NZ625532B2 - Compositions and methods for treating hepatitis c virus - Google Patents

Abstract

Provided is a composition and a unit dosage form comprising GS-7977 (sofosbuvir) and at least one pharmaceutically acceptable excipient. Preferably the composition and unit dosage form comprise a crystalline form of sofosbuvir. Sofosbuvir is useful in the treatment of hepatitis C.

Description

itions and s for Treating Hepatitis C Virus Field of the Invention Disclosed herein are a composition and unit dosage form for the treatment of tis C virus (HCV) infection comprising GS-7977 and at least one pharrnaceutically 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, ably 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 , 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 , the method is sufficient to e 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”) ion is a major health problem that leads to chronic liver disease, such as cirrhosis and hepatocellular oma, 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 US. Centers for Disease Control and Prevention, HCV is the most common blood-borne infection in the United States, with an estimated 3.2 million people (1.8%) chronically infected in the United States alone. (US. Centers for e Control and Prevention, Viral Hepatitis Surveillance — United States, 2010; US. Centers for Disease Control and Prevention, Morbidity and Mortality Weekly Report : 537-539 (May 6, 2011).) An estimated 150-180 million duals 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 SUBSTITUTE SHEET (RULE 26) al., J. Antimicrob. Chemother. (2010) 65: 2063-2069.) The viral disease is transmitted parenterally by contaminated blood and blood products, contaminated needles, or ly and ally from infected mothers 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 s a polyprotein of about 3,010 amino acids. The protein products of the HCV gene consist of the structural proteins C, E1, and E2, and the non-structural proteins NS2, NS3, NS4A and NS4B, and NSSA and NS5B. The nonstructural (“NS”) proteins are believed to provide the catalytic ery for viral replication. The NS3 protease releases NSSB, the RNA- IO dependent RNA polymerase, from the polyprotein chain. HCV NS5B polymerase is required for the synthesis of a double-stranded RNA from a -stranded'viral RNA that serves as a template in the replication cycle of HCV. ore, NSSB polymerase is considered to be an essential component in the HCV replication complex. (K. Ishi, et a1, Hepatology (1999) 29: 1227—1235; V. n, et al., Virology (1998) 249: 108-118.) Inhibition ofHCV NSSB polymerase ts formation of the double-stranded HCV RNA and therefore constitutes an attractive approach to the development of HCV—speCific antiviral therapies.

A number of ial molecular targets for drug development of direct acting antivirals as anti-HCV therapeutics have now been identified including, but not limited to, the NSZ-NS3 otease, the N3 protease, the N3 helicase, and the NS5B polymerase. The RNA—dependent RNA polymerase is essential for replication of the single—stranded, positive sense, RNA- genome, and this enzyme has elicited significant interest among medicinal chemists. r auxiliary protein ofHCV is referred to as NSSA. The NS5A nonstructural protein is a oprotein, with no apparent enzymatic activity; howeverit acts as a multifunctional regulator of cellular pathways, including host cell , immunity and innate immunity, and virus replication. (Appel et al., J.

Virol. (2005) 79: 3187-3194; Evans et al., Proc.Nat1. Acad. Sci. USA (2004) 101: 13038- 13043; Gale et al., Nature (2005) 436: 939-945; Gale et al., Virology (1997) 230: 217- 227; Ghosh et al., J. Gen. Virol. (1999) 80(Pt 5): 1179-1 183; 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 helix, where it is a part of the replication complex. (Blazar et al., J. Virol. (2004) 78: 11393-11400 and Penin et al., J. Biol. Chem. (2004) 279: 40835-40843.) Recent studies t that NSSA 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 n is azinc metalloprotein and domains 11 and III are in the inal region of the protein.

(Tellinghuisen et al., J. Biol. Chem. (2004) 279: 48576-48587 and Tellinghuisen et al., [0 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 specific sites, ily on serine residue within domains 11 and III, by host cell s.

(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. [5 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 y with pegylated interferon alfa—2a or pegylated interferon alfa—2b (collectively “peginterferon” or “PEG”) used alone or in combination with rin (“RBV”). The primary goal of treatment for chronic hepatitis C is a sustained Virologic response (“SVR”), which refers to an undetectable level of serum HCV RNA ined for a period oftime post—treatment. Host factors including age, body , race, and advanced fibrosis influence the outcome of treatment (Dienstag and McHutchison Gastroenterology 130: 231-264 and Missiha et al., Gastroenterology (2008) 134: 1699-1714), but are poor predictors of response. In contrast, viral factors like the genotype and the on-treatment pattern of viral response can be used to determine the likelihood oftreatment success and guide treatment duration dually, and they have proven to be very useful in clinical practice. (Ge et al., Nature (2009) 461: 399-401.) 2012/066605 In spite of an aging response in some patients to SOC treatment, the overall se to peginterferon/ribavirin combination therapy among patients infected with Hepatitis C virus is only about 50%. SVR rates are <50% for patients infected with genotype 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 s the time in which patients show evidence of complete viral suppression (negative HCV status) following the initiation of ent.

Peginterferon alfa—2a ("PEG-IFN—d-Za" or "peginterferon (it-2a"), marketed under the trademark PEGASYS®, is an antiviral stered 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 ofCHC in patients with compensated liver e not previously treated with eron alpha, in patients with histological evidence of cirrhosis and compensated liver disease, and in adults with CHC/HIV co-infection. Combination therapy using PEG-IFN- u-Za and ribavirin is recommended unless the patient has contraindication to or significant intolerance to ribavirin.

Peginterferon alfa—Zb ("PEG-IFN-d-Zb" or "peginterferon Ot-Zb”), marketed under the trademark PEGINTRON®, is also administered by aneous injection and is indicated for use alone or in combination with ribavirin to treat CHC in patients with compensated liver e. Like PEG-IFN—u-2a, PEG-lFN-a—Zb has undesirable side effects.

Ribavirin ("REV"), marketed under the trademark COPEGUS®, is a side analogue indicated for the ent ofCHC virus infection in combination with peginterferon in patients 5 years of age 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 have shown that ribavirin alone can normalize alanine ransferase (“ALT”) levels transiently during the course tment in some patients with CHC infections. However, these studies have reported that ribavirin alone did not reduce HCV RNA levels during or after therapy and did not produce any sustained gic response. (Di Bisceglie et al., Ann. Intern. Med. (1995) 123(12): 897— 903; Dusheiko et al., J. Hepatology (1996) 25: 591-598; Bodenheimer, Jr., et al., ' Hepatology (1997) 26(2): 7.) 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.) , ent of CHC using peginterferon alone or in combination with ribavirin several disadvantages. First and foremost, this therapy is not effective for many ts.

For instance, certain Phase 3 al trials using the combination of peginterferon ribavirin ed 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 e events. For instance, the boxed warning on the PEGASYS® label states that use peginterferon may cause or ate fatal or life—threatening neuropsychiatric, autoimmune, ischemic, and infectious disorders. (PEGASYS® (peginterferon alfa—2a) FDA-approved label, revised 09/2011.) onally, the boxed warning on the COPEGUS® label states that ribavirin adverse effects may e hemolytic anemia and that significant "teratogenic and embryocidal effects have been demonstrated in all animal s exposed to ribavirin." (COPEGUS® (ribavirin) FDA-approved label, revised 1.) Finally, the peginterferon/ribavirin treatment protocol is quite expensive.

VI Given these disadvantages, there has been a recognized need to develop new anti—HCV drug substances and treatment regimens.

The FDA recently approved two additional drug products for the treatment of genotype 1 CHC, boceprevir and telaprevir, both ofwhich are HCV NS3/4 protease inhibitors. Boceprevir, marketed under the trademark LIS®, is indicated for the treatment ofgenotype 1 CHC infection, in combination with interferon and ribavirin, in adult ts (218 years of age) with compensated liver e, including cirrhosis, who are previously untreated or who have failed us interferon and ribavirin therapy.

Telaprevir, marketed under the ark INCIVEK®, is indicated, in combination with interferon and ribavirin, for the treatment of genotype 1 CHC in adult patients with compensated liver disease, including cirrhosis, who are treatment-naive or who have been previously treated with interferon-based treatment, inclUding prior null responders, partial responders, and relapsers. Both boceprevir and telaprevir are approved for administration in ation with peginterferon and ribavirin only; neither is approved for monotherapy or for administration with ribavirin alone. EK® (telaprevir) FDA- approved label, revised 06/2012; VICTRELIS® (boceprevir) FDA-approved label, revised 07/2012.) The introduction of both boceprevir and telaprevir has increased the therapeutic options available to HCV-infected patients; however, both treatment regimens have 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 ation with peginterferon (x-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 clinicaE studies show that boceprevir used in IO 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 ent costs are quite expensive. Finally, use of boceprevir in combination with peginterferon and ribavirin is presently limited to those subjects infected with HCV genotype 1.

The telaprevir regimen (used in ation 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 of79% was reported for ts receiving telaprevir in combination with peginterferon and ribavirin for 12 weeks. (Jacobson et a1., New Engl. J. Med. (2011) 364: 2405-2416.) However, s reveal that about half ofthe treated ts 50 developed a skin rash or itching, and a small number of patients developed the severe Stevens-Johnson Syndrome, a life-threatening skin ion, 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 pe 1. gh 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 have disadvantages. Further, genotype 1 patients who fail therapy with boceprevir and/or telaprevir in combination with erferon and ribavirin may develop undesirable N83 protease inhibitor resistance. (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 d rates of viral hrough and/or viral resistance. In ular, there is a need for interferon-free ent regimens that are effective for treating CHC but result 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 nucleotide analog prodrug currently in Phase 2/Phase 3 trials for treatment of c HCV infection.

Several Phase 2 al trials have 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 s of these trials, along with the results if in vitro studies, revealed several potential and hereto unknown ages ofHCV 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 ion.

Summary Disclosed herein are a ition and unit dosage form for the treatment of hepatitis C virus (HCV) infection comprising GS-7977 and at least one pharmaceutically WO 82003 2012/066605 acceptable excipient, as well as methods for making said composition and unit dosage form.

Also disclosed herein is a method of ng a subject, preferably a human, _ infected with tis C virus, said method comprising administering to the Subject for a time period an effective amount of GS-7977 and an ive amount of ribavirin. In one aspect, the method comprises administering to the subject an interferon-free treatment regimen comprising an ive amount of GS-7977 and an effective amount of rin.

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 ofthe time period.

Brief Description of the Drawings Figure 1. Plot of Mean HCV RNA (loglo IU/mL) versus time during treatment and for up to 12 weeks after the end of treatment (“BOT”) for HCV GT2/GT3 treatment-naive 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 1b, 1a, 2a, 2b, 3a, 4a, 50 and 5a NSSB harboring the $282T mutation (compared to the corresponding wild-type) d with GS-7977 or ribavirin.

Figure 3. Percentage of wild—type at $282 position in HCV replicons before and afier treatment with GS—7977, ribavirin, and a combination of GS-7977 and ribavirin in long-term passaging study (15-30 days).

Detailed Description Definitions The phrase "a" or "an" entity as used herein refers to one or more of that entity; for 50 example, a compound refers to one or more compounds or at least one compound. As such, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein.

The term "about" (also ented by “~”) has its plain and ordinary meaning of ”approximately" except as related to an amount of GS-7977, an amount ofribavirin, or an amount ofHCV RNA. As related to an amount of GS—7977, an amount of ribavirin, or an amount ofHCV RNA, the qualifier "about" reflects the standard mental error.

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 stance occurs and instances in which it does not.

The term "subjec " 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 ofthe HCV ion in a subject.

The term "undetectable amount" refers to an amount ofHCV RNA, as determined by the assay methodology described herein, that is less than the limit of detection ("LOD") of about 15 IU/mL.

A sustained virologic response (SVR) for a patient d according to one of the treatment regimens described herein is defined as a patient who completes the HCV treatment regimen and who has an undetectable amount ofHCV RNA (i.e., < about 15 {0 IU/mL) for a period oftime post—treatment as measured in accordance with the assay methodology described herein. SVR-N is the abbreviation for sustained virologic response N weeks after completion of one of the HCV treatment ns disclosed herein. For example, SVR-4 is the abbreviation for sustained gic response 4 weeks after completion of one of the HCV treatment regimens disclosed herein.

The term ration" or "dosage form" is intended to include both solid and liquid formulations of the active compound and one skilled in the art will iate that an active ingredient can exist in different preparations depending on the desired dose and pharmacokinetic ters.

The term “unit dosage form” refers to a physically discrete unit containing a predetermined quantity of the active compound. Preferred unit dosage forms are those - ning a daily dose or unit daily sub-dose, or an appropriate fraction thereof, ofGS- 7977.

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

RVR is the abbreviation for rapid Virologic response'and refers to an undetectable level ofHCV RNA in the blood at week 4 of treatment. The occurrence ofRVR has been reported to be tive of ultimate SVR for a full treatment course of 48 weeks with peginterferon/ribavirin combination treatment in HCV GT-l 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.

QlD means that the dose is administered four times a day.

The highest activities of alanine aminotransferase (ALT) are found in hepatocytes and ed (skeletal and cardiac) muscle cells. Increased serum ALT activity can any hepatocellular injury or necrosis of striated muscle. With cell injury or death, ALT escapes from the cytosol. In addition, release ofALT from the cytosol can occur secondary to cellular necrosis or as a result of cellular injury with membrane damage.

Determination ofALT activity is a relatively sensitive indicator of hepatic damage.

Mechanisms of increased activity ofALT in serum include enzyme release from damaged cells or induction of enzyme activity, such as increased enzyme synthesis from drug administration. m, et al., Aliment col Ther. 2006 Oct 15; 24(8) 1133- 1149).

The interleukin 28B (ILZSB) gene encodes a ne distantly related to type I interferons and the IL—lO family. The ILZ8B gene, interleukin 28A (ILZSA), and interleukin 29 (ILZ9) are three closely related cytokine genes that form a cytokine gene cluster on a chromosomal region mapped to 19q13; sion of the cytokines encoded by the three genes can be induced by viral infection. All three cytokines have been shown to interact with a heterodimeric class II cytokine receptor that consists of interleukin 10 receptor, beta (lLlORB), and interleukin 28 receptor, alpha (IL28RA). (National Center for Biotechnology Information, Entrez Gene Entry for IL28B, Gene ID: 282617, updated on -2010.) Body mass index (“BMI”) is a measurement based on a '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 ofBMI are kg/mz.

LCD is the abbreviation for limit of detection. As used herein with regard to IO HCV RNA measurements, in one aspect LCD 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 IU/mL to about 20 IU/mL. In a particularly preferred embodiment, the LCD is about 15 IU/mL.

GT is the abbreviation for genotype. [5 IU is the abbreviation for international unit, which is a measure of the amount of a substance based on biological activity or .

There are several recognized HCV Genotypes (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11), which can be further categorized by different sub-types: 1 (la, 1b, and 1c), 2 (2a, 2b, 20), 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 (11a). Genotype 1 is the inant form found in North and South America, , Asia, Australia, and New Zealand. Genotypes 2 and 3' are also widely distributed hout North America, Europe, Australia, East Asia and some portions of . In some ns of Africa, Genotype 4 predominates, while in others (such as South Africa) genotype 5 predominates. The method disclosed herein is contemplated to be independently effective for the treatment of each of the HCV genotypes, 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 ted interferon to the subject.

GS—7977, (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)(2,4-dioxo-3,4- dihydropyrimidin—l l)fluoro-3 -hydroxy—4—methyltetrahydrofuran yl)methoxy)(phenoxy)phosphoryl)amino)propanoate, available from Gilead Sciences, Inc., is described and d in US. Patent No. 7,964,580. (See also US 016251, US 2010/0298257, US 2011/0251 152 and US 107278.) GS-7977 has the structure: >R o mil—owl“P‘ 0 ifO OPh HO a’F GS-7977 can be crystalline or amorphous. Examples of preparing lline 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.

Polymorphic Forms 1-6 of GS-7977 disclosed in US 2010/0298257 and/or US 2011/0251152 have the following characteristic X—ray powder diffraction (XRPD) pattern —values measured according to the XRPD s 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).

Polymorphic Forms 1 and 6 are alternatively 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 ition comprises polymorphic Form 6 of GS- 7977. It has been found that Form 6 has a melt onset of approximately 121°C and is not copic, with less than 0.2% moisture sorption at room temperature and 90% RH.

Form 6 is chemically stable when stored under opened conditions at 40°C/75% RH for 30 days.

In one aspect, 7 is substantially free from its corresponding phosphorous- based diastereomer (S)-isopropyl 2—(((R)-(((2R,3R,4R,5R)-5—(2,4-dioxo—3,4- dihydropyrimidin- 1 (2H)-yl)fluorohydroxymethy1tetrahydrofi1ran—2— yl)methoxy)(phenoxy)phosphoryl)amino)propanoate. In one embodiment, 7 is at least 95% free from its corresponding phosphorous-based diastereomer. In another embodiment, GS-7977 is at least 97% flee from its corresponding orous-based diastereomer. In another embodiment, GS—7977 is at least 99% free from its corresponding phosphorous—based reomer. In a further embodiment, GS-7977 is at least 99.9% free from its corresponding phosphorous-based diastereomer.

Ribavirin, 1-B-D-ribofuranosyI—1H-1,2,4-triazolecarboxamide, is described in the Merck Index (12th n), monograph no. 8365. (See also US Patent No. 4,530,901.) As used herein, "treatment" or "treating" is an approach for obtaining beneficial or desired clinical results. Beneficial 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 detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected al if not receiving treatment. "Treatment" is an intervention performed with the ion of preventing the development or ng the pathology of a disorder. The term "treatment" of an HCV infection, as used herein, also includes treatment or prophylaxis of [5 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 ed to a composition for the ent of hepatitis C virus (HCV) comprisingva) GS-7977, and b) a ceutically acceptable excipient.

In a first aspect ofthe first embodiment, the composition for the treatment of HCV comprises from about 25% to about 35% w/w of GS-7977. In r 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 one subembodiment, the composition comprises about 30% w/w of GS-7977. In another subembodiment, the composition comprises about 33% w/w ofGS— 7977. In another subembodiment, the composition ses about 33.33% w/w of GS- 7977.

In a second aspect of the first embodiment, the composition comprises lline GS—7977. In one subembodiment, the composition comprises crystalline GS-7977 having XRPD 20—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) .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, .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 29- reflections (°) 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 20- reflections (°) at about: (1) 5.2, 7.5, 9.6, 16.7, 18.3, and 22.2; or (2) XRPD 20—reflections [0 (°) at about: 5.0, 7.3, 9.4, and 18.1. In another preferred subembodiment, the composition ses 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 lline GS-7977 having XRPD 20- ions (°) 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 pharrnaceutically 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 m ate, ium ate, dry starch, calcium sulfate, cellulose, compressible , confectioner's sugar, dextrates, dextrin, dextrose, dibasic calcium phosphate dihydrate, yl palmitostearate, hydrogenated vegetable oil (type I), inositol, kaolin, lactose, magnesium carbonate, magnesium oxide, maltodextrin, ol, microcrystalline cellulose, polymethacrylates, potassium chloride, powdered cellulose, powdered sugar, pregelatinized starch, sodium de, sorbitol, starch, sucrose, sugar s, 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 ose, starch, tribasic calcium phosphate, and combinations f. In r 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, ose, a cation exchange resin, cellulose, gums, citrus pulp, colloidal silicon e, corn starch, croscarmellose sodium (e.g., Ac—Di— Sol®), crospovidone, guar gum, s aluminum silicate, an ion ge resin (e.g., polyacrin potassium), magnesium aluminum silicate, methyl ose, microcrystalline cellulose, modified cellulose gum, d corn starch, montrnorillonite clay, natural sponge, polyacrilin potassium, potato starch, powdered cellulose, povidone, pregelatinized starch, sodium te, 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 red subembodiment, the disintegrant is selected fiom the group consisting of croscarmellose sodium (e.g., Ac—Di- Sol®), crospovidone, microcrystalline cellulose, modified corn starch, povidone, pregelatinized , sodium starch glycolate, and combinations f. 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 colloidal silicon dioxide, talc, starch, starch derivatives, and combinations thereof. In a preferred subembodiment, the glidant comprises colloidal silicon dioxide.‘ ' In another subembodiment, the lubricant is selected from the group consisting of calcium stearate, glyceryl monostearate, yl palmitostearate, hydrogenated castor oil, hydrogenated ble oil, light l oil, magnesium stearate, mineral oil, polyethylene , sodium benzoate, sodium lauryl sulfate, sodium stearyl fumarate, c acid, talc, zinc stearate, and combinations thereof. In a preferred subembodiment, the lubricant is selected from the group consisting of calcium stearate, magnesium stearate, hylene glycol, sodium stearyl fumarate, stearic acid, talc, and combinations thereof. In another preferred subembodiment, the lubricant is magnesium stearate.

In r subembodiment, the pharmaceutically acceptable excipient ses: a) about 55% w/w to about 65% w/w of a t; b) about 2.5 % w/w to about 7.5% w/w of a disintegrant; 0) about 0.25% w/w to about 0.75% w/w of a glidant; and (I) 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 disintegrant; 0) about 0.5% w/w a glidant; and (1) about 1.5% w/w a lubricant.

In another preferred subembodiment, the pharmaceutically able excipient comprises a) about 60% w/w a diluent comprising mannitol and/or microcrystalline cellulose; b) about 5 %w/w of croscarrnellose sodium; 0) about 0.5% w/w of colloidal silicon dioxide; and d) about 1.5% w/w ofmagnesium stearate. In another preferred subembodiment, the pharmaceutically acceptable excipient comprises a) about 30% w/w ofmannitol and about 30% w/w of microcrystalline cellulose; b) about 5 % w/w of croscarmellose sodium; 0) about 0.5% w/w of colloidal silicon dioxide; and (1) about 1.5% w/v'v ofmagnesium stearate.

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 film coat composition, n the aqueous film coat ition 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, ypropylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, cellulose 50 acetate phthalate, sodium ethyl cellulose sulfate, carboxymethyl cellulose, polyvinylpyrolidone, zein, and an acrylic polymer (e.g., methacrylic acid/methacrylic acid ester copblymers 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 ition ses about w/w to about 35% w/w of crystalline GS—7977; about 30% w/w ofmannitol and about % w/w ofmicrocrystalline cellulose; about 5 %w/w of croscarmellose sodium; about 0.5% w/w of dal silicon dioxide; and about 1.5% w/w ofmagnesium stearate. In one odiment, 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 ose; about 5 %w/w of croscarmellose sodium; about 0.5% w/w of colloidal silicon dioxide; and about 1.5% w/w of magnesium stearate. In another subembodiment, the composition comprises about 30% w/w of crystalline 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 colloidal silicon dioxide; and about 1.5% w/w of ium stearate. 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 ofmannitol and about 30%,w/w of microcrystalline cellulose; about 5 %W/w of croscarmellose ; about 0.5% w/w of colloidal silicon dioxide; and about 1.5% w/w of magnesium stearate. 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 ol and about 30% w/w of microcrystalline ose; about %w/w of croscarmellose sodium; about 0.5% w/w of colloidal silicon dioxide; and about 1.5% w/w of magnesium stearate. In another odiment, the composition comprises about 33% w/w of crystalline GS—7977; about 30% w/W of mannitol and about % w/w of microcrystalline cellulose; about 5 %w/w of croscarmellose sodium; about 0.5% w/w of colloidal silicon e; and about 1.5% w/w of magnesium stearate. 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, 180,188, 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 colloidal silicon dioxide; and about 1.5% w/w ofmagnesium stearate. 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 ofmicrocrystalline cellulose; about 5 %w/w of croscarmellose sodium; about 0.5% w/w of colloidal silicon e; and about 1.5% w/w of magnesium stearate.

In another subembodiment, the ition further comprises a coating agent.

A second embodiment is ed 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 GS- 7977 having XRPD 20-reflections (°) at about: (1) 5.2, 7.5, 9.6, 16.7, 18.3, and 22.2; (2) .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 -reflections (°) at about: (1) 5.0 and 7.3; or (2) 6.1 and 12.7. In one red l0 subembodiment, the unit dosage form ses crystalline GS-7977 having XRPD 20- reflections (°) 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 7 having XRPD 20—reflections (°) at about 6.1, 8.2, .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 [5 red subembodiment, the composition comprises crystalline GS-7977 having XRPD -reflections (°) at about: 5 .0 and 7.3. In a further preferred subembodiment, the ition 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 able 50 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 ting of calcium carbonate, dicalcium phosphate, dry starch, calcium sulfate, cellulose, ssible sugars, confectioner‘s sugar, dextrates, n, dextrose, c calcium phosphate dihydrate, glyceryl palmitostearate, hydrogenated vegetable oil (type I), inositol, , lactose, magnesium carbonate, magnesium oxide, maltodextrin, mannitol, microcrystalline cellulose, polymethacrylates, potassium chloride, powdered cellulose, powdered sugar, pregelatinized starch, sodium chloride, ol, starch, sucrose, sugar spheres, talc, tribasic m phosphate, and combinations thereof. In a preferred subembodiment, the diluent is selected from the group consisting of dicalcium phosphate, cellulose, ssible 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 ations thereof.

In another odiment, 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, colloidal silicon dioxide, corn starch, croscarmellose sodium (e.g., Ac-Di- Sol®), crospovidone, guar gum, hydrous aluminum te, an ion exchange resin (e.g., polyacrin potassium), magnesium aluminum silicate, methyl cellulose, rystalline cellulose, modified cellulose gum, modified corn starch, rillonite 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 f. In a preferred odiment, the disintegrant is selected from the group consisting of croscarmellose sodium (e.g., Ac-Di- Sol), crospovidone, microcrystalline cellulose, modified corn starch, povidone, pregelatinized starch, sodium starch glycolate, and combinations thereof. In r preferred subembodiment, the disintegrant is croscarmellose sodium (e.g., Ac-Di-Sol).

In another subembodiment, the glidant is selected from the group consisting of colloidal silicon dioxide, talc, starch, starch derivatives, and combinations f. In a preferred subembodiment, the glidant comprises colloidal silicon dioxide.

In another subembodiment, the lubricant is selected from the group consisting of calcium stearate, glyceryl monostearate, yl palmitostearate, hydrogenated castor oil, hydrogenated vegetable oil, light mineral oil, magnesium stearate, mineral oil, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, sodium stearyl fumarate, stearic acid, talc, zinc stearate, and combinations thereof. In a preferred subembodiment, the lubricant is ed from the group consisting of calcium stearate, magnesium stearate, polyethylene glycol, sodium stearyl fumarate, stearic acid, talc, and ations thereof. ‘In r preferred subembodiment, the lubricant is magnesium stearate.

In another subembodiment, the pharmaceutiCally able excipient comprises: a) about 660 mg to about 780 mg of a diluent; b) about 30 mg to about 90 mg of a disintegrant; 0) about 3 mg to about 9 mg of a glidant; and d) about 15 mg to about 21 mg of a lubricant. In a preferred subembodiment, the pharmaceutically acceptable excipient comprises a) about 710 mg to about 720 mg of a t; b) about 60 mg of a disintegrant; 0) about 6 mg of a glidant; and (1) 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 egrant; 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 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 colloidal silicon dioxide; and (1) about 18 mg ofmagnesium te. In another preferred subembodiment, the pharmaceutically acceptable excipient ses a) about ' 716 mg ofa t comprising mannitol and/or microcrystalline cellulose; b) about 60 mg of croscarmellose ; c) about 6 mg of colloidal silicon dioxide; and (1) about 18 mg of magnesium stearate. In r preferred odiment, 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 50 dal silicon dioxide; and d) about 18 mg ofmagnesium stearate.

In a third aspect ofthe 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 rming 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 among hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, cellulose acetate phthalate, sodium ethyl cellulose sulfate, carboxymethyl cellulose, [0 polyvinylpyrolidone, zein, and an acrylic polymer (e.g., methacrylic acid/methacrylic acid ester copolymers such as methacrylic acid/methylmethacrylate mers, 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 g agent. In another subembodiment, the unit dosage comprises about 36 [5 mg to about 48 mg of a coating agent. In another subembodiment, the unit dosage comprises about 36 mg of a coating agent. In r subembodiment, the unit dosage comprises about 36 mg of a coating agent that further ses a taste-masking agent.

In a fourth aspect ofthe second ment, the unit dosage form comprises about 400 mg of crystalline GS-7977; about 360 mg of mannitol and about 356 mg of microcrystalline cellulose; about 60 mg of croscarmellose sodium; about 6 mg of dal silicon dioxide; and about 18 mg of magnesium stearate. 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, '1 8.0, 18.8, 19.4, 19.8, 20.1, 20.8, 21.8, and 23.3; about 360 mg ofmannitol and about 356 mg of rystalline cellulose; about 60 mg of croscarmellose ; about 6 mg of dal silicon dioxide; and about 18 mg of magnesium stearate. In another subembodiment, the unit dosage form comprises about 400 mg of crystalline 7 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 50 6 mg of colloidal silicon e; and about 1-8 mg of magnesium stearate.

WO 82003 In a fifth aspect of the second embodiment, the unit dosage form comprises a e or a tablet. In one odiment, the unit dosage form comprises a tablet. In another subembodiment, the unit dosage form ses atablet and further comprises a coating agent.

With respect to the coating agent, film-forming pelymers are typically provided in either aqueous or organic solvent—based ons 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 on or dispersion by the user by admixing with the aqueous vehicle.

It will be appreciated that the aqueous film coat composition further ses 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 l and/or a ketone. Examples of an alcohol e 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 st cohesive strength and thus mechanical properties. The ents are also selected to provide good 50 deposition of the film—forming polymer onto the surface ofthe unit dosage form, such that a nt and nt 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 11 Purple and Opadry II Yellow). [5 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 25— % w/w of 7, and b) a pharmaceutically acceptable excipient is administered to the subject in combination with ribavirin.

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

A fourth embodiment is directed to a method oftreating a subject infected with hepatitis C virus comprising administering to the subject a unit dosage form sing 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 mg of GS—7977, and b) a pharmaceutically acceptable excipient is stered to the subject in combination with ribavirin.

In a secondaspect ofthe fourth embodiment, the subject is a human.

Tablet ation The choice of particular types and s of ents, and tabletting technique employed depends on the further ties 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 ofPharmacy 2006, let edition, 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 ist may modify the formulations within the teachings of the specification to e numerous formulations for a particular route of administration without rendering compositions containing GS—7977 unstable or compromising their therapeutic ty.

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 s comprise granules comprising GS-7977 and one or more - of a diluent, a disintegrant, a glidant, and a lubricant, n the granules are mixed with 50 one or more of a diluent, a egrant, a glidant, and a lubricant to form a granulation mixture that is compressed to form tablets.

A fifth embodiment is directed to a process for preparing a tablet composition comprising about 400 mg of 7, said process comprising blending an intragranular composition and an extragranular composition to obtain a blended composition; ssing the d 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 disintegrant, an ranular t, and an intragranular ant; and the extragranular composition comprises a first extragranular diluent, optionally a second extragranular diluent, an extragranular disintegrant, an extragranular glidant, and an extragranular lubricant, wherein the first intragranular diluent, the second intragranular diluent, the first extragranular diluent, and the second extragranular diluent are the same or different, the ranular disintegrant and the extragranular egrant are the same or different, the intragranular glidant and the extragranular t are the same or different, and the intragranular lubricant and the extragranular lubricant are the same or different.

In a second aspect ofthe fifih embodiment, the intragranular ition comprises GS-7977, a first intragranular diluent, an ranular disintegrant, an intragranular glidant, and an intragranular ant; and the extragranular composition comprises a first extragranular diluent, a second extragranular diluent, an extragranular disintegrant, an ranular glidant, and an extragranular lubricant, 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 ranular glidant are the same or different, and the intragranular lubricant and the extragranular lubricant are the same or different.

In a third aspect of the fifth embodiment, the intragranular composition comprises (ES-7977, a first intragranular diluent, a second intragranular diluent, an intragranular disintegrant, an intragranular glidant, and an intragranular lubricant; and the extragranular ' ition comprises a first extragranular diluent, an extragranular disintegrant, an extragranular t, and an extragranular lubricant, n 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 ranular lubricant and the extragranular lubricant are the same or different.

A fourth aspect of the fifth embodiment comprises at least one of the following steps: (1) Sitting/Blending: GS-7977 and pharmaceutically acceptable excipients are sified 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 sified through a 20-mesh , added to a blender, and blended for a first blending time period to produce an l blend. In one , the first blending time period ranges from about 5 to about 30 minutes. tely, an intragranular ant is passed through a 20-mesh screen, mixed with a n 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 1 minute to about 10 s. In another aspect, the second blending time period is from about 1 minute to about 5 s. In another , 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 ofthe formulation process increases. (2) Dry Granulation: (A) Roller Compaction: GS-7977 and pharmaceutically acceptable excipients are passed through a roller compactor to product compacts. Compacts are then milled (below) to achieve granules. In one non—limiting example, a blend comprising GS-7977, intragranular excipients, and lubricant, is passed through a roller compactor'until granulation is achieved. The non—limiting example has the 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 (preparation of /sifted granule): 7 and aceuticaly acceptable excipients are milled and/or sifted. In one non- limiting example, after GS—7977 and intragranular excipients have passed through the roller compactor, the al is passed/forced through a 20-mesh screen using a Comill or Fitz Mill, and then sified with a 60 mesh screen. In this miting 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)'Fina_l Blending: Granules comprising GS—7977 and intragranular excipients that have been milled/sifted are blended with extragranular excipients in a final blending.

In one non-limiting example, the milled/sified granules comprising GS-7977 and intragranular ients are added to a blender (e.g., a double-cone blender, a bin [0 blender, or a l 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 addedto the blend.

The mixture is d 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 s using a tablet press (e.g., a Globe Pharma Mini Press). (5) Optionally, tablets are film-coated with a film—coating agent.

In a fifih aspect of the filth embodiment, GS-7977 is blended with ranular excipients comprising microcrystalline cellulose, mannitol, croscarmellose sodium and 50 colloidal silicon dioxide in a blender. The mixture is milled and blended with a portion of magnesium stearate, then dry granulated using a roller tor and mill. The resulting granules are then blended with extragranular excipients comprising microcrystalline ose, croscarmellose sodium, and'colloidal silicon dioxide. An additional portion of magnesium stearate is added and the ing 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 s comprising about 400 mg of 65-7977. The tablet cores are film—coated, and the resulting film-coated tablets are then packaged.

The embodiments described herein may be d 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 edition, , 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 ations within the teachings of the specification to provide numerous formulations without rendering compositions ning GS—7977 le or compromising its therapeutic activity. The following non-limiting examples provide further guidance related to additional aspects of the disclosed methods and compositions.

Methods ofTreatment A sixth embodiment is directed to a method for treating a t infected with tis 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 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 7 is stered 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 r subembodiment, a daily dose of about 400 mg of GS— 7977 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 rin is a daily dose of about 1000 mg to about 1200 mg based on the subject’s body weight. In another subembodiment, the ive amount of ribavirin is a daily dose of 50 about 800 mg. In another subembodiment, the daily dose of ribavirin is administered to the subject QD, BID, TID, or QID. In a r 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 GS- 7977 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 stered 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.

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

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

In a r subembodiment, the t has an undetectable amount ofHCV RNA for at least 48 weeks after the end of the time period.

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

In a ninth aspect of the sixth ment, an effective amount of GS-7977 and an effective amount of rin are stered to the subject ing to an interferon— free treatment regimen. In one subembodiment, the interferon-free treatment regimen consists of stering 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 acceptable ent as disclosed herein.

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

In a first aspect ofthe seventh embodiment, the time period is selected fiom among from about 2 weeks to about 12 weeks, from about 3 weeks to about 12 weeks, 50. 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 odiment the time period is 12 weeks. In another odiment the time period is 8 weeks.

In a second aspect of 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 odiment, 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 ofGS- 7977 is administered to the subject QD.

In a third aspect ofthe 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 rent or alternative.

In a fourth aspect ofthe 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 r 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 rin 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 ofthe seventh embodiment, a daily dose of about 400 mg of GS— 7977 is administered to the subject in ation with a daily dose of about 800 mg to 50 about 1200 mg of ribavirin. In one subembodiment, a daily dose of about 400 mg of GS— 7977 is administered to the t 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 virin.

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

In a seventh aspect of the seventh embodiment, the subject has an undetectable amount ofHCV RNA for at least 24 weeks afier 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 ofHCV RNA for at least 48 weeks after the end of the time .

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

In a ninth aspect of the seventh ment, an effective amount of GS-7977 and an effective amount of ribavirin are administered to the t ing 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 ofGS—7977 £0 comprises a ition comprising GS-7977 and at least one pharmaceutically acceptable excipient as disclosed herein.

In an eleventh aspect ofthe seventh embodiment, the effective amount of GS- 7977 ses a unit dosage form comprising GS-7977 and at least one pharmaceutically acceptable excipient as disclosed herein. [5 An eighth ment 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 rin sufficient to produce an undetectable amount ofHCV RNA in the human for at least 12 weeks after the end ofthe time period.

In a first aspect ofthe eighth embodiment, the time period is ed 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 1 1 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 QED. 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 GS- 7977 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 ive amount of rin, 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 r subembodiment, the daily dose of rin 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 afifih aspect of the eighth embodiment, a daily dose of about 400 mg of GS— 7977 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 GS- 7977 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 ation 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 genotype 1, 2, 3, 4, 5, or 6, or any combination thereof. In one subembodiment, the subject is infected with HCV genotype 1, 2, or 3, or any combination thereof.

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

In an eighth aspect ofthe eighth embodiment, an effective amount of GS-7977 and an effective amount of rin are administered to the human ing to an interferon-free ent regimen. In one subembodiment, the interferon-free treatment n 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 68-7977 and at least one pharmaceutically acceptable excipient as disclosed herein.

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

A ninth embodiment {is directed to a method of ng 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 amount ofHCV RNA in the human that is less than about 15 IU/mL for at least 12 weeks after the end ofthe 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, fi‘om about 1 1 weeks to about 12 weeks, and about 12 weeks. In one subembodiment the time period is about 12 weeks. In r subembodiment the time period is about 8 weeks.

In a second aspect ofthe ninth embodiment, the effective amount of 7 is a daily dose selected from about 100 mg to about 800mg, 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 GS— 7977 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 rin wherein the administration is concurrent or alternative.

In a fourth aspect of the ninth embodiment, the ive amount of ribavirin is a daily dose selected from about 600mg 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 . In another subembodiment, the effective amount of ribavirin is a daily dose of 50 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 ninth embodiment, a daily dose of about 400 mg of GS- 7977 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 GS— 7977 is administered to the human in combination with a daily dose of about 800 mg of rin. 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 genotype 1, 2, 3, 4, 5, or 6, or any combination thereof. In one subembodiment, the human is infected with HCV genotype 1, 2, or 3, or any combination thereof.

In a h aspect of the ninth embodiment, the human has an amount ofHCV 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 ofHCV RNA less than about 15 IU/mL for at least 36 weeks after the end of the time . In r subembodiment, the ‘ human has an amount ofHCV RNA less than about 15 IU/mL for at least 48 weeks after the end of the time .

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 ing to an interferon— free treatment regimen. In one subembodiment, the interferon—free ent regiment consists of administering an effective amount ofGS-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 .

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.

WO 82003 2012/066605 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 ofthe tenth embodiment, about 400 mg of GS—7977 is stered 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 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. £0 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 QlD. In another subembodiment, a daily dose of about 1000 mg to about 1200 mg ofribavirin is administered to the human BID. In a further odiment, a daily dose of 1000 mg or 1200 mg of ribavirin is stered to the subject based on body weight.

In a fifth aspect of the tenth embodiment, about 800 mg of ribavirin is stered 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 BID.

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

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

In a further subembodiment, the human has an undetectable, amount ofHCV RNA for at least 48 weeks after the end of the time period.

In an eighth aspect ofthe tenth embodiment, the about 400 mg of GS-7977 comprises a ition comprising GS-7977 and at least one pharrnaceutically able 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 .

An eleventh embodiment is directed to a composition useful for the ent 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 ment, the composition does not comprise peginterferon.

In a second aspect ofthe eleventh embodiment, the effective amount of GS—7977 ses 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 mgof GS~ 7977 administered to the subject QD.

In a third aspect of the eleventh embodiment, the effective amount of ribavirin ses 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 ive amount of ribavirin is about 1000 mg to about 1200 mg stered 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 r 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, [0 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 7 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 [5 comprises about .400 mg of GS-7977 administered to the subject QD and about 800 mg of ribavirin stered to the t 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 stered to the subject BID In a fifth aspect of the eleventh embodiment, the composition is capable of providing an undetectable amount ofHCV RNA for at least 12 weeks after the end of a time period following treatment of a t infected with hepatitis C Virus for the time period. In one subembodiment, the composition is capable of providing an undetectable amountofHCV RNA for at least 24 weeks after the end of a time period ing ent of a subject infected with hepatitis C virus for the time period. In another subembodiment, the composition is capable of providing an undetectable amount ofHCV 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 r subembodiment, the composition is capable of providing an undetectable amount ofHCV 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. 2012/066605 In a sixth aspect of the eleventh embodiment, the composition is capable of ing less than about 15 IU/mL ofHCVRNA for at least 12 weeks after the end of a time period following treatment of a subject ed with tis C virus for the time period. In one subembodiment, the composition is capable of providing less than about IU/mL ofHCV RNA for at least 24 weeks after the end of a time period following treatment of a subject infected with tis C Virus for the time . 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 ofHCV RNA for at least 48 weeks after the end of a time period ing treatment of a subject infected with hepatitis C virus for the time period.

In a h aspect of the eleventh embodiment, the effective amount of GS-7977 comprises a unit dosage form comprising GS—7977 and at least one ceutically [5 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 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 ofthe twelfth embodiment, the use comprises 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, 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 ofthe twelfth embodiment, the effective amount 'ofGS-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 about400 mg. In one subembodiment, the daily dose of GS-7977 is administered to the subject QD, BID, TID, or QID. In another odiment, 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 GS- 7977, is stered to the subject QD.

In a third aspect of the h embodiment, an effective amount of GS—7977 is used in combination with an effective amount of rin, wherein the administration of GS—7977 and ribavirin is concurrent or ative.

In a fourth aspect ofthe 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 r 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 r 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 ofthe 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 7 isabout 400 mg QD and the effective amount of ribavirin is about 1000 mg to about 1.200 mg BID.

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

In a seventh aspect of the twelfth embodiment, the t has an undetectable amount ofHCV RNA for at least 12 weeks after the end of the time period. In one subembodiment, the subject has an undetectable amount ofHCV RNA for at least 24 weeks after the end of the time period. In r subembodiment, the subject has an undetectable amount ofHCV RNA for at least 36 weeks after the end of the time period.

In a further subembodiment, the t has an ctable amount ofHCV RNA for at least 48 weeks after the end of the time .

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

In a ninth aspect ofthe twelfih embodiment, the subject is a human.

In a tenth aspect ofthe 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 consists of administering an effective amount of GS-7977 and an effective amount of ribavirin to the subject for a time .

In an eleventh aspect of the twelfth embodiment, the effective amount of GS-7977 ses 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.

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

HCV pe PEGASYS® Dose* COPEGUS® Dose Genotypes l, 4 180 pg <75 kg = 1000 mg 48 weeks 275 kg = 1200 mg 48 weeks Genotypes 2, 3 Genotypes 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 FDA- approved label for PEGASYS® dated September 29, 2011 is incorporated by reference.

The daily dose ofCOPEGUS® 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., genotype), se to therapy, and tolerability of the regimen. Based on the foregoing, as well as the examples 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 of 1000 mg or 1200 mg depending on body weight. .

Based on the data ed herein, an effective amount of GS-7977 is 400 mg QD, which can also be administered BID, TID, or QID. It is also contemplated that an effective amount of GS-7977 can e 100 mg to 400 mg and all 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 stration is considered, which means that while the GS-7977 and ribavirin are administered during the same time period, the specific order of administration on a daily basis can be: GS-7977 followed by rin, GS—7977 and rin 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 ation with the oral (capsule or tablet form) strationof ribavirin. Of , other types of administration of both medicaments, as they become available, are contemplated, such as by nasal spray, by a buccal or sublingual administration dosage form, ermally, 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 ingredient 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 series of formulations containing polymorphic Form 1 7 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 ution times.

Considerations were also given to re sorption properties of the tablets due to the sensitivity of Form 1 Of GS-7977 to moisture.

All formulations were compressed into tablets at both high and low hardness levels. Formulation and tablet performance were determined by tablet egration time, content uniformity, and dissolution, as presented in Table 1A. [5 Table 1A. Formulation Compositions for GS—7977 Form 1 Tablets Using a Roller Compaction Process — Formulation Composition (% w/w) ”mu- Intragranular GS-7977 Microcrystalline 25-0 3373 ----- Cellulose —-—-—--_ IIIIIIICroscannellose 2.00 3.0 2.00 Colloidal 0.3 0.3 Stearate Extragranular Microcrystalline 49.0 31.9 31.8 15.3 21.0 ose - - Mannitol __ 153 5 8 Croscannellose I-IIIII2 0 2 0 2 00 Dicalcium III-III IIIIIColloidal 0.25 IIIIIIIIMagnesium 0.5 0.5 0.5 0.5 0.5 0.5 0.5 IIIIIIIITotal Tablet 400 300 300 300 300 300 300 IIIIIIIHardness (kp) 8.1 16.3 7.4 17.2 10.2 5.8 NA 5.4 12.1 8.2 5.1 IIIIIIIIIIIIIDisintegration 0:17 0:33 0:13 3:16 0:48 45:00 0:14 6:27 1:43 ~ 1:23 8:06 Dissolution @ 102 94 82 87 NA 95 64 45M IIIIIIIIIIII The results in Table 1A show that use of microcrystalline cellulose as the sole diluent (Formulations A, B1, B2) produced tablets with acceptable hardness, egration and dissolution, even without incorporating a disintegrant. In contrast, incorporation of mannitol as the sole diluent (Formulation C) without a disintegrant ed in lower compressibility and a longer egration time resulting in slower dissolution. When used in combination with rystalline 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. r, 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 1A support the use of formulations containing microcrystalline cellulose and mannitol/microcrystalline cellulose, in particular, as ts.

Formulations B2 and G2 in Table 1A prepared per a roller compaction/granulation process were r 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 lar sieve (Tri—Sorb®) dessicant. The data shown in Table 1B show a decrease in moisture level as the amount of ol is increased (concomitant with reduction in microcrystalline cellulose). [0 Table 1B. Stability Data for GS-7977 Form 1 Tablets Using a Roller Compaction Process HPLC Assay - Dissolution (% Dissolved)“ - Formulation Stability ' Unknown % img/deg Condition . at RRT 0.67 °C/60% 40 °C/75% RH so9 oo ( 8) 84 96 (6) .3) (3) °C/60% 92 96 RH (2) 40 °C/75% ) 4) ( RH 8O 90 §3 94 (5 5) (4 79 91 96 \o \i 4O °C/75% ) ( 2) RH (no 7 \ow dessicant) ) (2) 2) (3) WO 82003 “Dissolution method: USP Apparatus II (paddles) with 900 mL, pH 6.8 (50 mM sodium phosphate), 05% SLS, 75 rpm, 37 °C l GS- 7977 400 mg Tablets Formulations (Tablets A and B) comprising GS-7977 polymorphic Form 1 were prepared by dry granulation. The formulations contained GS-7977 (polymorphic Form 1) (33.33%), mannitol (30.00%), microcrystalline cellulose (29.67%), croscarmellose sodium (5.00%), colloidal n dioxide (0.50%), and ium stearate (1.50%), as described in Table 2.

Table 2. GS-7977 Polymorphic Form 1 400 mg Tablet Compositions % w/w of 400 mg tablet GS—7977 (Form 1) ' 3333 33.33 rmellose Sodium _ FD&C Red 40 Aluminum Lake FD&C Blue 2 Aluminum Lake 0.10 Colloidal Silicon Dioxide 0.25 Microcrystalline Cellulose 29.67 29.12 —»_— Colloidal Silicon Dioxide 0.25 0.25 Magnesium Stearate Tablets ning about 400 mg of 7 (Form 1) per tablet and an Opadry II purple film coating (Tablet A in Table 2) were prepared as s: (1) A composition comprising GS—7977 (Form 1) and the intragranular excipients (mannitol, croscarmellose sodium, and colloidal silicon dioxide) was sifted through a 20- mesh screen and added to a r (V—shell blender) and blended for about 10—15 minutes to obtain an initial blend. Separately, the intragranular magnesium te 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 ranular blend. (2) tely, the extragranular excipients microcrystalline cellulose, croscarmellose sodium, and colloidal n 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, colloidal silicon dioxide, and magnesium stearate was passed through a roller compactor equipped with a 20—mesh (0.84 mm) milling screen on the granulator and both - and 60-mesh (0.25 mm) screens on the separator until ation was achieved. The roller compactor parameters were: (i) 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. Ribbons were produced using flat straight- grooved rollers. Upon passing through the roller compactor, the material was ' passed/forced through a 20-mesh screen and then sifted with a 60 mesh screen. Granules were sorted into three categories e, 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 to the final ng step.

Material that passed through the 60 mesh screen was considered ‘fine’ and was re- circulated h the roller cOmpactor. This process was repeated until a m amount (e.g., less than 20%) of fines remained. 2012/066605 (4) The milled/sifted granules from step (3) and the sified extragranular excipients (microcrystalline cellulose, mannitol, croscarmellose sodium and silicon dioxide) from step (2) were added to a blender ll blender) and blended for about 15 minutes. tely, magnesium stearate was passed through a 20-mesh screen. The magnesium stearate was added to the blender and blended for about 5 s 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 GS— 7977. As needed, a 15% w/w aqueous suspension for ating comprising polyvinyl alcohol (Opadry II ) was prepared and applied to achieve a target weight gain of 3% (range: 2—4%). The coating suspension was sprayed at 300 g/min/4 guns (range: 200- 400 g/min/4 guns) at a target pan speed of 5 rpm (range: 4—8 rpm) and an exhaust ature of46 i5 °C. The GS-7977 tablets were packaged with 30 tablets and 1 gram of desiccant per bottle. ’ Uncoated tablets comprising 400 mg of 7 (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 containing 7 polymorphic Form 6 (33.33%), mannitol (30.00%), microcrystalline cellulose (29.67%), croscarmellose sodium (5.00%), colloidal silicon dioxide (0.50%), and magnesium stearate (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 ofForm 6. In addition, incorporation of a large tion of the excipients into the intragranular composition decreased the ial fOr powder segregation and the variability in the blend, and improved. the tablet content uniformity for the Tablet C formulation.

Table 3. GS-7977 Polymorphic Form 6 400 mg Tablet Composition Intragranular Components GS-7977 (Form 6) Mannitol Microcrystalline Cellulose Croscarmellose Sodium Colloidal Silicon Dioxide Magnesium Stearate Extragranular ents Microcrystalline Cellulose Croscarmellose Sodium dal Silicon Dioxide Magnesium Stearate Coating Agent The Tablet C formulation was prepared by blending the ranular components listed in Table 3, other than magnesium stearate (i.e., GS-7977, microcrystalline ose, mannitol, croscarmellose sodium and colloidal silicon dioxide) in a blender.

The mixture was milled, blended with the intragranular magnesium stearate and dry granulated using a roller compaction s train and mill. The resulting-ribbons were milled through a milling screen and then blended with the extragranular excipients (microcrystalline cellulose, croscarmellose sodium, colloidal silicon dioxiade, magnesium [0 stearate) 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 sion for the film-coating s was prepared and applied to achieve a target weight gain of 3%.

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, theresults ofwhich are presented in Table 4.

Table 4. Moisture and Stability Data for GS—7977 400 mg Tablets Tablet A Tablet B Tablet C Moisture HPLC Assay Moisture HPLC Assay HPLC (% w/w) (% w/w) Assay % % % % GS—7977 GS-7977 Impurity 7 100 99.5 0.05 101.7 101.7 0.05' 101.1 101.1 0.04 100.9 The results in Table 4 show that the exemplary tablet itions described herein exhibit stability to both moisture and degradation.

The dissolution profile (75 RPM, Apparatus II (Paddle), Phosphate buffer pH 6.8 900 mL) of tablets having the Tablet B formulation was tested lly 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 (:1: RSDa) min 30 min 45 min 60 min —--- 40°C/75%RH 87:1:3 99i2 101i2 100:1:3 aRSD = Relative Standard Deviation In Vitro Antiviral Synergyfor the ation ofGS-977 and Ribavirin The ral effect ofGS-7977 in ation with ribavirin was evaluated using the HCV genotype 1a 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 FBS, 100 units/mL penicillin, 100 ug/mL streptomycin, and 0.1 mM non- essential amino acids. Replicon cells were maintained in 0.5 mg/mL cin®. The cells were passaged every 3-4 days before reaching confluency. All compounds were supplied in 100% DMSO and compound serial dilutions were performed in 100% DMSO. To each well of a 11 plate was added 90 uL of cell culture medium (without Geneticin®) containing 2000 suspended HCV replicon cells and 0.4 ”L of compound solution. The DMSO concentration ofthe final assay wells was 0.44%. The plates were incubated for 3 days at 37°C with 5% C02 and 85% humidity.

For the CCso assay, the media in the ll plate was aspirated and the wells were washed four times With 100 ML 1 X PBS each. A volume of 50 uL 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 ature before the fluorescence signal (excitation 490 nm, emission 520 nm) was measured.

ECso assays were performed in the same wells as CCso assays. The calcein—PBS solution was aspirated and a volume of20 uL of Dual-Glo® luciferase buffer was added to each well. The plate was incubated for 10 minutes at room temperature and a volume of 20 uL of a solution ning a 1:100 e of Dual-Glo® Stop & Glo® substrate and Dual—Glo® Stop & Glo® buffer was added to each well. The plate was incubated at room temperature 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. ard et a1., MacSyngergyTM 11, Version 1.0, University of Michigan, Ann Arbor (1993).) Two-compound synergy definitions are provided in Table 6: Table 6. mpound Synergy Definitions I Synergy/Antagonism Volume (nM2%) Interaction l -50 and>-100 _ GS-7977 in combination with ribavirin showed a synergy volume of .3 i 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 t combined drug concentrations (320 nM GS-7977, 1600 nM ribavirin, 14.0 i 4.4 % inhibition on cell growth). (See also Hebner et al., 63ml Annual Meeting ofthe American Association for the Study of Liver Diseases, Poster 1875, Nov. 12, 2012.) These findings support the potential of GS-7977 stered in combination with ribavirin to achieve ed viral suppression compared to GS—7977 or ribavirin monotherapy.

In Vitro Susceptibility ofSZ82T Mutants to GS—7977, Ribavirin, and the Combination of GS- 7977 and Ribavirin 'In vitro s have shown that SZ82T is the primary on selected by GS— 7977 in HCV genotype 1a, 1b and 2a replicon cells. (Lam et al., J. Virology (2011) 85(23): 12334—12342; Lam et al., Antimicrob. Agents Chemother. (2012) 56(6): 3359- 3368.) $282T mutations in NSSB were created by site-directed mutagenesis in 1a—H77, lb con-1, and 2a JFHl sub—genomic ons. 1b con-l-based chimeric ons containing 2b, 3a, 4a, 5a, or 6a NSSB were also engineered to harbor the S282T mutation.

(See Wong et al., Virology (2012) 429:57-62.) ation capacities and drug susceptibilities of SZ82T to G5-7977 and ribavirin were determined in transient replicon assays. The susceptibilities of S282T and wild—type (WT) NSSB to GS—7977 and ribavirin were further studied by ing the mixture of 50% $282T and 50% WT in GT2a in the ce ofGS—7977 and ribavirin individually and in combination. Relative percentages of mutant and WT Were assessed by deep sequencing.

Introduction of the NSSB SZ82T mutation into lb, la, 2a, 2b, 3a, 4a, and 5a HCV replicons resulted in reduced susceptibility to GS—7977 for all seven pes, producing a 2- to 16-fold increase in EC50 values compared to the ype from the corresponding pes. Surprisingly, the S282T replicons were 3- to 10-fold ensitive to treatment with ribavirin than their corresponding wild—type for these seven genotypes.

EC50 values were not calculated for genotype 6a $282T mutants due to low signal—to- noise ratios; the genotype 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 8282T Mutants in Genotype 1-6 Replicons —S-7977 Ribavirin hese chimeric replicons carry NSSB from genotypes 2b, 3a, 4a; however, the NSSA sequence in all of hese chimeric replicons is derived from genotype 1b.

'ECSO 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 ofWT than $282T, resulting in a population that was 92% mutant S282T over fifteen days. Ribavirin alone suppressed S282T more than WT, ing in a population that was 96% WT after fifteen days. The combination of GS- 7977 and ribavirin also preferentially inhibited SZ82T over WT, resulting in a population that was 91% WT following thirty days oftreatment. The results of the passaging study are presented in Figure 3. (See also Han et al., 63rd Annual Meeting of the American Association for the Study of Liver Diseases, Foster 1078, Nov. 11, 2.012.) Thus, while the SZ82T replicon has been shown to confer reduced susceptibility to GS—7977 in vitro, the mutant replicon has trated increased susceptibility to rin over the wile—type, suggesting that treatment ofCHC with the ation of GS-7977 and ribavirin may result in reduced Viral breakthroughs and incidence of resistance ed to monotherapy with GS-7977 alone. The hypersensitivity of SZ82T mutants to ribavirin may provide an additional advantage to combination treatment comprising GS—7977 and ribavirin, in terms of preventing or delaying the emergency of S282T s.

Quantification ofHCVRNA in Hunian Clinical s Quantitative HCV RNA testing for clinical trials-was performed using the Roche COBAS® AmpliPrep/COBAS® HCV TaqMan® assay using a rdized, automatic RNA extraction system and standardized controls and calibrators. The established LOD of the assay was 15 TU/mL (defined by a 95% hit rate with WHO Standards). HCV RNA levels were measured using serum samples.

US 2010/0226885 (US 12/376,180), which is incorporated by reference, also discloses a method for measuring r a patient has achieved an HCV negative status 'using RT-PCR to measure HCV RNA levels. ent Regimens — P7977-0221 and PROTON al 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 2012/066605 ribavirin in treatment-naive GT1 HCV subjects for 4 weeks, followed by up to an additional 44 weeks of treatment with SOC peginterferon and ribavirin. High RVR (88- 94%) was observed for all three GS—7977 ent groups. Following discontinuatiou 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 83.3%, respectively, for patients receiving a 200 mg GS—7977 treatment n, 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 GS- 7977, peginterferon, and rin at daily dosage levels of 200 mg and 400 mg of GS- 7977 for 12 weeks, followed by up to an additional 36 weeks of treatment with SOC erferon and ribavirin. A greater number of subjects experienced viral breakthrough after cessation ofthe GS—7977 200 mg dosagelevel while still receiving peginterferon/ribavirin treatment compared to no viral breakthroughs after ion of the GS—7977 400 mg dosage level while still receiving erferon/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 £0 The g Phase 2a ELECTRON clinical study evaluated GS—7977 400 mg QD for 8 or 12 weeks in combination with or without ribavirin and/or erferon in ts with GT1, GT2 or GT3 HCV infection. Preliminary data demonstrates 100% SVR-12 for treatment-naive GT2 or GT3 HCV ts treated with a combination of GS—7977 and ribavirin, regardless of the presence of peginterferon, as Well as 84% SVR~ 12 for treatment-naive GT1 HCV patients ing combination treatment with GS—7977 and ribavirin. In comparison, only 60% oftreatment-naive 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 te arms, with abbreviated ons of peginterferon for 4, 8, or 12 weeks in treatment-naive 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-naive); 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 (180 ug weekly) weeks 1-8 only/ PEG (180 ug weekly) weeks 1-12 (GT2/GT3 treatment-naive).

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 7, RBV and PEG: Group 5: GS-7977 (400 mg QD) monotherapy for 12 weeks (GT2/GT3 treatment- na'l've); Group 6: GS—7977 (400 mg QD) with PEG (180 ug weekly) and RBV (1000/1200 mg BID) for 8 weeks (GT2/GT3 treatment—naive); 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 ON trial, two additional erferon—free regimens were explored in treatment-naive 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—naive); and Group 9: 7 (400 mg QD) with RBV (1000/1200 mg BID) for 12 weeks (GT2/GT3 ent—experienced).

In Part 4 of the ELECTRON trial, two further peginterferon-free regimenswere [5 added: Group 10: GS~7977 (400 mg QD) with RBV (1000/1200 mg BID) for 8 weeks (GT2/GT3 treatment-naive); and Group 11: GS-7977 (400 mg QD) with RBV (800 mg BID) for 12 weeks (GT2/GT3 treatment-naive).

Null responders were defined as patients with <2 loglo IU/mL decline 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 erferon and ribavirin: (1) < 2 loglo IU/mL decline from baseline HCV RNA, (2) 2 logo IU/mL reduction in HCV RNA, but HCV RNA > limit ofquantitation (“LOQ”) at end of treatment, and (3) HCV RNA < LOQ at end of treatment but subsequent 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 ized in Tables 8A and 8B, below.

Table 8A. ELECTRON t Demographics (Groups 1-5) GS-7977 GS—7977 7 GS—7977 GS-7977 NO RBV NO PEG 4 Wks PEG 8- Wks PEG 12 Wks PEG NO PEG GT2/GT3 Tx—Naive (Group 1) s 2, 3, 4) (Group 5) "m11 10 ( 49» ‘ ge (Mean, range) 47 47 49 46 43 (35-53) (29-66) (29-66) ) (22-57) 1: M1 (Mean, range) (kg/m2) 28 26 25 24 26 (23.7-35.7) (21.3-32.2) (18.1-32.5) (20 8-284) (18.2-39.4) I CV RNA (Mean, SD) 6.7 (0.42) 6.6 (0.52) 6.4 (0.57) 6.3 (0 76) 5.7 (0.89) logm IU/mL) 6.7 6.6 6.4 6.4 5.7 I CV RNA (Median, range) (6.6-7.3) (5.8-7.3) (5.1-7.0) (5.2 7 1) (4.6-7.3) 4 :7 L28B CC/CT/TT 5/4/1 4/4/1 4/4/2 4/5/2 2/6/2 L28B CC (11, %) 4 (40) 4 (36) 2 (20) Table 8B. ELECTRON Patient Demographics (Groups 6-9) GS—7977 ' GS-7977 GS-7977 RBV RBV RBV NO PEG NO PEG NO PEG 12 Wks 12 Wks 12 Wks GT2/GT3 GT1 GT1 GT2/GT3 ve Null Tx—Naive ”Ix-Experienced Group 6) (Group 7) (Group 8) (Group 9) .5-3C!‘CD'1 AZV 10 -—25 25 I ale (n, %) 50 —m 76 Race sian, %) I: M1 (Mean,‘range) 24.8 (21-349) 40.0) CV RNA (Mean, SD) 6 1 6.8 6.1 6.5 logm 1U/mL) W) —_—m L28B CC/CTfTT 3/6/1 2/5/3 11/12/2 L28B CC(n,%) 3(30) 11(44) 11(44) A summary ofthe patient results for treatment-naive HCV GT2/GT3 Groups 1—5 as related to the percentage of patients having an amount ofHCV RNA below the limits of detection (LOD) is provided in Table 9. 2012/066605 Table 9. ELECTRON Groups 1-5 Patient Results 00---— From Table 9 it can be seen that all treatment-naive HCV GT2 and GT3 patients d with GS-7977 and RBV for 12 weeks (Groups 1-4) had no detectable amount of HCV RNA during the entire treatment period (with or without PEG); All such patients treated with a combination of GS-7977 and RBV (with or t PEG) had no detectable amount ofHCV RNA at 12 weeks and at 24 weeks after the termination of treatment.

Table 9 also reveals that all HCV GT2/GT3 treatment-naive patients receiving 12 weeks ofGS-7977 (400 mg QD) monotherapy (Group 5) had no detectable amount of HCV RNA during the entire treatment period. However, only 60% of the patients receiving GS-7977 monotherapy ed SVR-l2 and SVR—24.

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

Table 10 provides the mean HCV RNA values (Ioglo lU/mL) for treatment-naive HCV GT2/GT3 patients (N=10) for time oftreatment (12 weeks) up to 12 weeks after treatment (W24) for patients receiving a combination of 400 mg QD of GS—7977 and 1000/1200 mg BID (based on weight) ofRBV (Group 1). Table 10 also provides the mean HCV RNA values (loglo TU/mL) for treatment—naive HCV GT2/GT3 patients (N=10) for the time of treatment (12 weeks) for patients receiving a 12-week regimen of 400 mg QD ofGS—7977 only (Group 5). The terms "D1 (6 hr)" and "D1 (12 hr)" refer to the ed measurements made 6 hrs and 12 hrs, respectively, on day 1 ing 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 (longU/mL) GS-7977 NO. RBV (Group 5) aInitial ing Values for patients. bDay 1 results 6 hrs after dosing. 0Day 1 results 12 hrs after dosing.

The data in Table 10 and Figure 1 clearly show that treatment ofHCV GT2/GT3 treatment-naive ts with a combination of 68-7977 and RBV (in the amounts noted above) results in mean HCV RNA levels below the limit of detection during weeks 4-12 of the treatment , as well as SVR-12. This data also shows that the mean HCV RNA value is below the limit of detection 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 ing cessation of treatment compared to patients who received monotherapy with GS-7977 (Group 5).

These results demonstrate that the combination of 7 and ribavirin is advantageous in that patients can be treated for HCV without receiving peginterferon treatment and achieve a high rate of .

A summary ofthe inary patient results for all nine fiilly reported cohorts of the ELECTRON trial as related to the percentage of patients having an amount ofHCV RNA below the limits of detection (LCD) is summarized in Table 11.

Table 11. ELECTRON Groups 1-9 Patient Results Genotype 1 Genotype 2/3 ' Genotype 2/3 Treatment Naive Null Genotype 1 Treatment Responders Experienced 7 GS-7977 GS-7977 GS-7977 68-7977 63—7977 68-7977 Time RBV RBV NO RBV RBV RBV RBV (Wks) NO PEG PEG NO PEG NO PEG NO PEG NO PEG 12 wks 12 wks 12 weeks 12 weeks 12 weeks 12- weeks (Group 1) s 2, 3, 4) (Group 5) (Group 7) (Group 8) (Group 9) (N= 10) (N=30) (N=10) (N = 10) (N = 25) (N = 25) - n (%) n (%) n (%) _-w» 5 (50) w» w» w» 1°00» mm» woo» mm» 2500» 1°00» 2500» -—— W» 1000» 250°» W» 100°» 190» W» woo» W6» The data in Table 11 demonstrate an SVR-l2 rate of 100% for treatment-naive ts with HCV GT2/GT3 (Groups 1-4, 6) when treated with a combination of GS— 7977 (400 mg QD) and RBV, regardless ofthe presence of peginterferon. The data in Table 11 also demonstrates an SVR-12 rate of 84% for patients with HCV GT1 (Group 8) treated with a combination of GS—7977 and RBV in the absence of erferon. In contrast, monotherapy with GS-7977 (Group 5) for GT2/GT3 treatment-naive ts produced an SVR—12 rate of 60%. 1.0 All patients enrolled in Group 10 (8 weeks of 7 + ribavirin combination therapy in treatment-naive GT2/GT3 HCV ts) 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 side—effects normally associated with peginterferon may be avoided. Table 12 presents adverse events reported in at least 15% of the subjects in any treatment group for ELECTRON Groups 19.

Table 12. ON Groups 1-9 Adverse Events Reported in at Least 15% of Subjects in Any Treatment Group GS-7977 GS—7977 GS-7977 GS-7977 RBV PEG NO RBV RBV NO PEG RBV NO PEG PEG 12 wks 12 wks 12 wks 8 wks N=70 N=30 N=10 N=10 (Groups 1, 7, 8, 9) (Groups 2, 3, 4) (Group 5) (Group 6) 1 AB: in (%) ' .lood and Lymphatic System Disorders Anemia ao5'Q?a-a U5-9,D.-a(/2 Nausea Diarrhoea Abdominl Pain Flatulence 8::053a8919..Humgs:m%a § >E.-at2:HN5’.O:3 Fatigue lrritability Pyrexia ‘ Chills - Injection Site Erythema .- Axillary Pain “—— 5wo1-0—.8 (/1 Q- ’5"mI'V‘Er.O:1In Upper atory Tract Infection ism and Nutrition Disorders Decreased Appetite usculoskeletal and Connective Tissue =a0"1a.QU) _—_—- _—--_ ' espiratory, Thoracic and Mediastinal 18 (26) 15 (50) 3 (30) 5 (50) II isorders Gropharyngeal Pain 5 (7) 3 (10) 2 (20) 1 (10) Skin and Subcutaneous Tissue 31 (44) 25 (83) 3 (30) 8 (80) Disorders _———- ___—- —'---— The data in Table 12 reveal that lower incidence rates (%) were reported for a number oftypes of adverse events for treatment ns involving the combination of 7 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 ed for the interferon-free treatment regimens combining GS—7977 and ribavirin: blood and lymphatic system disorders (including anemia); pain and chills; metabolism and nutrition disorders (including decreased appetite); musculoskeletal and connective tissue disorders (including myalgia, back pain and arthralgia); nervous system [0 disorders ding headache and dizziness); psychiatric disorders (including insomnia); . respiratory, thoracic and mediastinal ers (including dyspnoea); and skin and aneous tissue disorders (including pruritis, dry skin and alopecia).

The data in Table 13, below, reveals reduced frequencies of Grade 3 and Grade 4 logic abnormalities for eron—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 Hematologic Abnormalities Laboratory GS-7977 .GS-7977 7 GS-7977 GS-7977 GS-7977 GS-7977 Abnormalities RBV RBV NO RBV RBV RBV RBV RBV NO PEG PEG NO PEG PEG NO PEG NO PEG NO PEG 12 wks 12 wks 12 weeks 8 weeks 12 weeks '12 weeks 12 weeks (Group 1) (Groups 2, 3, 4) (Group 5) (Group 6) (Group 7) (Group 8) (Group 9) (N= 10) (N=30) (N=10) (N= 10) (N=25) (N=25) n (%) n (%) n (%) n (%) n 0%) n (%) Alanine aminotransferase 1 (3) - 1 (4) Hemoglobin 1(3) 1 (10) 1 (10) Lymphocytes 3 (10) o 0 0 0 o o w» 0 i _0 5(17) 0 ’1 2(20) 0 0 0 Grade 4 0 5 (17) 0 White blood cells Grade3 0 6(20) 0 o o l 0 I 0 1(10) 0 _[ 0 _I 0 1 1(10) I 0 0 Additional results, not shown here, show a rapid ization ofALT levels in all 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 .

GS—7977 Resistance in Human Clinical Studies To date, no virologic breakthrough has been. observed during treatment with GS— 7977, suggesting a-high barrier to resistance. Across the 0221, PROTON, ELECTRON (Groups 1-9) and ATOMIC Phase 2 human clinical studies oftreatment regimens involving GS—7977 alone or in combination with ribavirin and/or peginterferon, 53 out of 621 patients have experienced viral relapse after cessation of GS containing treatment. Population sequencing of the viral relapse samples showed that SZ82T was detected in only one ofthe 53 patients, who was GT2b and ed 4 weeks after completion of 12 weeks of 7 monotherapy. Deep sequencing revealed 99% $282T in this GT2b patient at relapse. Population and clonal ypic analysis demonstrated that the GT2b containing sample was 8- to 13-fold less susceptible to GS-7977 compared to ponding baseline virus. For the other 52 ts experiencing relapse, deep sequencing at baseline and relapse showed no $282T, and no specific NSSB mutation at other residues was identified by population or deep sequencing as being associated with GS-7977 resistance. (See also Svarovskaia et al., 63rd Annual Meeting of the an Association for the Study of Liver Diseases, Poster 753, Nov. 1 1, 2012.) The ing illustrates that GS—7977 has a high resistance barrier. Notably, the S282Tmutation has not been observed in any patient receiving a treatment regimen combining GS-7977 and ribavirin.

Concordance ofSVR-4 with SVR—]2 and SVR—24for Treatment Regimens Combining GS- 7977 with Ribavirin and Optionally erferon Florian et al. have reported that SVR-12 and SVR-24 were concordant across a large population database ofHCV clinical trials ing trials involving peginterferon/ribavirin combination treatment and treatment regimens combining peginterferon, ribavirin and telaprevir or boceprevir, with SVR—12 having a positive predictive 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 oftreatment with GS-7977, either alone or in combination with rin and optionally peginterferon, were ted. Only patients treated for at least 12 weeks with 400 mg GS-7977 who 50 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 SVR- 12 and SVR-24 across all regimens for patients who achieved SVR—4 and for whom post- treatment 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., GS- 7977 Phase 2 Trials: Concordance of SVR4 with SVR12 and SVR24 in HCV Genotypes 1-3, EASL (April 18-22, 2012).) The foregoing suggests that the SVR data presented herein may have tive value for -term SVR rates including SVR-24, SVR-36 and SVR-48.

The compositions and unit dosage forms sing GS-7977 disclosed herein provide good stability to moisture and degradation, as well as desirable dissolution and disintegration profiles. They may be used to treat HCV infection optionally in combination with ribavirin, peginterferon or any other ral agent. ‘ Additionally, the foregoing data illustrate that GS-7977 administered in combination with rin (with or without peginterferon) elicited a rapid decline 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 GS- 7977, including when combined with ribavirin and optionally peginterferon. SVR-12 was 100% for HCV GT2 and GT3 treatment-naive patients who received a combination of GS-7977 and rin for 12 weeks and 84% for HCV GT1 treatment-naive patients who received a combination ofGS-7977 and rin for 12 weeks, compared to 60% SVR- 12 for HCV GT2 and GT3 treatment-naive patients who received GS-7977 alone. Given that ribavirin, alone, has been shown to have little to no effect on HCV RNA levels in human al trials, the foregoing clinical and in vitro data demonstrates that the combination of 7 and ribavirin produces a synergistic reduction 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 s, ting that interferon-free treatment with a combination of GS-7977 and ribavirin may offer advantages over ent regimens involving peginterferon.

Even further, in vitro results showing that HCV replicons with the SZ82T mutation, which show reduced susceptibility to 68-7977, display increased susceptibility to ribavirin t that the ation of GS—7977 and ribavirin may provide a treatment regimen resulting in reduced rates of resistance compared to monotherapy with GS-7977. Thus far, the S282T mutation has not been observed in a patient receiving GS- 7977 and ribavirin combination therapy, compared to the ation of the mutation in one patient receiving GS-7977 monotherapy.

The ability to provide effective therapy t peginterferon according to the methods described herein has the potential to significantly improve therapeutic options for individuals living with HCV infection.

The foregoing ption of the present invention provides illustration and description, but is not intended to be exhaustive or to limit the ion to the precise one disclosed. ations and variations are possible in light of the above teachings or may be acquired from practice of the invention. 1001480432

Claims (23)

What is claimed is:

1. A pharmaceutical composition comprising: 25% to 35% w/w of a crystalline compound having the structure: O N O O P O HO F ; and 5 55% w/w to 65% w/w of a diluent consisting of mannitol and microcrystalline cellulose; wherein the lline compound has XRPD 2θ-reflections (±0.2°) at 6.1 and 12.7.

2. The pharmaceutical composition of claim 1, further comprising: 2.5 % w/w to 7.5% w/w of a disintegrant; 0.25% w/w to 0.75% w/w of a glidant; and 10 1.25% w/w to 1.75% w/w of a lubricant.

3. The pharmaceutical composition of claim 1 or claim 2, wherein the diluent ts of 30% w/w of mannitol and 30% w/w of rystalline cellulose.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the composition comprises 33% w/w of the crystalline compound. 15 5. A ceutical composition comprising: 25% to 35% w/w of a crystalline compound having the structure: N O O P O HO F 30% w/w of mannitol; 30% w/w of microcrystalline cellulose; 1001480432 2.5 % w/w to 7.5% w/w of croscarmellose sodium; 0.25% w/w to 0.75% w/w of dal silicon dioxide; and 1.25% w/w to 1.75% w/w of magnesium stearate, wherein the crystalline compound has XRPD 2θ-reflections (±0.2°) at 6.1 and 12.7.

5

6. A pharmaceutical composition comprising: 33% w/w of a crystalline nd having the structure: O N O O P O HO F 30% w/w of mannitol; 30% w/w of microcrystalline cellulose; 10 5% w/w of croscarmellose ; 0.5% w/w of colloidal silicon dioxide; and 1.5% w/w of magnesium stearate, n the crystalline compound has XRPD 2θ-reflections (±0.2°) at 6.1 and 12.7.

7. The ceutical composition of claim 6, comprising an intragranular portion 15 comprising: 33% w/w of the crystalline compound; 30% w/w of mannitol; 25% w/w of microcrystalline cellulose; 2.5% w/w of croscarmellose sodium; 20 0.45% w/w of colloidal silicon dioxide; and 0.75% w/w of ium stearate.

8. The pharmaceutical composition of claim 7, further comprising an extragranular portion comprising: 5% w/w of microcrystalline cellulose; 25 2.5% w/w of croscarmellose sodium; 0.05% w/w of colloidal silicon dioxide; and 1001480432 0.75% w/w of magnesium stearate.

9. The pharmaceutical composition of any one of claims 1 to 8, ated as a tablet.

10. Use of a pharmaceutical composition according to any one of claims 1 to 8 in the 5 manufacture of a medicament for the treatment of a hepatitis C virus infection in a human in need thereof.

11. The use of claim 10, wherein the pharmaceutical ition is to be administered to the human for 12 weeks.

12. The use of claim 10, wherein the pharmaceutical composition is to be 10 administered to the human in combination with ribavirin.

13. The use of claim 10, wherein the human is infected with HCV genotype 1, 2, 3, 4 or any combination thereof.

14. The use of claim 13, wherein the human is ed with HCV genotype 4.

15. A unit dosage form comprising: 15 400 mg of a crystalline compound having the structure: O N O O P O HO F ; and 660 mg to 780 mg of a t consisting of mannitol and rystalline cellulose, wherein the crystalline compound has XRPD 2θ-reflections (±0.2°) at 6.1 and 12.7.

16. A unit dosage form comprising: 20 400 mg of a crystalline compound having the structure: 0432 O N O O P O HO F 360 mg of mannitol; 356 mg of microcrystalline cellulose; 60 mg of croscarmellose sodium; 5 6 mg of colloidal silicon dioxide; and 18 mg of magnesium stearate. wherein the crystalline compound has XRPD 2θ-reflections (±0.2°) at 6.1 and 12.7.

17. The unit dosage form of claim 16, formulated as a tablet.

18. Use of a unit dosage form according to any one of claims 15 to 17 in the 10 manufacture of a medicament for the treatment of a hepatitis C virus infection in a human in need thereof.

19. The use of claim 18, wherein the pharmaceutical ition is to be administered to the human for 12 weeks.

20. The use of claim 18, wherein the unit dosage form is to be administered to the 15 human in combination with ribavirin.

21. The use of claim 18, wherein the unit dosage form is administered to the human in combination with ribavirin as part of an interferon-free treatment regimen.

22. The use of claim 18, wherein the human is infected with HCV pe 1, 2, 3, 4 or any combination thereof. 20

23. The use of claim 22, n the human is infected with HCV genotype 4.