioxide; and

    d) about 1.5% w/w of magnésium stéarate.
21. 21. The composition according to claim 1, wherein the composition comprises:

    a) about 33% w/w of crystalline GS-7977;

    b) about 30% w/w of mannitol and about 30% w/w of microcrystalline cellulose;

    c) about 5 %w/w of croscarmellose sodium;

    d) about 0.5% w/w of colloïdal silicon dioxide; and

    e) about 1.5% w/w of magnésium stéarate.
22. 22. The composition according to claim 21, wherein the crystalline GS-7977 has XRPD 20-reflections (°) at about:
23. 23.3.

    42. The unit dosage form according to claim 40, wherein the crystalline GS-7977 has XRPD 20-reflections (°) at about:

    23.3.

    28. The unit dosage form according to claim 25, wherein the crystalline GS-7977 has XRPD 20-reflections (°) at about (1) 5.0 and 7.3; or (2) 6.1 and 12.7.

    29. The unit dosage form according to claim 24, wherein the at least one pharmaceutically acceptable excipient comprises at least one of a diluent, a disintegrant, a glidant, and a lubricant.

    30. The unit dosage form according to claim 29, wherein the at least one pharmaceutically acceptable excipient comprises a diluent selected from the group consisting of dicalcium phosphate, cellulose, compressible sugars, dibasic calcium phosphate dehydrate, lactose, mannitol, microcrystalline cellulose, starch, tribasic calcium phosphate, and combinations thereof.

    31. The unit dosage form according to claim 30, wherein the diluent is selected from the group consisting of mannitol, microcrystalline cellulose, and combinations thereof.

    32. The unit dosage form according to claim 29, wherein the at least one pharmaceutically acceptable excipient comprises a disintegrant selected from the group consisting of croscarmellose sodium, crospovidone, microcrystalline cellulose, modified corn starch, povidone, pregelatinized starch, sodium starch glycolate, and combinations thereof.

    33. The unit dosage form according to claim 32, wherein the disintegrant is croscarmellose sodium.

    34. The unit dosage form according to claim 29, wherein the at least one pharmaceutically acceptable excipient comprises a glidant selected from the group consisting of colloïdal silicon dioxide, talc, starch, starch dérivatives, and combinations thereof.

    35. The unit dosage form according to claim 34, wherein the glidant is colloïdal silicon dioxide.

    36. The unit dosage form according to claim 29, wherein the at least one pharmaceutically acceptable excipient comprises a lubricant selected from the group consisting of calcium stéarate, magnésium stéarate, polyethylene glycol, sodium stearyl fiimarate, stearic acid, talc, and combinations thereof.

    37. The unit dosage form according to claim 36, wherein the lubricant is magnésium stéarate. ,

    38. The unit dosage form according to claim 24 further comprising a coating agent.

    39. The unit dosage form according to claim 24, wherein the at least one pharmaceutically acceptable excipient comprises:

    a) about 660 mg to about 780 mg of a diluent;

    b) about 30 mg to about 90 mg of a disintegrant;

    c) about 3 mg to about 9 mg of a glidant; and

    d) about 15 mg to about 21 mg of a lubricant.

    ©<

    »·

    40. The unit dosage form according to claim 24, wherein the unit dosage form comprises:

    a) about 400 mg of crystalline GS-7977;

    b) about 360 mg of mannitol and about 356 mg of microcrystalline cellulose;

    c) about 60 mg of croscarmellose sodium;

    d) about 6 mg of colloïdal silicon dioxide; and

    e) about 18 mg of magnésium stéarate.

    41. The unit dosage form according to claim 40, wherein the crystalline GS-7977 has XRPD 20-reflections (°) at about:

    23.3.

    27. The unit dosage form according to claim 25, wherein the crystalline GS-7977 has XRPD 20-reflections (°) at about:

    23. The composition according to claim 21, wherein the crystalline GS-7977 has XRPD 20-reflections (°) at about:

    23.3.

    23.3.

    23.3.
24. 24. A unit dosage form comprising:

    a) about 400 mg of GS-7977; and r · -r

    b) at least one pharmaceutically acceptable excipient.

    25. The unit dosage form according to claim 24 comprising crystalline GS-7977.

    26. The unit dosage form according to claim 25, wherein the crystalline GS-7977 has XRPD 20-reflections (°) at about:
25. 25 55. The use according to claim 54, wherein the unit dosage form according to claim 24 is administered to the human in combination with ribavirin as part of an interferon-free treatment regimen.