Classifications

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  • A61K31/4025—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil not condensed and containing further heterocyclic rings, e.g. cromakalim
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  • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
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  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
  • A61K31/4436—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a heterocyclic ring having sulfur as a ring hetero atom
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61K31/5375—1,4-Oxazines, e.g. morpholine
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Definitions

• This invention relates to combinations of therapeutic molecules useful for treating hepatitis C virus infection.
• the present invention relates to methods, uses, dosing regimens, and compositions.
• Hepatitis is a disease occurring throughout the world. Hepatitis is generally of viral nature, although, if considered a state of chronic inflammation of the liver, there are other known, non-infectious causes. Viral hepatitis is by far the most common form of hepatitis.
• the U.S. Centers for Disease Control has estimated that at least 1.8% of the U.S. population has serologic evidence of HCV infection, in the majority of cases associated with chronic active infection.
• HCV is a positive-stranded RNA virus belonging to the Flaviviridae family and has closest relationship to the pestiviruses that include hog cholera virus and bovine viral diarrhea virus.
• the HCV genome is a single-stranded, positive-sense RNA of about 9,600 bp coding for a polyprotein of 3009-3030 amino acids, which is cleaved co- and post-translationally by cellular and two viral proteinases into mature viral proteins (core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B).
• core E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B.
• the structural proteins, E1 and E2 are believed to be embedded into a viral lipid envelope and form stable heterodimers.
• the structural core protein is believed to interact with the viral RNA genome to form the nucleocapsid.
• the nonstructural proteins designated NS2 to NS5 include proteins with enzymatic functions involved in virus replication and protein processing including a polymerase, protease, and helicase. HCV replicates through the
• HCV is a genetically diverse virus. Within a single infected patient, many variant viruses can be identified, leading to the description ‘viral swarm’, or viral quasispecies. Within the global human population, HCV is also genetically diverse, with at least 6 major ‘genotypes’ identified (Genotypes 1-6), and numerous subtypes (i.e., HCV Genotype 1a and 1b). HCV genotypes are defined by genomic phylogenetic analysis, and diagnosed (in a given patient) by HCV RNA sequence-based diagnostic assays.
• the main route of infection with HCV is blood exposure.
• the magnitude of the HCV infection as a health problem is illustrated by the prevalence among high-risk groups. For example, in some surveys, 60% to 90% of hemophiliacs and more than 80% of intravenous drug abusers in western countries had chronic HCV infection. For intravenous drug abusers, the prevalence varies from about 28% to 80% depending on the population studied.
• the proportion of new HCV infections associated with blood or blood product transfusion has been markedly reduced due to pharmaceutical advances and widespread use of sensitive serologic and RNA detection assays used to screen blood donors, however, a large cohort of aging, chronically infected persons is already established.
• PEG-IFN ⁇ 1 ⁇ or PEG-IFN ⁇ 1b pegylated interferon- ⁇
• PEG-IFN ⁇ 1 ⁇ or PEG-IFN ⁇ 1b pegylated interferon- ⁇
• SVR Sustained Virologic Response
• treatment with PEG-IFN+RBV is not well tolerated, with an adverse event profile that includes flu-like symptoms, thrombocytopenia, anemia, and serious psychiatric side effects. While treatment with the current standard of care is suboptimal, many patients are precluded from ever starting therapy due to comorbidities common in HCV-infected populations, including psychiatric disorders, advanced liver disease, and substance abuse.
• Ribavirin is a nucleoside analog antiviral drug. Ribavirin is typically taken orally (by mouth) twice a day. The exact mechanism for ribavirin is unknown. However, it is believed that when ribavirin enters a cell it is phosphorylated; it then acts as an inhibitor of inosine 5′-monophosphate dehydrogenase (IMPDH). IMPDH inhibitors such as ribavirin reduce the intracellular synthesis and storage of guanine, a nucleotide “building block” necessary for DNA and RNA production, thus inhibiting viral replication. IMPDH inhibitors also interfere with the reproduction of rapidly proliferating cells and cells with a high rate of protein turnover.
• IMPDH inosine 5′-monophosphate dehydrogenase
• ribavirin monotherapy has little effect on HCV RNA levels, but is associated with a decline in serum alanine transferase (ALT). This observation suggests that ribavirin may not be acting as an antiviral agent, but rather as a modulator of immune system function. Ribavirin is only approved for use, for HCV infection, in combination with IFN.
• PEG-IFN/ribavirin therapy is considered the ‘standard-of-care’ treatment for chronic HCV infection.
• the standard of care is, however, expected to change rapidly in the near future with approval of direct acting antiviral agents which will, initially, be used in combination with PEG-IFN/ribavirin.
• HCV genotype 1 is more resistant to therapy than types 2 and 3.
• many current treatments for HCV produce unwanted side effects.
• new anti-viral therapies that produce fewer unwanted side-effects, that are more effective against a range of HCV genotypes, or that have less complicated dosing schedules, i.e. that require administration of agents fewer times during a day.
• compositions and therapeutic methods that are useful for treating viral infections (e.g. HCV).
• Certain compositions and methods of the invention produce fewer unwanted side-effects, are more effective against a range of HCV genotypes, reduce the potential for viral rebound due to resistance selection and have shortened less complicated dosing schedules than currently available therapies.
• the invention provides a composition comprising two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof.
• the invention provides a method of treating an HCV infection in a human, comprising administering two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof to the human.
• the invention provides a method for ameliorating one or more symptoms of an HCV infection in a human, comprising administering two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof to the human.
• the invention provides a method for reducing viral load in a human with HCV, comprising administering two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof to the human.
• the invention provides a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents in a human, comprising administering two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof to the human.
• the invention provides the use of two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof in medical therapy.
• the invention provides the use of two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof for the prophylactic or therapeutic treatment of a viral (e.g. HCV) infection.
• a viral infection e.g. HCV
• the invention provides the use of a composition of the invention for the prophylactic or therapeutic treatment of a viral (e.g. HCV) infection.
• a viral infection e.g. HCV
• the invention provides the use of two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof to prepare a medicament for treating a viral (e.g. HCV) infection in a human.
• a viral infection e.g. HCV
• the invention provides the use of a composition of the invention to prepare a medicament for treating a viral (e.g. HCV) infection in a human.
• a viral infection e.g. HCV
• the invention provides the use of two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof to prepare a medicament for ameliorating one or more symptoms of a viral (e.g. HCV) infection in a human.
• a viral infection e.g. HCV
• the invention provides the use of a composition of the invention to prepare a medicament for ameliorating one or more symptoms of a viral (HCV) infection in a human.
• HCV viral
• the invention provides the use of two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof to prepare a medicament for reducing viral load in a human.
• the invention provides the use of a composition of the invention to prepare a medicament for reducing viral load in a human.
• the invention provides the use of two or more compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof to prepare a medicament for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents in a human.
• the invention provides the use of a composition of the invention to prepare a medicament for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents in a human.
• the invention provides a composition comprising two, three, four or five Combination Compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof, provided that Compound 1 and Compound 2 are not the only Combination Compounds in the composition and further provided that Compound 1 and Compound 3 are not the only Combination Compounds in the composition.
• the invention provides a composition comprising Compound 1 and Compound 6.
• the invention provides a composition comprising Compound 1, Compound 3 and Compound 6.
• the invention provides a composition comprising Compound 3 and Compound 5.
• the invention provides a composition comprising Compound 3 and Compound 6.
• the invention provides a composition comprising Compound 3, Compound 5 and Compound 6.
• compositions further comprise one or more pharmaceutically acceptable diluents or carriers.
• the invention provides that the foregoing compositions are formulated as a unit dosage form for once daily administration.
• the invention provides that the foregoing compositions are formulated for oral administration.
• the invention provides that the foregoing compositions formulated as a tablet.
• the invention provides a method of treating an HCV infection in a human, comprising administering two or more Combination Compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof to the human, provided that Compound 1 and Compound 2 are not the only Combination Compounds administered and further provided that Compound 1 and Compound 3 are not the only Combination Compounds administered.
• the invention provides a method for ameliorating one or more symptoms of an HCV infection in a human, comprising administering two or more Combination Compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof to the human, provided that Compound 1 and Compound 2 are not the only Combination Compounds administered and further provided that Compound 1 and Compound 3 are not the only Combination Compounds administered.
• the invention provides a method for reducing viral load in a human with HCV, comprising administering two or more Combination Compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof to the human, provided that Compound 1 and Compound 2 are not the only Combination Compounds administered and further provided that Compound 1 and Compound 3 are not the only Combination Compounds administered.
• the invention provides a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents in a human, comprising administering two or more Combination Compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof to the human, provided that Compound 1 and Compound 2 are not the only Combination Compounds administered and further provided that Compound 1 and Compound 3 are not the only Combination Compounds administered.
• the invention provides that the methods for treating an HCV infection in a human, for ameliorating one or more symptoms of an HCV infection in a human for reducing viral load in a human with HCV, and for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents in a human further comprise administering an interferon to the human.
• the invention provides methods for treating an HCV infection in a human, for ameliorating one or more symptoms of an HCV infection in a human for reducing viral load in a human with HCV, and for reducing emergence of HCV quasispecies wherein an interferon is not administered to the human.
• the invention provides that the methods for treating an HCV infection in a human, for ameliorating one or more symptoms of an HCV infection in a human for reducing viral load in a human with HCV, and for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents in a human further comprise administering ribavirin to the human.
• the invention provides that the methods for treating an HCV infection in a human, for ameliorating one or more symptoms of an HCV infection in a human for reducing viral load in a human with HCV, and for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents in a human further comprise administering one or more additional agents selected from ribavirin, an interferon, alpha-glucosidase 1 inhibitors, hepatoprotectants, TLR-7 agonists, cyclophilin inhibitors, HCV viral entry inhibitors, HCV maturation inhibitors, and HCV IRES inhibitors to the human.
• additional agents selected from ribavirin, an interferon, alpha-glucosidase 1 inhibitors, hepatoprotectants, TLR-7 agonists, cyclophilin inhibitors, HCV viral entry inhibitors, HCV maturation inhibitors, and HCV IRES inhibitors to the human.
• the invention provides for use of two or more Combination Compounds selected from Combination Compounds Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof in medical therapy, provided that Compound 1 and Compound 2 are not the only Combination Compounds selected and further provided that Compound 1 and Compound 3 are not the only Combination Compounds selected.
• the invention provides for use of two or more Combination Compounds selected from Combination Compounds Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof for the prophylactic or therapeutic treatment of an HCV infection, provided that Compound 1 and Compound 2 are not the only Combination Compounds selected and further provided that Compound 1 and Compound 3 are not the only Combination Compounds selected.
• the invention provides for use of two or more Combination Compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof to prepare a medicament for treating an HCV infection in a human, provided that Compound 1 and Compound 2 are not the only Combination Compounds selected and further provided that Compound 1 and Compound 3 are not the only Combination Compounds selected.
• the invention provides for the use of two or more Combination Compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof to prepare a medicament for ameliorating one or more symptoms of an HCV infection in a human, provided that Compound 1 and Compound 2 are not the only Combination Compounds selected and further provided that Compound 1 and Compound 3 are not the only Combination Compounds selected.
• the invention provides for the use of two or more Combination Compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof to prepare a medicament for reducing viral load in a human, provided that Compound 1 and Compound 2 are not the only Combination Compounds selected and further provided that Compound 1 and Compound 3 are not the only Combination Compounds selected.
• the invention provides for the use of two or more Combination Compounds selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and pharmaceutically acceptable salts thereof to prepare a medicament for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents in a human, provided that Compound 1 and Compound 2 are not the only Combination Compounds selected and further provided that Compound 1 and Compound 3 are not the only Combination Compounds selected.
• compositions and methods of the invention may provide “synergy” and “synergistic effects”, i.e. the effect achieved when the active ingredients (including two or more Combination Compounds) are used together is greater than the sum of the effects that results from using the compounds separately.
• compositions and methods of the invention are beneficial because they provide treatments for a wide range of HCV genotypes and because they cause fewer or less serious side effects than current HCV therapies (e.g. treatments that include the administration of interferon).
• Compounds refers to Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• Compound 1 may also be referred to as 5-((6-(2,4-bis(trifluoromethyl)phenyl)pyridazin-3-yl)methyl)-2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridine or 5H-imidazo[4,5-c]pyridine, 5-[[6-[2,4-bis(trifluoromethyl)phenyl]pyridazin-3-yl]methyl]-2-(2-fluorophenyl).
• Compound 2 may also be referred to as (2R,6S,13aR,14aS,16aS)-2-(8-chloro-2-(2-(isopropylamino)thiazol-4-yl)-7-methoxyquinolin-4-yloxy)-6-(cyclopentyloxycarbonylamino)-5,16-dioxooctadecahydrocyclopropa[e]pyrrolo[1,2- ⁇ ][1,4]diazacyclopentadecin-14a-yl(2,6-difluorobenzyl)phosphinic acid.
• ribavirin refers to:
• Ribavirin is also referred to as 1- ⁇ -D-ribofuranosyl-1H-1,2,4-Triazole-3-carboxamide, 1- ⁇ -D-ribofuranosyl-1,2,4-triazol-3-carboxyamide; 1- ⁇ -D-Ribofuranosyl-1,2,4-triazole-3-carboxamide; COPEGUS (Roche); DRG-0028; HSDB 6513; ICN 1229; MegaRibavirin (e.g.
• ribavirin in formulations of 100 mg of ribavirin/mL); NSC 163039; RAVANEX (BioPartners); REBETOL (Schering-Plough; Aesca; Bayer Schering Pharma; Essex; Pfizer; Trading Pharma; Zuellig Pharma); Ribamide; RIBAMIDIL (Biopharma, Russia); RIBASPHERE (Three Rivers Pharmaceuticals); Ribavarin; Ribavirina; Tribavirin; VILONA (Valeant Pharmaceuticals; ICN Pharmaceuticals); VIRAMID (ICN Pharmaceuticals; Alfa Wassermann); VIRAZOLE (Valeant Pharmaceuticals); and VIRIZADOLE (Uci-farma, Sao Bernardo do Campo, Sao Paulo, Brazil).
• ribavirin includes analogs of ribavirin, including taribavirin (VIRAMIDINE, ICN 3142).
• interferon includes 1) interferons, e.g., pegylated rIFN-alpha 2b (PEG-Intron, Merck & Co., Inc.), pegylated rIFN-alpha 2a (PEGASYS, Hoffmann-La Roche Inc.), rIFN-alpha 2b (INTRON® A, Merck & Co., Inc.), rIFN-alpha 2a (Roferon®-A, Hoffmann-La Roche Inc.), interferon alpha (MULTIFERON® Viranative AB Corporation, OPC-18, Alfaferone, Alfanative, subalin), interferon alfacon-1 (Valeant), interferon alpha-nl (WellferonTM, Glaxo Wellcome), interferon alpha-n3 (ALFERON®-Hemispherx Biopharma, Inc.), interferon-beta-1a (AVONEX® Biogen Idec, DL-8234 Daiichi Pharmaceutical Co.
• interferon-omega (omega DUROS®, Alza Corporation, Intarcia Therapeutics, Inc.; Biomed 510, Intarcia Therapeutics, Inc.), albinterferon alpha-2b (ALBUFERON®, Human Genome Sciences, INC.), IFN alpha-2b XL, BLX-883 (LOCTERON®, Biolex Therapeutics, INC.), DA-3021, glycosylated interferon alpha-2b (AVI-005), PEG-INFERGEN®, Amgen, Inc., Pegylated interferon lambda-1 (type III) (PEGylated IL-29), and BELEROFON®, Nautilus Biotech.
• Combination therapy means compositions or methods or uses or the like that incorporate two or more of the Combination Compounds.
• Combination therapy may also incorporate other active ingredients in addition to the two or more of the Combination Compounds including, but not limited to: ribavirin, an interferon, an alpha-glucosidase 1 inhibitor, a hepatoprotectant, a Toll-like receptor (TLR)-7 agonist, a cyclophilin inhibitor, an HCV viral entry inhibitor, an HCV maturation inhibitor, and an HCV IRES inhibitor.
• active ingredient means a component of a combination therapy that a exerts or is capable of exerting a pharmaceutical effect including any of the Combination Compounds, ribavirin, an interferon, an alpha-glucosidase 1 inhibitor, a hepatoprotectant, a TLR-7 agonist (such as Compound 8), a cyclophilin inhibitor, an HCV viral entry inhibitor, an HCV maturation inhibitor, and an HCV IRES inhibitor.
• treating when used in the context of treating a disease, means slowing or stopping the progression of a disease, or ameliorating at least one symptom of a disease, more preferably ameliorating more than one symptom of a disease.
• an HCV patient may experience an improvement in one or all of the following symptoms that can be associated with HCV infection: fever, headache, muscle aches, jaundice, fatigue, loss of appetite, nausea, vomiting and diarrhea.
• Treatment of a hepatitis C virus infection can include reducing the HCV viral load in an HCV infected human being.
• Certain of the compounds described herein contain one or more chiral centers, or may otherwise be capable of existing as multiple stereoisomers.
• the scope of the present invention includes mixtures of stereoisomers as well as purified enantiomers or enantiomerically/diastereomerically enriched mixtures. Also included within the scope of the invention are the individual isomers of the compounds represented by the formulae shown herein, as well as any wholly or partially equilibrated mixtures thereof.
• the present invention also includes the individual isomers of the compounds represented by the formula shown herein as mixtures with isomers thereof in which one or more chiral centers are inverted. Stereochemical definitions and conventions used herein generally follow S. P.
• optically active compounds Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light.
• the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s).
• the prefixes d and I or (+) and ( ⁇ ) are employed to designate the sign of rotation of plane-polarized light by the compound, with ( ⁇ ) or I meaning that the compound is levorotatory.
• a compound prefixed with (+) or d is dextrorotatory.
• a specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture.
• a 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process.
• the terms “racemic mixture” and “racemate” refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
• the present invention encompasses combinations of two or more of the Combination Compounds.
• Table I showing possible two-way (Combinations 1-21), three-way (Combinations 22-56), four-way (Combinations 57-92) and five-way (Combinations 93-113) combinations of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 of the invention is provided below:
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 1 and further comprising a second compound selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 2, Compound 3, Compound 4, Compound 5 or Compound 6.
• the second compound is not Compound 2 or Compound 3.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 2 and further comprising a second compound selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 4.
• the second compound is not Compound 1.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 3 and further comprising a second compound selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 4 or Compound 5 or Compound 6.
• the second compound is not Compound 1.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 4 and further comprising a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 2 or Compound 3 or Compound 6.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 5 and further comprising a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 3 or Compound 6.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 6 and further comprising a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3 or Compound 4 or Compound 5.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 7 and further comprising a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 1 and further comprising a second compound and a third compound each selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 3, or Compound 4, or Compound 5 or Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 2 and the third compound may be Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 2 and further comprising a second compound and a third compound each selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 3 and further comprising a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 4 or Compound 5 or Compound 6.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• the second compound may be Compound 5 and the third compound may be Compound 6.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 4 and further comprising a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3 or Compound 6.
• the second compound may be Compound 1 and the third compound may be Compound 2.
• the second compound may be Compound 1 and the third compound may be Compound 3.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 5 and further comprising a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 3 or Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 6 and further comprising a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3 or Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 2.
• the second compound may be Compound 1 and the third compound may be Compound 3.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound may be Compound 5.
• the second compound may be Compound 3 and the third compound may be Compound 5.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 7 and further comprising a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 1 and further comprising a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 3, Compound 4, Compound 5, or Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 2 and the third compound may be Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 2 and further comprising a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 3 and further comprising a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 4 or Compound 5 or Compound 6.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• the second compound may be Compound 5 and the third compound may be Compound 6.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 4 and further comprising a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3, or Compound 6.
• the second compound may be Compound 1 and the third compound may be Compound 2.
• the second compound may be Compound 1 and the third compound may be Compound 3.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 5 and further comprising a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 3 or Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 6 and further comprising a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3, or Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 2.
• the second compound may be Compound 1 and the third compound may be Compound 3.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound may be Compound 5.
• the second compound may be Compound 3 and the third compound may be Compound 5.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 7 and further comprising a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 1 and further comprising a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 3, Compound 4, Compound 5 or Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 2 and the third compound may be Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 2 and further comprising a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 3 and further comprising a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 4 or Compound 5 or Compound 6.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• the second compound may be Compound 5 and the third compound may be Compound 6.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 4 and further comprising a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3 or Compound 6.
• the second compound may be Compound 1 and the third compound may be Compound 2.
• the second compound may be Compound 1 and the third compound may be Compound 3.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 5 and further comprising a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 3 or Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 6 and further comprising a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3, and Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 2.
• the second compound may be Compound 1 and the third compound may be Compound 3.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound may be Compound 5.
• the second compound may be Compound 3 and the third compound may be Compound 5.
• compositions e.g. a pharmaceutical composition, the composition comprising Compound 7 and further comprising a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6.
• the Combination Compounds and other active ingredients can be in the form of a salt.
• the salts of the Combination Compounds and other active ingredients are pharmaceutically acceptable salts.
• Salts encompassed within the term “pharmaceutically acceptable salts” refer to non-toxic salts of the Combination Compounds and/or other active ingredients.
• Suitable pharmaceutically acceptable salts include inorganic acid addition salts such as chloride, bromide, sulfate, phosphate, and nitrate; organic acid addition salts such as acetate, galactarate, propionate, succinate, lactate, glycolate, malate, tartrate, citrate, maleate, fumarate, methanesulfonate, p-toluenesulfonate, and ascorbate; salts with acidic amino acid such as aspartate and glutamate; alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; ammonium salt; organic basic salts such as trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, and N,N′-dibenzylethylenediamine salt; and salts with basic amino acid such as lysine salt and arginine salt.
• the salts may be in some cases hydrate
• Combination Compounds and/or other active ingredients can be formulated with conventional carriers or excipients, which can be selected in accord with ordinary practice.
• Tablets typically contain excipients, glidants, fillers, binders and the like.
• Aqueous formulations can be prepared in sterile form, and when intended for delivery by other than oral administration generally will be isotonic. All formulations will optionally contain excipients such as those set forth in the Handbook of Pharmaceutical Excipients (1986), herein incorporated by reference in its entirety. Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and the like.
• the pH of the formulations ranges from about 3 to about 11, but is ordinarily about 7 to 10.
• the formulations of the invention both for veterinary and for human use, comprise at least one active ingredient, together with one or more acceptable carriers and optionally other therapeutic ingredients.
• the carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and physiologically innocuous to the recipient thereof.
• the formulations include those suitable for the administration routes set forth below.
• the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations generally can be found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.), herein incorporated by reference in its entirety. Such methods include the step of bringing into association an active ingredient with the carrier which constitutes one or more accessory ingredients.
• the formulations can be prepared by uniformly and intimately bringing into association one or more active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
• Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of an active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
• An active ingredient may also be administered as a bolus, electuary or paste.
• a tablet can made by compression or molding, optionally with one or more accessory ingredients.
• Compressed tablets may be prepared by compressing in a suitable machine an active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent.
• Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.
• the tablets may optionally be coated or scored and optionally can be formulated so as to provide slow or controlled release of an active ingredient.
• the formulations can be preferably applied as a topical ointment or cream containing an active ingredient(s) in an amount of, for example, 0.075 to 20% w/w (including active ingredient(s) in a range between 0.1% and 20% in increments of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc.), preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w.
• an active ingredient may be employed with either a paraffinic or a water-miscible ointment base.
• an active ingredient may be formulated in a cream with an oil-in-water cream base.
• the aqueous phase of the cream base may include, for example, at least 30% w/w of a polyhydric alcohol, i.e. an alcohol having two or more hydroxyl groups such as propylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400) and mixtures thereof.
• the topical formulations may desirably include a compound which enhances absorption or penetration of an active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethyl sulphoxide and related analogs.
• the oily phase of the emulsions of Combination Compounds and/or other active ingredients may be constituted from known ingredients in a known manner. While the phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat.
• the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax
• the wax together with the oil and fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
• Emulgents and emulsion stabilizers suitable for use in the formulation of the invention include Tween® 60 (ICI Americas Inc.), Span 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodium lauryl sulfate.
• the choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties.
• the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers.
• Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
• compositions according to the present invention comprise one or more active together with one or more pharmaceutically acceptable carriers or excipients and optionally other therapeutic agents.
• Pharmaceutical formulations containing active ingredients may be in any form suitable for the intended method of administration.
• tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared.
• Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation.
• Tablets containing an active ingredient in admixture with non-toxic pharmaceutically acceptable excipient which are suitable for manufacture of tablets are acceptable.
• excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium or sodium phosphate; granulating and disintegrating agents, such as maize starch, or alginic acid; binding agents, such as cellulose, microcrystalline cellulose, starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc.
• inert diluents such as calcium or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium or sodium phosphate
• granulating and disintegrating agents such as maize starch, or alginic acid
• binding agents such as cellulose, microcrystalline cellulose, starch,
• Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
• a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.
• Formulations for oral use may be also presented as hard gelatin capsules where an active ingredient(s) is mixed with an inert solid diluent, for example calcium phosphate or kaolin, or as soft gelatin capsules wherein an active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.
• an inert solid diluent for example calcium phosphate or kaolin
• an oil medium such as peanut oil, liquid paraffin or olive oil.
• Aqueous suspensions of the invention contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
• excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcelluose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate).
• a suspending agent
• the aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.
• Oil suspensions may be formulated by suspending an active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
• the oral suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol.
• Sweetening agents, such as those set forth herein, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.
• Dispersible powders and granules of the invention suitable for preparation of an aqueous suspension by the addition of water provide an active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives.
• a dispersing or wetting agent and suspending agents are exemplified by those disclosed above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.
• the pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions.
• the oily phase may be a vegetable oil, such as olive oil or arachis oil, a mineral oil, such as liquid paraffin, or a mixture of these.
• Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate.
• the emulsion may also contain sweetening and flavoring agents.
• Syrups and elixirs may be formulated with sweetening agents, such as glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.
• sweetening agents such as glycerol, sorbitol or sucrose.
• Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.
• compositions of the invention may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension.
• a sterile injectable preparation such as a sterile injectable aqueous or oleaginous suspension.
• This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned herein.
• the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane-diol or prepared as a lyophilized powder.
• a non-toxic parenterally acceptable diluent or solvent such as a solution in 1,3-butane-diol or prepared as a lyophilized powder.
• acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
• sterile fixed oils may conventionally be employed as a solvent or suspending medium.
• any bland fixed oil may be employed including synthetic mono- or diglycerides.
• fatty acids such as oleic acid may likewise be used in the preparation of injectables.
• a time-release formulation intended for oral administration to humans may contain approximately 1 to 1000 mg of active material compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95% of the total compositions (weight:weight).
• the pharmaceutical composition can be prepared to provide easily measurable amounts for administration.
• an aqueous solution intended for intravenous infusion may contain from about 3 to 500 ⁇ g of an active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 mL/hr can occur.
• Formulations suitable for administration to the eye include eye drops wherein an active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for an active ingredient.
• An active ingredient is preferably present in such formulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10% particularly about 1.5% w/w.
• Formulations suitable for topical administration in the mouth include lozenges comprising an active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising an active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising an active ingredient in a suitable liquid carrier.
• Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.
• Formulations suitable for intrapulmonary or nasal administration have a particle size for example in the range of 0.1 to 500 ⁇ m (including particle sizes in a range between 0.1 and 500 ⁇ m in increments such as 0.5 ⁇ m, 1 ⁇ m, 30 ⁇ m, 35 ⁇ m, etc.), which is administered by rapid inhalation through the nasal passage or by inhalation through the mouth so as to reach the alveolar sacs.
• Suitable formulations include aqueous or oily solutions of an active ingredient.
• Formulations suitable for aerosol or dry powder administration may be prepared according to conventional methods and may be delivered with other therapeutic agents such as compounds heretofore used in the treatment or prophylaxis of infections as described herein.
• Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to an active ingredient such carriers as are known in the art to be appropriate.
• Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
• the formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use.
• Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described.
• Preferred unit dosage formulations can be those containing a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.
• compositions of Combination Compounds and/or other active ingredients may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
• Combination Compounds and other active ingredients can also be formulated to provide controlled release of an active ingredient to allow less frequent dosing or to improve the pharmacokinetic or toxicity profile of an active ingredient. Accordingly, the invention also provided compositions comprising two or more of the Combination Compounds formulated for sustained or controlled release.
• the effective dose of an active ingredient depends at least on the nature of the condition being treated, toxicity, whether the compound is being used prophylactically (lower doses) or against an active disease or condition, the method of delivery, and the pharmaceutical formulation, and can be determined by the clinician using conventional dose escalation studies.
• compositions of the invention can be formulated to provide effective doses.
• the composition may comprise from 1.0 mg to 100 mg, from 5 mg to 40 mg, from 30 mg to 50 mg, or 20 mg or 40 mg and can be adapted to be administered one or more times daily to a human being in need thereof in combination with any one or more of Compound 2, Compound 3, Compound 6, Compound 4, Compound 5 and Compound 7.
• the composition may comprise from 25 mg to 800 mg, from 50 mg to 400 mg, or from 60 mg to 300 mg or from 70 mg to 200 mg or may be 150 mg and can be adapted to be administered one or more times daily to a human being in need thereof in combination with any one or more of Compound 1, Compound 3, Compound 6, Compound 4, Compound 5 and Compound 7.
• the composition may comprise from 10 mg to 1000 mg, or 50 to 400 mg, or 100 mg to 400 mg or 200 mg to 400 mg and can be adapted to be administered one or more times daily to a human being in need thereof in combination with any one or more of Compound 1, Compound 2, Compound 6, Compound 4, Compound 5 and Compound 7.
• the composition may comprise from 25 mg to 400 mg or from 25 mg to 200 mg can be adapted to be administered one or more times daily to a human being in need thereof in combination with any one or more of Compound 1, Compound 2, Compound 3, Compound 6, Compound 5 and Compound 7.
• the composition may comprise from 50 mg to 1000 mg or 100 mg to 750 mg can be adapted to be administered one or more times daily to a human being in need thereof in combination with any one or more of Compound 1, Compound 2, Compound 3, Compound 6, Compound 4 and Compound 7.
• the composition may comprise from 1 mg to 500 mg or from 3 mg to 300 mg or from 3 mg to 200 mg or from 3 mg to 100 mg or from 10 mg to 90 mg or from 30 mg to 90 mg can be adapted to be administered one or more times daily to a human being in need thereof in combination with any one or more of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 7.
• the composition may comprise from 100 micrograms up to 3000 mg, from 25 mg up to 2000 mg, or from 50 mg up to 1000 mg and can be adapted to be administered one or more times daily (e.g.
• Dosages for Compounds 1-7 that are co-administered may need to be adjusted to account for potential drug-drug interactions.
• Compound 1 affects drug metabolizing systems
• Compound 2 appears to have the effect of increasing the exposure of Compound 1 approximately 2-3 ⁇ . Therefore, a dose reduction (e.g. 2 ⁇ -3 ⁇ ) of Compound 1 would be anticipated when Compound 1 is combined with Compound 2.
• a dose reduction e.g. 2 ⁇ -3 ⁇
• Compound 2 appears to have the effect of increasing the exposure of Compound 6 approximately 5 ⁇ , so dose reduction (e.g. 3 ⁇ -5 ⁇ ) of Compound 6 would be anticipated when Compound 6 is dosed with Compound 2. Therefore, a 10 mg dose of Compound 6 when coadministered with Compound 2 approximate to a 30 mg dose.
• the two or more Combination Compounds may be administered in conjunction with Ribavirin in amounts of about 800 mg, 1000 mg or 1200 mg per day in single or multiple dosages (e.g. about 400 mg, 500 mg or 600 mg twice daily).
• Combination Compounds may be used in the dosages set forth above.
• One aspect of the present invention includes Compound 1 for use in a method of treating HCV infections, wherein compound 1 is used in combination with a second compound selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 3, Compound 4, Compound 5 or Compound 6.
• the second compound may also be Compound 4, Compound 5 or Compound 6.
• Another aspect of the present invention includes Compound 2 for use in a method of treating HCV infections, wherein compound 2 is used in combination with a second compound selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 4.
• Another aspect of the present invention includes Compound 3 for use in a method of treating HCV infections, wherein compound 3 is used in combination with a second compound selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 4 or Compound 5 or Compound 6.
• the second compound may also be Compound 6.
• Another aspect of the present invention includes Compound 4 for use in a method of treating HCV infections, wherein Compound 4 is used in combination with a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 2 or Compound 3 or Compound 6.
• Another aspect of the present invention includes Compound 5 for use in a method of treating HCV infections, wherein Compound 5 is used in combination with a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6 and Compound 7.
• the second compound may be Compound 1, Compound 3 or Compound 6.
• Another aspect of the present invention includes Compound 6 for use in a method of treating HCV infections, wherein Compound 6 is used in combination with a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3 or Compound 4.
• Another aspect of the present invention includes Compound 7 for use in a method of treating HCV infections, wherein Compound 7 is used in combination with a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6.
• Another aspect of the present invention includes Compound 1 for use in a method of treating HCV infections, wherein compound 1 is used in combination with a second compound and a third compound each selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 3, or Compound 4, or Compound 5 or Compound 6.
• the second compound may be Compound 4, or Compound 5 or Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 2 and the third compound may be Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• Another aspect of the present invention includes Compound 2 for use in a method of treating HCV infections, wherein compound 2 is used in combination with a second compound and a third compound each selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• Another aspect of the present invention includes Compound 3 for use in a method of treating HCV infections, wherein compound 3 is used in combination with a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 4 or Compound 5 or Compound 6.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• the second compound may be Compound 5 and the third compound may be Compound 6.
• Another aspect of the present invention includes Compound 4 for use in a method of treating HCV infections, wherein Compound 4 is used in combination with a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3 or Compound 6.
• the second compound may be Compound 1 and the third compound may be Compound 2.
• the second compound may be Compound 1 and the third compound may be Compound 3.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• Another aspect of the present invention includes Compound 5 for use in a method of treating HCV infections, wherein Compound 5 is used in combination with a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 3 or Compound 6.
• Another aspect of the present invention includes Compound 6 for use in a method of treating HCV infections, wherein Compound 6 is used in combination with a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3 or Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 2.
• the second compound may be Compound 1 and the third compound may be Compound 3.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 2 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound
• Another aspect of the present invention includes Compound 7 for use in a method of treating HCV infections, wherein Compound 7 is used in combination with a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6.
• Another aspect of the present invention includes Compound 1 for use in a method of treating HCV infections, wherein compound 1 is used in combination with a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 3, Compound 4, Compound 5, or Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 2 and the third compound may be Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• Another aspect of the present invention includes Compound 2 for use in a method of treating HCV infections, wherein compound 2 is used in combination with a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• Another aspect of the present invention includes Compound 3 for use in a method of treating HCV infections, wherein compound 3 is used in combination with a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 4 or Compound 5 or Compound 6.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• the second compound may be Compound 5 and the third compound may be Compound 6.
• Another aspect of the present invention includes Compound 4 for use in a method of treating HCV infections, wherein Compound 4 is used in combination with a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3, or Compound 6.
• the second compound may be Compound 1 and the third compound may be Compound 2.
• the second compound may be Compound 1 and the third compound may be Compound 3.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• Another aspect of the present invention includes Compound 5 for use in a method of treating HCV infections, wherein Compound 5 is used in combination with a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6 and Compound 7.
• the second compound may be Compound 1.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• Another aspect of the present invention includes Compound 6 for use in a method of treating HCV infections, wherein Compound 6 is used in combination with a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3, or Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 2.
• the second compound may be Compound 1 and the third compound may be Compound 3.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 2 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound may be Compound 5.
• Compound 5 Compound 7
• Another aspect of the present invention includes Compound 7 for use in a method of treating HCV infections, wherein Compound 7 is used in combination with a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6.
• Another aspect of the present invention includes Compound 1 for use in a method of treating HCV infections, wherein compound 1 is used in combination with a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 3, Compound 4, Compound 5 or Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 2 and the third compound may be Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• Another aspect of the present invention includes Compound 2 for use in a method of treating HCV infections, wherein compound 2 is used in combination with a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• Another aspect of the present invention includes Compound 3 for use in a method of treating HCV infections, wherein compound 3 is used in combination with a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 4 or Compound 5 or Compound 6.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• the second compound may be Compound 5 and the third compound may be Compound 6.
• Another aspect of the present invention includes Compound 4 for use in a method of treating HCV infections, wherein Compound 4 is used in combination with a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3 or Compound 6.
• the second compound may be Compound 1 and the third compound may be Compound 2.
• the second compound may be Compound 1 and the third compound may be Compound 3.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• Another aspect of the present invention includes Compound 5 for use in a method of treating HCV infections, wherein Compound 5 is used in combination with a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 3 or Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• Another aspect of the present invention includes Compound 6 for use in a method of treating HCV infections, wherein Compound 6 is used in combination with a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3, or Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 2.
• the second compound may be Compound 1 and the third compound may be Compound 3.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 2 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound may be Compound 5.
• Compound 5 Compound 7
• Another aspect of the present invention includes Compound 7 for use in a method of treating HCV infections, wherein Compound 7 is used in combination with a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6.
• One aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 1 and further comprising administering a second compound selected from the group consisting of comprising Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 3, Compound 4, Compound 5 or Compound 6.
• the second compound may also be Compound 4, Compound 5, Compound 6 or Compound 7
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 2 and further comprising administering a second compound selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 4.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 3 and further comprising administering a second compound selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 4 or Compound 5 or Compound 6.
• Another aspect of the present invention includes a method for ameliorating one or more symptoms of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 4 and further comprising administering a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 2 or Compound 3 or Compound 6.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 5 and further comprising administering a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 3 or Compound 6.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 6 and further comprising administering a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3 or Compound 4.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 7 and further comprising administering a second compound selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 1 and further comprising administering a second compound and a third compound each selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 3, or Compound 4, or Compound 5 or Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 2 and the third compound may be Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 2 and further comprising administering a second compound and a third compound each selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 3 and further comprising administering a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 4 or Compound 5 or Compound 6.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• the second compound may be Compound 5 and the third compound may be Compound 6.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 4 and further comprising administering a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3 or Compound 6.
• the second compound may be Compound 1 and the third compound may be Compound 2.
• the second compound may be Compound 1 and the third compound may be Compound 3.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 5 and further comprising administering a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 3 or Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 6 and further comprising administering a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3 or Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 2.
• the second compound may be Compound 1 and the third compound may be Compound 3.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 2 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 5.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 7 and further comprising administering a second compound and a third compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 1 and further comprising administering a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 3, Compound 4, Compound 5, or Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 2 and the third compound may be Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 2 and further comprising administering a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 3 and further comprising administering a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 4 or Compound 5 or Compound 6.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• the second compound may be Compound 5 and the third compound may be Compound 6.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 4 and further comprising administering a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3, or Compound 6.
• the second compound may be Compound 1 and the third compound may be Compound 2.
• the second compound may be Compound 1 and the third compound may be Compound 3.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 5 and further comprising administering a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 3 or Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 6 and further comprising administering a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3, or Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 2.
• the second compound may be Compound 1 and the third compound may be Compound 3.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 2 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 5.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 7 and further comprising administering a second compound, a third compound and a fourth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 1 and further comprising administering a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 3, Compound 4, Compound 5 or Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 2 and the third compound may be Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 2 and further comprising administering a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 3 and further comprising administering a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 4, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 4 or Compound 5 or Compound 6.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 4 and the third compound may be Compound 6.
• the second compound may be Compound 5 and the third compound may be Compound 6.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 4 and further comprising administering a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3 or Compound 6.
• the second compound may be Compound 1 and the third compound may be Compound 2.
• the second compound may be Compound 1 and the third compound may be Compound 3.
• the second compound may be Compound 1 and the third compound may be Compound 6.
• the second compound may be Compound 2 and the third compound may be Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 5 and further comprising administering a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 6 and Compound 7.
• the second compound may be Compound 1 or Compound 5 or Compound 6.
• the second compound may be Compound 3 and the third compound may be Compound 6.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 6 and further comprising administering a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 7.
• the second compound may be Compound 1, Compound 2, Compound 3, and Compound 4.
• the second compound may be Compound 1 and the third compound may be Compound 2.
• the second compound may be Compound 1 and the third compound may be Compound 3.
• the second compound may be Compound 1 and the third compound may be Compound 4.
• the second compound may be Compound 2 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 4.
• the second compound may be Compound 3 and the third compound may be Compound 5.
• Another aspect of the present invention includes a method for ameliorating one or more symptom of HCV infection in a human, a method for reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, and a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents, each method comprising administering Compound 7 and further comprising administering a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6.
• Two or more of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 and any other components of a combination therapy can be adapted to be administered by any route appropriate to the condition to be treated. Suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) and the like. It will be appreciated that the preferred route may vary with, for example, the condition of the recipient.
• a synergistic effect may be attained when the active ingredients are: (1) co-formulated (e.g. in a unitary dosage form) and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen.
• a synergistic effect may be attained when the compounds are administered or delivered sequentially, e.g., in separate tablets, pills or capsules, or by different injections in separate syringes.
• an effective dosage of each active ingredient is administered sequentially, i.e. serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.
• Co-administration of a Combination Compound with one or more Combination Compounds generally refers to simultaneous or sequential administration of one or more Combination Compounds, such that therapeutically effective amounts of two or more Combination Compounds are present in the body of the patient.
• Combination Compounds e.g. two, three or four Combinations Compounds
• co-formulated Combination Compounds may be co-administered with one or more additional Combination Compounds.
• Co-administration also includes administration of unit dosages of the Combination Compounds before or after administration of unit dosages of one or more other active ingredients, for example, administration of two or more Combination Compounds within seconds, minutes, or hours of the administration of one or more other active ingredients.
• a unit dose of a Combination Compound can be administered first, followed within seconds or minutes by administration of a unit dose of a second Combination Compound, followed within seconds or minutes by administration of a unit dose of one or more other active ingredients.
• a unit dose of one or more other active ingredients can be administered first, followed within seconds or minutes by administration of a unit dose of a Combination Compound, followed within seconds or minutes by administration of a unit dose of a second Combination Compound.
• the Combination Compounds may be administered one after another within seconds, minutes, or hours (e.g. 1-12 hours) of each other and the one or more additional active ingredients may be administered before, during or after the administration of the Combination Compounds.
• the Combination Compounds are co-formulated, they can be administered simultaneously, or before or after the administration of one or more additional active ingredients.
• the combination therapy may be administered as separate dosage forms with each active ingredient, administered together or separately, sequentially or concurrently, and close in time or remote in time to each other.
• the course of treatment can extend, for example, from about 12 weeks to about 48 weeks, or longer, for example, from about 12 weeks to about 24 weeks.
• the present invention includes a combination of therapeutically effective components to ameliorate at least one symptom of HCV infection in a human being including, but not limited to, nausea, vomiting, loss of appetite, fatigue, jaundice, vomiting, diarrhea, dehydration, abdominal pain, cirrhosis of the liver.
• the use of combination therapy is effective to reduce the viral load of HCV viral particles present in the body of the infected person by a statistically significant amount. Viral load can be measured, for example, by measuring plasma HCV RNA levels using, for example, the COBAS TaqMan HCV assay (Roche Molecular Systems).
• an HCV infected person who is treated with the Combination Compounds in accordance with the present invention experiences an improvement in one or all of the symptoms associated with the HCV infection.
• One aspect of the present invention provides for compositions, methods, uses and the like for the treatment of HCV comprising administering two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin, without administering one or more interferons.
• This aspect of the invention may be particularly useful because it allows for the effective treatment of HCV without the side effects associated with the administration of one or more interferon.
• the combined amount of ribavirin and Combination Compounds or pharmaceutically acceptable salts thereof, optionally with one or more additional agents, is effective to treat HCV infection.
• Another aspect of the present invention includes a method for ameliorating one or more symptoms of HCV infection in a human comprising: administering two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin, without concurrent administration of one or more interferon.
• the present invention does not foreclose the potential for dosing one or more interferon. Rather, the present invention may be used in conjunction with another therapy that, in fact, includes one or more interferon.
• An aspect of the present invention includes efficacious treatment of HCV with ribavirin without the need for one or more interferon.
• Another aspect of the present invention includes a method for reducing viral load in a human diagnosed with HCV comprising: administering two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin, but not one or more interferon.
• Another aspect of the present invention includes a method for treating HCV in a human subject consisting essentially of administration of ribavirin in conjunction with two or more of the Combination Compounds or pharmaceutically acceptable salts thereof.
• Another aspect of the present invention includes a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents comprising: administering two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin, without concurrent administration of one or more interferon.
• another aspect of the present invention includes a composition, e.g. a pharmaceutical composition for ameliorating one or more symptom of HCV infection in a human comprising two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin, without one or more interferon.
• a composition for reducing viral load in a human diagnosed with HCV comprising two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin, but not one or more interferon.
• Another aspect of the present invention includes a composition for treating HCV in a human subject consisting essentially of ribavirin in conjunction with two or more of the Combination Compounds or pharmaceutically acceptable salts thereof.
• compositions for ribavirin-based HCV therapy comprising two or more of the Combination Compounds or pharmaceutically acceptable salts thereof, with the proviso that said composition does not include one or more interferon.
• compositions for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents comprising two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin, without one or more interferon.
• the compositions may include compositions in which Compound 1 and Compound 2 are not the only Combination Compounds and in which Compound 1 and Compound 3 are not the only Combination Compounds.
• another aspect of the present invention includes use of: two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin, without one or more interferon, in the manufacture of a medicament for ameliorating one or more symptoms of HCV infection in a human; as well as use of: two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin, but not one or more interferon, in the manufacture of medicament for reducing viral load in a human diagnosed with HCV; as well as use of ribavirin in conjunction with two or more of the Combination Compounds or pharmaceutically acceptable salts thereof in the manufacture of a medicament for treating HCV in a human subject, wherein said use does not include use of one or more interferon; as well as use of two or more of the Combination Compounds or pharmaceutically acceptable salts thereof, in the manufacture of a medicament for ribavirin-based HCV therapy, wherein said use avoids administration of one or more interferon; as well as use of two or more of
• Another aspect of the present invention includes a combination comprising ribavirin and two or more of the Combination Compounds or pharmaceutically acceptable salts thereof, which combination is substantially free of one or more interferon.
• the combination may occur as separate dosage forms with each active ingredient, administered together or separate, sequentially or concurrently, and close in time or remote in time to each other.
• the combination may include combinations in which Compound 1 and Compound 2 are not the only Combination Compounds and in which Compound 1 and Compound 3 are not the only Combination Compounds.
• kits comprising: ribavirin, two or more of the Combination Compounds and instruction regarding a treatment regimen to treat, reduce viral load, or delay onset or progression of HCV wherein the treatment regimen includes administration of the two or more of the Combination Compounds and ribavirin without administration of one or more interferon.
• a kit may also include packaging, such as a blister pack.
• such a kit may provide for individual prescription and dosing of each component as separately packaged pharmaceutics, but when combined with the instruction regarding a treatment regimen to treat, reduce viral load, or delay onset or progression of HCV, such is intended to be within the scope of the present invention.
• kits may include kits in which Compound 1 and Compound 2 are not the only Combination Compounds and in which Compound 1 and Compound 3 are not the only Combination Compounds.
• compositions comprising: ribavirin; two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and one or more pharmaceutically acceptable carriers.
• the pharmaceutical composition may be a unitary dosage form.
• the compositions may include compositions in which Compound 1 and Compound 2 are not the only Combination Compounds and in which Compound 1 and Compound 3 are not the only Combination Compounds.
• the combination therapy with Ribavirin may be administered as separate dosage forms with each active ingredient administered (including the Combination Compounds), may be administered together (e.g., in the form of a unit dosage, such as a tablet) or separately, sequentially or concurrently, and close in time or remote in time to each other. If administered separately, each compound may be administered with the other(s) at the same time, or either before or after such administration of the other(s).
• the active ingredients can be administered daily.
• a daily dosage of the active ingredients is administered in separate sub-doses, such as one, two, three or four times per day.
• the daily dosage of Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin may be administered once per day.
• the present invention includes compositions, methods, uses and the like for the treatment of HCV comprising administering two or more Combination Compounds or a pharmaceutically acceptable salt thereof; and ribavirin, but not one or more interferon, the present invention does not foreclose the potential for dosing one or more interferon to the human. Rather, the present invention may be used in conjunction with another therapy for another indication that, in fact, includes one or more interferon.
• compositions, methods, uses and the like comprising administering two or more of the Combination Compounds or pharmaceutically acceptable salts thereof and ribavirin, and one or more interferon for treatment of HCV.
• the administration of more interferon may be in temporal relation to the administration of the Combination Compounds and ribavirin.
• Another aspect of the present invention includes a method for ameliorating one or more symptoms of HCV infection in a human comprising administering two or more of the Combination Compounds or pharmaceutically acceptable salts thereof, ribavirin, and one or more interferons.
• Another aspect of the present invention includes a method for reducing viral load in a human diagnosed with HCV comprising: administering two or more of the Combination Compounds or pharmaceutically acceptable salts thereof along with ribavirin and one or more interferons.
• Another aspect of the present invention includes a method of ribavirin-based HCV therapy comprising administering two or more of the Combination Compounds or pharmaceutically acceptable salts thereof along with ribavirin, and one or more interferons.
• Another aspect of the present invention includes a method for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents comprising: administering two or more of the Combination Compounds or pharmaceutically acceptable salts thereof along with ribavirin and one or more interferons.
• Another aspect of the present invention includes use of two or more of the Combination Compounds or pharmaceutically acceptable salts thereof ribavirin, and one or more interferons, in the manufacture of a medicament for ameliorating one or more symptoms of HCV infection in a human.
• Another aspect of the present invention includes use of two or more of the Combination Compounds or pharmaceutically acceptable salts thereof along with ribavirin and one or more interferons, in the manufacture of medicament for reducing viral load in a human diagnosed with HCV.
• Another aspect of the present invention includes use of ribavirin in conjunction with two or more of the Combination Compounds or pharmaceutically acceptable salts thereof in the manufacture of a medicament for treating HCV in a human subject, wherein said use includes use of one or more interferons.
• Another aspect of the present invention includes use of two or more of the Combination Compounds or pharmaceutically acceptable salts thereof, in the manufacture of a medicament for ribavirin-based HCV therapy, wherein said use includes administration of one or more interferon.
• Another aspect of the present invention includes use of two or more of the Combination Compounds or pharmaceutically acceptable salts thereof, ribavirin, and one or more interferons in the manufacture of a medicament for reducing emergence of HCV quasispecies with resistance to coadministered oral antiviral agents.
• Another aspect of the present invention includes a combination comprising ribavirin and two or more of the Combination Compounds or pharmaceutically acceptable salts thereof, which combination includes one or more interferons.
• kits comprising: ribavirin, two or more of the Combination Compounds and one or more interferon; and instructions regarding a treatment regimen to treat, reduce viral load, or delay onset or progression of HCV wherein the treatment regimen includes administration of the two or more of the Combination Compounds and ribavirin and administration of one or more interferon.
• a kit may also include packaging, such as a blister pack.
• such a kit may provide for individual prescription and dosing of each component as separately packaged pharmaceutics, but when combined with the instruction regarding a treatment regimen to treat, reduce viral load, or delay onset or progression of HCV, such is intended to be within the scope of the present invention.
• Another aspect of the present invention includes a pharmaceutical composition
• a pharmaceutical composition comprising: two or more of the Combination Compounds or pharmaceutically acceptable salts thereof, ribavirin, and one or more interferon; and one or more pharmaceutically acceptable carriers.
• the pharmaceutical composition may be a unitary dosage form.
• the combination therapy with Ribavirin and one or more interferons may be administered as separate dosage forms with the one or more interferons administered to the patient and each of the remaining active ingredients to be employed in the combination therapy (including the Combination Compounds) are administered together (e.g., in the form of a unit dosage, such as a tablet) or separately, sequentially or concurrently, and close in time or remote in time to each other.
• each active ingredient may be administered with the other(s) at the same time, or either before or after such administration of the other(s).
• the active ingredients can be administered daily.
• a daily dosage is administered in separate sub-doses, such as one, two, three or four times per day.
• non-limiting examples of suitable combinations include the combinations of two or more of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 (including, but not limited to, combinations in which Compound 1 and Compound 2 are not the only Combination Compounds and in which Compound 1 and Compound 3 are not the only Combination Compounds) with one or more additional active ingredients including HCV NS3 protease inhibitors, alpha-glucosidase 1 inhibitors, hepatoprotectants, nucleoside or nucleotide inhibitors of HCV NS5B polymerase, non-nucleoside inhibitors of HCV NS5B polymerase, HCV NS5A inhibitors, TLR-7 agonists, cyclophilin inhibitors, HCV IRES inhibitors, HCV entry inhibitors, HCV maturation inhibitors, and pharmacokinetic enhancers, as well as other drugs for treating HCV. More specifically, one or more compounds of the present invention may be combined with one or more compounds selected from the
• Compound 1 can be prepared using synthetic methods and intermediates like those described in U.S. Pat. No. 7,754,720. Compound 1 can also be prepared as described in the following Example.
• the intermediate compound 104 was prepared as follows.
• TCCA trichloroisocyanuric acid
• Compound 2 can be prepared using synthetic methods and intermediates like those described in U.S. Ser. No. 12/202,319 (US 20100051763 A1). Compound 2 can also be prepared as described in the following Example.
• Phosphinate ester 206 (23.7 g, 24.05 mmol) was dissolved in CH 3 CN (240 mL) and cooled to 0° C. Iodotrimethylsilane (17.4 mL, 122.3 mmol) was added at a fast drop-wise pace followed by, after 10 min, 2,6-lutidine (17.0 mL, 146.4 mmol). The reaction mixture was slowly warmed to room temperature and stirred for 1 h then cooled back down to 0° C. and 2,6-lutidine (11.1 mL, 95.6 mmol) followed by MeOH (24 mL) were added.
• the macrocyclic olefin 204 (7.34 g, 8.42 mmol) was dissolved in ethylacetate (105 mL) and rhodium on alumina (5% wt, 2.945 g, 0.40 wt %) was added. The system was evacuated and flushed with H 2 (1 atm, 3 ⁇ ). To the system, after 3 h, was added more rhodium on alumina (5% wt, 842 mg, 0.10 wt %) and evacuated and flushed with H 2 (1 atm, 3 ⁇ ). After an additional 1 h the suspension was filtered and concentrated in vacuo providing 6.49 g of reduced macrocycle 205 in 88% crude yield. LCMS (M+1): 872.04.
• the brosylate macrocycle 205 (6.49 g, 7.67 mmol) was dissolved in N-methylpyrrolidinone (25.0 mL) and 8-chloro-2-(2-isopropylamino-thiazol-4-yl)-7-methoxy-quinolin-4-ol 207 (2.564 g, 7.33 mmol) followed by Cs 2 CO 3 (4.40 g, 13.50 mmol) were added. The mixture was heated to 65° C. for 6 h then diluted with ethylacetate (200 mL) and washed with LiCl (5%, 250 mL).
• the crude ester 213 (3.18 g, 5.07 mmol) was dissolved in THF (25 mL), H 2 O (25 mL), and then MeOH (6 mL) and LiOH (660 mg, 25.4 mmol) was added.
• the reaction mixture was stirred at room temperature for 1 h and diluted with EtOAc.
• the reaction mixture was acidified to pH 2 with 1 N HCl and the two layers were separated.
• the aqueous layer was extracted with EtOAc (2 ⁇ ).
• the combined organic layers were washed with brine, dried with Na 2 SO 4 concentrated and dried under vacuum to give 3.09 g of acid 201.
• the solution of the bromoketone obtained from the previous reaction was suspended in i-propanol (270 mL) and isopropylisourea (9.4 g, 0.080 mol).
• the reaction mixture was heated at 72° C. for 32 hours.
• LCMS analysis of the reaction demonstrated complete conversion to the desired product.
• the reaction was allowed to cool to room temperature to allow for the product to precipitate out of the solution.
• the reaction was further cooled to 0° C. for 12 hours before filtration.
• the filtrate was washed with ether and dried on lypholizer to provide 8.03 g of compound 207 as an orange solid.
• Compound 3 can be prepared using synthetic methods and intermediates like those described in U.S. Ser. No. 12/215,605 (US 20090257978 A1). Compound 3 can also be prepared as described in the following Example.
• the intermediate compound 315 was prepared as follows.
• N-t-Boc-cis-4-Hydroxy-L-Proline methyl ester 303 (100.0 g, 407.7 mmol) and DABCO (1.5 eq, 68.6 g, 611.6 mmol) were dissolved in anhydrous toluene (200 mL) in a 2 L three necked round bottom flask with a mechanical stirrer and an addition funnel. After cooling the solution to 0° C. under N 2 , A solution of 4-Bromo-benzenesulfonyl chloride (1.3 eq, 135.6 g, 530.0 mmol) in 300 mL of toluene was added through addition funnel over 60 minutes.
• the reaction mixture was stirred at room temperature for 3 h, with monitoring by LCMS.
• the reaction mixture was concentrated to remove dichloromethane under reduced pressure and the white solid that formed was filtered off.
• the remaining DMF solution was diluted with ethyl acetate (1 L), washed successively with 3% LiCl (aq) (3 ⁇ 650 mL), sat'd NH 4 Cl (2 ⁇ 500 mL), 0.5N HCl (aq) (2 ⁇ 600 mL), brine (500 mL), sat'd NaHCO 3 (3 ⁇ 500 mL), and brine (500 mL).
• the resulting organic fraction was dried (MgSO 4 ) and concentrated to afford compound 305 (111 g).
• Methyl ester 314 (23.6 g, 26 mmol) was dissolved in glacial acetic acid (200 ml), 1.4 N HCl in H 2 O (75 ml) was added to the solution. The mixture was stirred at 60° C. for 1 hour. Upon completion of the reaction, the mixture was concentrated to remove the solvents, coevaporated with toluene ( ⁇ 2) to remove residual acetic acid. The residue was then dissolved in EtOAc (500 ml) and sat. NaHCO 3 aqueous solution (enough to neutralize the mixture) while monitoring CO 2 evolution. The organic layer was washed with brine, dried (Na 2 SO 4 ) and concentrated in vacuo.
• Compound 4 can be prepared as described in the following Example.
• Diastereomeric mixture 414 was dissolved in heptane and isopropanol (70%:30%, 230 mg in 4.5 mL of the mixed solvents) and subjected to chiral column separation under the following conditions:
• Solvent system 70% heptane and 30% isopropanol
• the intermediate diastereomeric mixture 414 was prepared as follows.
• Compound 5 can be prepared as described in the following Example.
• the intermediate compound 508 was prepared as follows.
• (R)-4-methylcyclohex-3-enecarboxylic acid 503 (371 mg, 2.65 mmol), azeotropically dried by evaporation from toluene, was treated with potassium phosphate tribasic (1.13 g, 7.94 mmol), suspended in dichloromethane (7.6 mL) and treated with dimethylformamide (4 drops). The reaction mixture was cooled to 0° C. and treated dropwise with oxalyl chloride (0.75 mL, 7.9 mmol). The reaction mixture was allowed to warm to ambient temperature while stirring for 2 hours. After filtering the solids, the solution was concentrated, treated with hexanes and concentrated again to afford (R)-4-methylcyclohex-3-enecarbonyl chloride 504 as a light yellow oil which was used immediately in the next step.
• Trimethylsulfoxonium chloride (79 mg, 0.62 mmol) in DMSO (1.5 mL) was treated with sodium hydride (21 mg, 60% oil dispersion, 0.53 mmol) and stirred at ambient temperature for 10 min.
• the orange solution was treated with 5% citric acid until pH 3 and partitioned between water and ethyl acetate.
• the organic layer was separated and the aqueous was extracted again with ethyl acetate.
• the combined organics were washed with 5% LiCl, water and brine, and dried over sodium sulfate.
• Compound 6 can be prepared using synthetic methods and intermediates like those described in U.S. Ser. No. 12/779,023 (US 20100310512 A1). Compound 6 can also be prepared as described in the following Example.
• the crude material was used in the next step without further purification.
• the crude material was dissolved in DMF (1.5 mL) and DIEA (53.4 mg, 0.414 mmol) was added.
• the reaction was stirred at room temperature. After 20 minutes, the reaction was diluted with EtOAc and was washed with aqueous bicarbonate solution, aqueous LiCl solution (5%), brine, and was dried over sodium sulfate.
• the intermediate compound 614 was prepared as follows.
• the reaction mixture was allowed to warm to RT and stirred for 110 h (about 5 days) after which the reagents were quenched with saturated aqueous ammonium chloride ( ⁇ 150 mL).
• the contents of the flask were slowly poured into a 2 L sep funnel containing saturated aqueous sodium bicarbonate (800 mL).
• the aqueous phase was extracted three times with 300 mL ethyl acetate.
• the combined organics were dried over magnesium sulfate and concentrated to provide a mixture of Compounds 603 and 604.
• the crude material from sub-part b was dissolved in 3:1:1 THF/water/acetone (165 mL) then treated with N-methylmorpholine-N-oxide (3.45 g, 29.4 mmol) and osmium tetroxide (4 wt % in water, 5 mL, 0.818 mmol). After stirring at RT for 7 h, the reagents were quenched with 1 M aqueous sodium thiosulfate ( ⁇ 100 mL). The contents of the flask were then poured into a 1 L sep funnel containing water ( ⁇ 300 mL). The aqueous phase was extracted three times with 300 mL dichloromethane.
• the reaction was cooled to room temperature and poured onto ice containing sodium bicarbonate. A solid formed and was collected via filtration. The crude material was taken into EtOAc and was washed with aqueous HCl (1 M) and brine. The solution was dried over sodium sulfate.
• 2,7-Dibromo-9,9-difluoro-9H-fluorene 608 (372 mg, 1.04 mmol), Pd(PPh 3 ) 4 (30.0 mg, 0.026 mmol), PdCl 2 (PPh 3 ) 2 (18.2 mg, 0.026 mmol), As(PPh 3 ) 3 (5.0 mg) were dissolved in dioxane (10 mL) under an argon atmosphere. Ethoxyvinyl-tributyl tin (376.4 mg, 1.04 mmol) was added. The mixture was heated for 140 minutes at 85° C. (oil bath). The reaction was cooled to room temperature.
• N-bromo succinimide (177 mg, 1.0 mmol) was added followed by water (2 mL). The reaction was stirred at room temperature for 3 hours, after which the majority of the dioxane was removed in vacuo. The crude reaction mixture was diluted with EtOAc and was washed with water. All volatiles were removed in vacuo. Toluene was added and all volatiles were removed in vacuo for a second time. The crude material was dissolved in DMF/MeCN (2 mL, 1:1) at room temperature.
• the intermediate compound 613 can be prepared as follows
• reaction mixture was concentrated down and purified by flash column chromatography (silica gel, 20 to 80% ethyl acetate/hexane) to give 3-(6-Bromo-1H-benzoimidazol-2-yl)-2-aza-bicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester 618 (0.383 g, 72%) as an orange foam.
• Compound 7 can be prepared using synthetic methods and intermediates like those described in U.S. Pat. No. 7,429,572. Compound 7 can also be prepared as described in the following Example.
• the organic phase is filtered and concentrated in vacuo.
• the residue is dissolved in IPA (ca. 20 kg) and heptane (14.2 kg) is added.
• the solution is heated to about 74-75° C. to produce a clear solution, then about 5 L is removed by distillation.
• the resulting solution is cooled slowly to RT. A precipitate is formed at about 42-43° C. Cooling is continued slowly to 5° C. then stirred overnight.
• the resulting solid is filtered and the filtrate is washed with IPA/heptane (1:8) mixture (13.4 kg), and dried under vacuum at about 60-70° C. to afford 1.295 kg (86.65%) of compound 7 which is 99.45% pure by HPLC.
• the intermediate compound 706 can be prepared as follows.
• ketone 702 (138.91 g, 57.5% from cytidine) as a white solid and 22 g of unreacted starting material, 701, as a yellow solid.
• Compound 8 can be prepared using synthetic methods and intermediates like those described in U.S. Ser. No. 12/632,194. Compound 8 can also be prepared as described in the following Example.
• the intermediate compound 807 was prepared as follows.
• reaction mixture containing compound 803 was added to the reaction mixture containing compound 802 at 0° C.
• the reaction mixture was stirred until LC/MS indicated the consumption of compound 802, approximately 45 minutes.
• a saturated solution of NH 4 Cl (50 mL) was added.
• the layers were separated, and the aqueous layer was extracted with EtOAc (2 ⁇ 30 mL).
• the combined organic layers were dried over MgSO 4 , filtered, and concentrated under vacuum. Purification by silica gel chromatography provided 2.11 g of compound 804.
• HTBS High Throughput Replicon Assay
• Replicon cells harboring H77 (genotype 1a) or Conl (genotype 1b) HCV RNA and Renilla luciferase reporter were seeded in 384-well black plates at a density of 1.6 ⁇ 103 cells per well in 90 ⁇ L of DMEM culture medium, excluding G-418. Compounds were serially diluted in 100% DMSO and added to cells at a 1:225 dilution, achieving a final concentration of 0.44% DMSO in a total volume of 904 with a Biotek ⁇ Flow Workstation. Cell plates were incubated at 37° C. with 5% CO 2 for 3 days, after which culture media were removed and cells were assayed for luciferase activity as a marker for replication level.
• HCV NS3 Protease IC 50 Determination HCV NS3 protease activity was monitored using a fluorescence resonance energy transfer (FRET) depsipeptide substrate (RET 51, Anaspec, San Jose, Calif.) based on the method of Taliani, Taliani M, Bianchi E, Narjes F, Fossatelli M, Urbani A, Steinkuhler C, et al. A continuous assay of hepatitis C virus protease based on resonance energy transfer depsipeptide substrates. Anal Biochem 1996; 240 (1):60-7, herein incorporated by reference with regard to performing such assay.
• FRET fluorescence resonance energy transfer
• NS3 protease domains were pre-incubated at 37° C. for 10 minutes with 20 ⁇ M isogenic NS4A peptide cofactors (Sigma, St. Louis, Mo.), in 40% glycerol buffer with 50 mM HEPES pH 7.5 and 10 mM DTT.
• Compounds were diluted serially 1:3 in DMSO, incubated with the enzyme/cofactor mixture for 10 minutes and reactions were started by the addition of 2 ⁇ M RET 51 substrate (final concentration). Fluorescence increase was measured continuously over one hour using a Victor3 V fluorescence plate reader (Perkin Elmer, Waltham, Mass.).
• NS3 protease is complexed with NS4A peptide and then incubated with serial dilutions of the compounds (DMSO used as solvent). Reactions are started by addition of dual-labeled peptide substrate and the resulting kinetic increase in fluorescence is measured. Non-linear regression of velocity data is performed to calculate IC 50 s.
• Activity is initially tested against genotype 1b protease. Depending on the potency obtained against genotype 1b, additional genotypes (1a, 2a, 3) and or protease inhibitor resistant enzymes (D168Y, D168V, or Al 56T mutants) may be tested. BILN-2061 is used as a control during all assays. Compounds of the Examples were evaluated in this assay and were found to have IC 50 values of less than about 1 ⁇ M.
• Huh-luc cells stably replicating Bartenschlager's 1389luc-ubi-neo/NS3-3′/ET genotype 1b replicon
• DMSO is used as solvent
• Replicon copy number is measured by bioluminescence and non-linear regression is performed to calculate EC 50 s.
• Parallel plates treated with the same drug dilutions are assayed for cytotoxicity using the Promega CellTiter-Glo cell viability assay.
• compounds may be tested against a genotype 1a replicon and/or inhibitor resistant replicons encoding D168Y or Al 56T mutations.
• BILN-2061 is used as a control during all assays. Compounds of the Examples were evaluated in this assay and were found to have EC 50 values of less than about 5 ⁇ M.
• Replicon assays are conducted in normal cell culture medium (DMEM+10% FBS) supplemented with physiologic concentrations of human serum albumin (40 mg/mL) or ⁇ -acid glycoprotein (1 mg/mL). EC 50 s in the presence of human serum proteins are compared to the EC 50 in normal medium to determine the fold shift in potency.
• DMEM+10% FBS normal cell culture medium
• human serum albumin 40 mg/mL
• ⁇ -acid glycoprotein 1 mg/mL
• the inhibition of mammalian proteases including Porcine Pancreatic Elastase, Human Leukocyte Elastase, Protease 3, and Cathepsin D are measured at K m for the respective substrates for each enzyme.
• IC 50 for each enzyme is compared to the IC 50 obtained with NS3 1b protease to calculate selectivity.
• MT4 cells are treated with serial dilutions of compounds for a five day period.
• Cell viability is measured at the end of the treatment period using the Promega CellTiter-Glo assay and non-linear regression is performed to calculate CC 50 .
• Huh-luc cultures are incubated with compound at concentrations equal to EC 50 .
• cells are washed 2 ⁇ with cold medium and extracted with 85% acetonitrile; a sample of the media at each time-point is also extracted.
• Cell and media extracts are analyzed by LC/MS/MS to determine the molar concentration of compounds in each fraction.
• Solubility is determined by taking an aliquot of 10 mM DMSO stock solution and preparing the compound at a final concentration of 100 ⁇ M in the test media solutions (PBS, pH 7.4 and 0.1 N HCl, pH 1.5) with a total DMSO concentration of 1%.
• the test media solutions are incubated at room temperature with shaking for 1 hr.
• the solutions are then centrifuged and the recovered supernatants are assayed on the HPLC/UV. Solubility can be calculated by comparing the amount of compound detected in the defined test solution compared to the amount detected in DMSO at the same concentration.
• the stability of compounds after 1 hour incubation in the test media at 37° C. is also determined.
• hepatocyte suspensions 100 ⁇ L, 80,000 cells per well
• Cryopreserved hepatocytes are reconstituted in the serum-free incubation medium.
• the suspension is transferred into 96-well plates (50 ⁇ L/well).
• the compounds are diluted to 2 ⁇ M in incubation medium and then are added to hepatocyte suspensions to start the incubation. Samples are taken at 0, 10, 30 and 60 minutes after the start of incubation and reaction can be quenched with a mixture consisting of 0.3% formic acid in 90% acetonitrile/10% water.
• the concentration of the compound in each sample is analyzed using LC/MS/MS.
• the disappearance half-life of the compound in hepatocyte suspension is determined by fitting the concentration-time data with a monophasic exponential equation. The data is also scaled up to represent intrinsic hepatic clearance and/or total hepatic clearance.
• the compounds are added to the S9 suspension to start the incubation.
• Samples are taken at 0, 10, 30, and 60 minutes after the start of incubation.
• the concentration of the compound in each sample is analyzed using LC/MS/MS.
• the disappearance half-life of the compound in S9 suspension is determined by fitting the concentration-time data with a monophasic exponential equation.
• Both forward (A-to-B) and reverse (B-to-A) permeability is measured.
• Caco-2 monolayers are grown to confluence on collagen-coated, microporous, polycarbonate membranes in 12-well Costar Transwell® plates.
• the compounds are dosed on the apical side for forward permeability (A-to-B), and are dosed on the basolateral side for reverse permeability (B-to-A).
• the cells are incubated at 37° C. with 5% CO 2 in a humidified incubator. At the beginning of incubation, at 1 hr and 2 hr after incubation, a 200- ⁇ L aliquot is taken from the receiver chamber and replaced with fresh assay buffer. The concentration of the compound in each sample is determined with LC/MS/MS. The apparent permeability, Papp, is calculated.
• Plasma protein binding is measured by equilibrium dialysis. Each compound is spiked into blank plasma at a final concentration of 2 ⁇ M. The spiked plasma and phosphate buffer is placed into opposite sides of the assembled dialysis cells, which is then rotated slowly in a 37° C. water bath. At the end of the incubation, the concentration of the compound in plasma and phosphate buffer is determined. The percent unbound is calculated using the following equation:
• Cf and Cb are free and bound concentrations determined as the post-dialysis buffer and plasma concentrations, respectively.
• Each compound is incubated with each of 5 recombinant human CYP450 enzymes, including CYP1A2, CYP2C9, CYP3A4, CYP2D6 and CYP2C19 in the presence and absence of NADPH.
• Serial samples can be taken from the incubation mixture at the beginning of the incubation and at 5, 15, 30, 45 and 60 min after the start of the incubation.
• the concentration of the compound in the incubation mixture is determined by LC/MS/MS.
• the percentage of the compound remaining after incubation at each time point is calculated by comparing with the sampling at the start of incubation.
• Compounds are incubated for up to 2 hour in plasma (rat, dog, monkey, or human) at 37° C. Compounds are added to the plasma at final concentrations of 1 and 10:g/mL. Aliquots are taken at 0, 5, 15, 30, 60, and 120 min after adding the compound. Concentration of compounds and major metabolites at each timepoint are measured by LC/MS/MS. Biological data (antiviral potency [EC 50 ] was determined using a Renilla luciferase (RLuc)-based HCV replicon reporter assay—HCV 1b RLuc) for Compound 6 is 0.0045 nM.
• RLuc Renilla luciferase
• Compound 1 and Compound 2 were synthesized by Gilead Sciences (Foster City, Calif.).
• HCV genotype 1b replicon cells Huh-luc were obtained from Reblikon (Mainz, Germany). The replicon in these cells is designated 1389luc-ubi-neo/NS3-3′/ET and encodes a selectable resistance marker (neomycin phosphotransferase) as well as the firefly luciferase reporter gene.
• Huh-luc cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; GIBCO, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, Utah) and 0.5 mg/mL of G-418 (GIBCO). Cells were passaged twice a week and maintained at subconfluent levels.
• DMEM Dulbecco's Modified Eagle's Medium
• FBS fetal bovine serum
• G-418 G-418
• Replicon cells were seeded in 96-well plates at a density of 5 ⁇ 10 3 cells per well in 100 ⁇ L of DMEM culture medium, excluding G-418.
• Compounds 1 and 2 were serially diluted 1:3 in 100% DMSO (Sigma). These serial dilutions were added to the cells at a 1:200 dilution to achieve a final concentration of 0.5% DMSO in a total volume of 200 mL. Plates were incubated at 37° C. for 3 days, after which culture media were removed and cells were lysed and assayed for luciferase activity using a commercial luciferase assay (Promega, Madison, Wis.).
• Replicon cells were seeded in 96-well plates at a density of 5 ⁇ 10 3 cells per well in 100 ⁇ L of culture medium.
• Compounds 1 and 2 were serially diluted in 100% DMSO as described above and added in a matrix format to 96-well plates, achieving a defined set of different drug concentrations and ratios in a final volume of 2004 and a final DMSO concentration of 0.5%.
• the EC 50 value was selected as the midpoint for the concentration range tested.
• Cells were incubated for three days and analyzed for luciferase expression as indicated above. For the combination study, two independent experiments were performed in triplicate.
• the software calculates theoretical inhibition assuming an additive interaction between drugs (based on the Bliss Independence model) and quantifies statistically significant differences between the theoretical and observed inhibition values. Plotting these differences in three dimensions results in a surface where elevations in the Z-plane represent antiviral synergy and depressions represent antiviral antagonism between compounds. The calculated volumes of surface deviations are expressed in nM 2 %. Per Prichard and Shipman, combination effects are defined as:
• Compound 1 and Compound 3 were synthesized by Gilead Sciences (Foster City, Calif.). Ribavirin and IFN- ⁇ were purchased from Sigma (St. Louis, Mo.).
• HCV genotype 1b replicon cells Huh-luc were obtained from Reblikon (Mainz, Germany). The replicon in these cells is designated 1389luc-ubi-neo/NS3-3′/ET and encodes a selectable resistance marker (neomycin phosphotransferase) as well as the firefly luciferase reporter gene.
• Huh-luc cells were maintained in Dulbecco's Modified Eagle Medium (D-MEM) with GlutaMAXTM (Invitrogen, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, Utah) and 0.5 mg/mL of G-418 (Invitrogen). Cells were passaged twice a week and maintained at subconfluent levels.
• Replicon cells were seeded in 96-well plates at a density of 5 ⁇ 10 3 cells per well in 100 ⁇ L of DMEM plus 10% FBS culture medium, excluding G-418. Compounds were serially diluted 1:3 in 100% DMSO (Sigma). These serial dilutions were added to the cells at a 1:200 dilution to achieve a final concentration of 0.5% DMSO in a total volume of 200 mL. Plates were incubated at 37° C. for 3 days, after which culture media were removed and cells were lysed and assayed for luciferase activity using a commercial luciferase assay (Promega, Madison, Wis.).
• Replicon cells were seeded in 96-well plates at a density of 5 ⁇ 10 3 cells per well in 10:L of culture medium, excluding G-418.
• Compound 3 and other compounds were serially diluted in 100% DMSO as described above and added in a matrix format to 96-well plates, achieving a defined set of different drug concentrations and ratios in a final volume of 200:L and a final DMSO concentration of 0.5%.
• the EC 50 value was selected as the midpoint for the concentration range tested.
• Ribavirin which did not have a selective antiviral effect, a top dose of 6.2:M was selected since this was approximately 3-fold below the concentration at which cytotoxicity started to be observed.
• Cells were incubated with drugs for three days and analyzed for luciferase expression as indicated above. For each combination study, two independent experiments were performed in triplicate.
• Compound 1 Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 were synthesized by Gilead Sciences (Foster City, Calif.). Puromycin, IFN- ⁇ and Ribavirin were purchased from Sigma (St. Louis, Mo.). Calcein AM was purchased from Anaspec (Fremont, Calif.).
• the HCV genotype 1a replicon cell line used in this study was described previously.
• the cells were grown in cell culture medium containing Dulbecco's Modified Eagle Medium (DMEM) with GlutaMAX (Gibco, Carlsbad, Calif., Cat#10569-044), supplemented with 10% FBS (HyClone, Logan, Utah, Cat#SH30071.03), 100 Units/mL Penicillin, 100:g/mL Streptomycin (Gibco, Carlsbad, Calif., Cat#15140-122), and 0.1 mM non-essential amino acids (Gibco, Carlsbad, Calif., Cat#11140-050).
• Replicon cells were maintained in 0.5 mg/mL Geneticin (Invitrogen, Carlsbad, Calif., Cat#10131-035) to prevent the loss of HCV replicon. The cells were passaged every 3-4 days before reaching confluency.
• one compound was serially diluted in nine steps of 1:2 dilutions toward the horizontal direction with the other compound serially diluted in seven steps of 1:2 dilutions toward the vertical direction. This achieved a defined set of different drug concentrations and ratios. For each individual drug, the EC 50 value was selected as the midpoint for the concentration range tested. All serial dilutions were performed in four replicates per compound within the same 384-well plate. 100% DMSO was added into column 1-2 of each serial dilution 384-well plate. A HCV protease inhibitor ITMN-191 at 100 ⁇ M was added into column 23 as a control of 100% inhibition of HCV replication while puromycin at 10 mM was added into column 24 as a control of 100% cytotoxicity.
• the HCV replicon assay was a multiplex assay which can assess both cytotoxicity and anti-replicon activity from the same well.
• the CC 50 assay was performed first.
• the media in the 384-well cell culture plate was aspirated and the wells were washed four times with 100 ⁇ l ⁇ PBS each, using a Biotek ELX405 plate washer.
• a volume of 50 ⁇ L. of a solution containing 400 nM calcein AM (Anaspec, Fremont, Calif., Cat#25200-056) in 1 ⁇ PBS was added to each well of the plate with a Biotek ⁇ Flow Workstation.
• the plate was incubated for 30 minutes at room temperature before the fluorescence signal (excitation 490 nm, emission 520 nm) was measured with a Perkin Elmer Envision Plate Reader.
• EC 50 assay was performed in the same wells as CC 50 assay.
• the calcein-PBS solution in the 384-well cell culture plate was aspirated with a Biotek ELX405 plate washer.
• a volume of 20 ⁇ L of Dual-Glo luciferase buffer (Promega, Madison, Wis., Cat#E298B) was added to each well of the plate with a Biotek ⁇ Flow Workstation. The plate was incubated for 10 minutes at room temperature.
• a volume of 20 ⁇ L of a solution containing 1:100 mixture of Dual-Glo Stop & Glo substrate (Promega, Madison, Wis., Cat#E313B) and Dual-Glo Stop & Glo buffer (Promega, Madison, Wis., Cat#E314B) was then added to each well of the plate with a Biotek ⁇ Flow Workstation. The plate was then incubated at room temperature for 10 minutes before the luminescence signal was measured with a Perkin Elmer Envision Plate Reader.
• X c is the fluorescence signal from the compound-treated well
• M B is the average fluorescence signal from puromycin-treated wells
• M D is the average fluorescence signal from DMSO-treated wells.
• the anti-HCV replication activity was determined by the luminescence signal generated from the reporter renilla luciferase of the HCV replicon. The percent inhibition on HCV replicon was calculated using equation 1, where X c is the luminescence signal from compound-treated well; M B is average luminescence signal from the ITMN-191-treated wells; M D is the average luminescence signal from DMSO-treated wells.
• the CC 50 values were determined as the testing compound concentration that caused a 50% decrease of cell viability.
• the EC 50 values were determined as the testing compound concentration that caused a 50% decrease in HCV replication. Both CC 50 and EC 50 values were obtained using Pipeline Pilot 5.0 software package (Accelrys, San Diego, Calif.) by nonlinear regression fitting of experimental data to equation 2:
• y is the observed % inhibition of HCV replicon at x concentration of compound
• d is estimated response at zero compound concentration
• a is estimated response at infinite compound concentration
• c is the mid-range concentration (CC 50 or EC 50 );
• b is the Hill slope factor.
• Moderate antagonism ⁇ 50 and > ⁇ 100 nM 2 %
• Synergy and antagonism volumes (nM 2 %) calculated from deviations from additive surface are summarized in Table VII. At 95% confidence interval, the mean synergy and antagonism volumes are between 25 and ⁇ 25 nM 2 % when Compound 4 was combined with IFN- ⁇ , Compound 2 and Compound 6, indicative of additive interaction with those compounds. Furthermore, Compound 4 shows synergy volumes in the range of 25 to 50 nM 2 % when combined with Compound 1, Compound 5 or Compound 3, suggesting minor synergistic interaction.
• Compound 4 shows an antagonism volume of ⁇ 43 nM 2 % when combined with Ribavirin, suggesting a minor antagonistic interaction.
• the Ribavirin concentration that shows the highest antagonism with Compound 4 is around 0.5 to 1 ⁇ M, which is about 10-fold lower than the steady-state plasma concentration of Ribavirin (6-11 ⁇ M) observed in human at a dose of 800 mg/day.
• the antagonistic interaction between Ribavirin and Compound 4 is minimal across a wide range of Compound 4 concentrations (0-0.44 ⁇ M). Therefore, the observed minor antagonism between Ribavirin and Compound 4 in the in vitro replicon system is unlikely to have clinical significance.
• Compound 4 in a diastereomeric mixture was tested in combination with the current standard of care (IFN- ⁇ /Ribavirin), as well as Gilead Sciences' internal developmental candidates Compound 1 and Compound 5 (non-nucleoside NS5B inhibitors), Compound 2 and Compound 3 (NS3 protease inhibitors), and Compound 6 (NS5A inhibitor).
• Compound 4 showed additive antiviral activity in combination with IFN- ⁇ , Compound 2 and Compound 6 and minor synergy with Compound 1, Compound 5 and Compound 3.
• the antiviral activity of Compound 5 was tested in GT-1b Huh-lunet cells (using substantially the same methods as in the assays for Compound 4) in combination with the internal developmental compounds Compound 1, Compound 2 and Compound 3 (NS3 protease inhibitors), Compound 6 (NS5A inhibitor), Compound 4 (C-nuc NS5B inhibitor) and also the approved HCV therapeutics PEG-IFN- ⁇ and Ribavirin.
• Combination data were analyzed based on the Bliss Independence model using MacSynergy II software. Results of the combination assays were expressed as mean synergy and antagonism volumes (nM 2 ) calculated at 95% confidence from two independent experiments performed in triplicate. Combination effects are defined as:
• Compound 1 Compound 2, Compound 3, Compound 6 and Compound 7 were synthesized by Gilead Sciences (Foster City, Calif.). IFN- ⁇ and Ribavirin were purchased from Sigma (St. Louis, Mo.).
• HCV genotype 1b replicon cells Huh-luc were obtained from Reblikon (Mainz, Germany). The replicon in these cells is designated 1389luc-ubi-neo/NS3-37ET and encodes a selectable resistance marker (neomycin phosphotransferase) as well as the firefly luciferase reporter gene.
• Huh-luc cells were maintained in Dulbecco's Modified Eagle's Medium GlutaMax (DMEM; Invitrogen, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, Utah), 1 ⁇ penicillin/streptomycin, 1 ⁇ nonessential amino acids and 0.5 mg/mL of G-418 (all from Invitrogen, Carlsbad, Calif.). Cells were passaged twice a week and maintained at subconfluent levels.
• DMEM Dulbecco's Modified Eagle's Medium GlutaMax
• FBS fetal bovine serum
• penicillin/streptomycin 1 ⁇ nonessential amino acids
• G-418 all from Invitrogen, Carlsbad, Calif.
• Replicon cells were seeded in 96-well plates at a density of 7 ⁇ 10 3 cells per well in 100 ⁇ A of DMEM culture medium, excluding G-418. Compounds were serially diluted 1:2 in 100% DMSO. Serial dilutions were added to the cells at a 1:200 dilution to achieve a final concentration of 0.5% DMSO in a total volume of 200 ⁇ L. Plates were incubated at 37° C. for 3 days, after which culture media were removed and cells were lysed and assayed for luciferase activity using a commercial luciferase assay (Promega, Madison, Wis.).
• Replicon cells were seeded in 96-well plates at a density of 7 ⁇ 10 3 cells per well in 100 ⁇ L, culture medium, excluding G-418.
• Compound 6 and other compounds were serially diluted 1:2 in 100% DMSO and added in a matrix format to 96-well plates, achieving a defined set of different drug concentrations and ratios in a final volume of 200 ⁇ L, and a final DMSO concentration of 0.5%.
• the EC 50 value was selected as the midpoint for the concentration range tested.
• Cells were incubated for 3 days and analyzed for luciferase expression using a commercial luciferase assay (Promega). For each combination study, two independent experiments were performed in triplicate.
• Replicon cells were seeded and treated with drugs as described for the antiviral combination studies above. After three day incubation at 37° C., the culture media was removed and cells were lysed and assayed for cytotoxicity using a CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions. Relative Light Units were converted into percentages relative to the untreated controls (defined as 100%).
• luciferase levels in drug-treated samples were expressed as a percentage of those in untreated controls (defined as 100%).
• EC 50 values were calculated by nonlinear regression analysis of replicate data sets using XLfit 4 software (IDBS, Emeryville, Calif.).
• luciferase levels in drug-treated samples were expressed as a percentage of those in untreated controls (defined as 100%).
• Replicate data sets were then analyzed using the MacSynergy II program developed by Prichard and Shipman.
• the software (MacSynergyTM II, University of Michigan, Mich.) calculates theoretical inhibition assuming an additive interaction between drugs (based on the Bliss Independence model) and quantifies statistically significant differences between the theoretical and observed inhibition values. Plotting these differences in three dimensions results in a surface where elevations in the Z-plane represent antiviral synergy and depressions represent antiviral antagonism between compounds. The calculated volumes of surface deviations are expressed in nM 2 %. Per Prichard and Shipman, combination effects are defined as:
• HCV genotype 1b replicon cells Huh-luc were obtained from Reblikon (Mainz, Germany). The replicon in these cells is designated 1389luc-ubi-neo/NS3-3′/ET and encodes a selectable resistance marker (neomycin phosphotransferase) as well as the firefly luciferase reporter gene.
• Huh-luc cells were maintained in Dulbecco's Modified Eagle's Medium GlutaMax (DMEM; Invitrogen, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, Utah), 1 ⁇ penicillin/streptomycin, 1 ⁇ nonessential amino acids and 0.5 mg/mL of G-418 (all from Invitrogen, Carlsbad, Calif.). Cells were passaged twice a week and maintained at subconfluent levels.
• DMEM Dulbecco's Modified Eagle's Medium GlutaMax
• FBS fetal bovine serum
• penicillin/streptomycin 1 ⁇ nonessential amino acids
• G-418 all from Invitrogen, Carlsbad, Calif.
• Genotype 1b replicon cells were seeded in T-75 flasks at a cell density of 2.5 ⁇ 10 5 cells/ flask, excluding G418. Compounds were individually added to the cells at variable concentrations: Compound 6 was added at concentrations of either 1 pM, 2 pM, 4 pM, 6 pM, 8 pM, or 12 pM, Compound 4 was added at 125 nM, 250 nM, 375 nM, 500 nM or 1000 nM, Compound 1 was added at 1.25 nM, 2.5 nM, 5 nM, 2.75 nM or 10 nM, and Compound 3 was added at concentrations of 3.75 nM, 7.5 nM, 11.25 nM, 15 nM, 30 nM or 60 nM. Flasks were incubated at 37° C., media and compounds were refreshed every two days. After 6 days of incubation the replicon cells were collected for RNA extraction and replicon
• Genotype 1b replicon cells were seeded in T-75 flasks at a density of 2.5 ⁇ 10 5 cells/flask. Compounds were added to the T-75 flasks at the following concentrations: Compound 6 at 4 pM, Compound 4 at 1000 nM, Compound 1 at 10 nM, and Compound 3 at 26.25 nM. Flasks were incubated at 37° C. and compounds and media were refreshed every two days. All experiments were performed in duplicate and will be noted in as flask 1 and flask 2.
• the Qiagen One-step QRT-PCR kit was used according to manufacturer's protocol (Qiagen, Valencia Calif.).
• the genotype 1b HCV NS3 gene specific primers, forward primer NS3 — 180FL 5′-CGGCGGACTGTCTATCATGGTGC[FAM]G-′3(SEQ ID NO: 1) and reverse NS3 — 180 5′-GGTCCTGGTCCACATTGGTGT-′3(SEQ ID NO: 2) and 18S rRNA LUXTM [FAM] endogenous control primer set (115HM-01) were produced by Invitrogen corporation (Carlsbad, Calif.).
• the reverse transcriptase step the reactions were incubated at 44° C. for 30 min, the reverse transcriptase enzyme was then degraded by heating the sample to 94° C. for 10 min.
• the Q-PCR step included 38 cycles at 94° C. for 15 s and 58° C. for 30 s.
• RNA log drop is relative to the RNA levels in DMSO control treated replicon cells maintained for 6 days.
• the replicon RNA suppression by compounds Compound 6, Compound 4, Compound 1 and Compound 3 was determined in a 6 day assay as individual compounds and in various double, triple, and quadruple combinations.
• the replicon RNA log drop is relative to the RNA levels in DMSO control treated replicon cells maintained for 6 days alongside the treatment flasks.
• the ability of the various compound combinations to cure the cells from the HCV replicon was determined by colony formation. Colony formation occurred after compound treatment was removed and G418 pressure was returned for 14 days. If a compound combination completely cures the cell population from the HCV replicon no colonies will develop since the cells lack resistance to G418.
• results of these in vitro experiments indicate that combination of two compounds increases the viral RNA log drop over 6 day treatment and increases the rate of cured replicon cells.
• the dual combinations of Compound 6 with Compound 4 or Compound 3 results in larger replicon RNA log suppression and lowest number of uncured colonies compared to all other dual compound combinations.
• the combination of three or four compounds cures all replicon cells and the combination treatments suppress the replicon RNA levels to the assay limit of detection.
• HCV genotype 1a replicon cells Huh7-lunet were obtained from ReBLIkon GmbH (Mainz, Germany). The replicon in these cells is designated pCon1/SG-hRluc-Neo and encodes a selectable resistance marker (neomycin phosphotransferase) as well as the Renilla reniformis reporter gene. (Ref: Robinson M, et al. (2010) Novel Hepatitis C Virus Reporter Replicon Cell Lines Enable Efficient Antiviral Screening against Genotype 1a. Antimicrob. Agents Chemother. 54(8):3099-3106.).
• DMEM Dulbecco's Modified Eagle's Medium GlutaMax
• FBS fetal bovine serum
• penicillin/streptomycin 1 ⁇ penicillin/streptomycin
• lx nonessential amino acids all from Invitrogen, Carlsbad, Calif.
• Genotype 1a replicon cells were seeded in T-75 flasks at a cell density of 10 6 cells/ flask. Compounds were added to the cells at variable concentrations, corresponding to 1 ⁇ EC 50 , 2 ⁇ EC 50 , 3 ⁇ EC 50 , 10 ⁇ EC 50 , or 100 ⁇ EC 50 . Flasks were incubated at 37° C. and 5% CO 2 ; media and compounds were refreshed every three-four days. Cells were split every time when 90-95% confluence was reached. For each passage, 10 6 cells were maintained in a new flask with fresh media and compounds and at least 1 ⁇ 10 6 cells were collected and stored at ⁇ 80° C. for RNA extraction and subsequent HCV specific QRT-PCR analysis. Cells were plated on a 10-cm tissue culture dishes in the presence of G418 for at least 14 days to record colony formation of uncured replicon cells.
• MultiCode-RTx PCR technology primers targeting HCV 3′ UTR EraGen Biosciences, Madison, Wis.
• HCV detection and quantification Ref: Mulligan, E. K., et al (2009) Detection and Quantification of Hepatitis C Virus by MultiCode-RTx Real-Time PCR Targeting the HCV 3′ Untranslated Region. Journal of Clin Micro. 47 (8): 2635).
• RNA was used along with SuperScript III RT (Invitrogen, Carlsbad, Calif.), Titanium Taq DNA Polymerase (Clontech, Mountain View, Calif.), and 2 ⁇ ISOlution (EraGen Biosciences, Madison, Wis.).
• the assay was performed using Roche LightCycler 480 (Indianapolis, Ind.).
• the reverse transcriptase step the reactions were incubated at 50° C. for 15 min.
• the DNA polymerase was activated by heating the sample to 95° C. for 2 min.
• Q-PCR consisted of 50 cycles at 95° C. for 5 s, 58° C. for 10 s and 72° C. for 20 s.
• the replicon RNA suppression by compounds Compound 5, Compound 6, Compound 3 and Compound 1 was determined as individual compounds and in various double, triple, and quadruple combinations.
• the replicon RNA log drop is relative to the RNA levels in DMSO control treated replicon cells maintained alongside the treatment flasks.
• the ability of the various compound combinations to cure the cells from the HCV replicon was determined by colony formation. Colony formation occurred after compound treatment was removed and G418 pressure was returned for 14 days. If a compound combination completely cures the cell population from the HCV replicon no colonies will develop since the cells lack resistance to G418.
• results of these in vitro experiments indicate that combination of two compounds increases the viral RNA log drop and increases the rate of cured replicon cells.
• the combination of three or four compounds at 2 ⁇ EC50 or 3XEC50 cures all replicon cells and the combination treatments suppress the replicon RNA levels to the assay limit of detection.
• GT1 b and GT1 a replicons carrying Renilla Luciferase reporter were used to generate the mutant replicons.
• the mutations were introduced into the replicon construct by site-directed mutagenesis and confirmed by sequencing.
• Mutant replicon RNAs were generated from DNA by in vitro transcription and transfected into Huh-7 Lunet or Cl cells
• Huh-7 Cells following transfection of replicon RNA were seeded in 96-well plates at a density of 5 ⁇ 10 3 cells per well in 100 ⁇ l of DMEM culture medium. Compounds were serially diluted 1:3 in 100% DMSO (Sigma). These serial dilutions were added to the cells at a 1:200 dilution to achieve a final concentration of 0.5% DMSO in a total volume of 200 ⁇ L. Plates were incubated at 37° C. for 3 days, after which culture media were removed and cells were lysed and assayed for luciferase activity using a commercial luciferase assay (Promega, Madison, Wis.). EC 50 values were calculated using Prism.
• Table XVII and XVIII summarize the fold change in EC50 of the mutants compared to the corresponding wild-type GT1 b or GT1 a. For comparison, a fold change of 0-3 is considered “sensitive”, a fold change of 3-10 is considered “low”, a fold change of 10-50 is considered “medium” and a fold change of greater than 5 is considered “high”.
• All single NS3 PI-resistant mutants retain full susceptibility to Compound 6, Compound 4, Compound 1, IFN and RBV.
• the dual class mutants that confer resistance to Pl's and NS5A inhibitors were sensitive to Compound 1, Compound 4, Compound 5, IFN and RBV.
• the dual class mutants that confer resistance to Pl's and Compound 1 were sensitive to Compound 6, Compound 4, Compound 5, IFN and RBV.
• the triple class mutants that confer resistance to P1, NS5A and Compound 1 remained susceptible to Compound 4, Compound 5, IFN and RBV.
• This Clinical Example shows that the combination of Compound 1 and Compound 2 plus ribavirin is more effective at reducing HCV viral load, and suppressing HCV viral rebound, than the combination of Compound 1 plus Compound 2 without ribavirin.
• Subjects in Arm 1 received Compound 2 at 75 mg+Compound 1 at 40 mg, both administered twice daily (BID) (double regimen) and subjects in Arm 2 received Compound 2 at 75 mg+Compound 1 at 40 mg, both administered BID, and plus ribavirin, also administered BID (triple regimen) for 28 days.
• Plasma HCV RNA was monitored approximately twice weekly to gauge virologic response in relation to the protocol-specified criteria for early initiation of PEG/RIBA. From preliminary analysis of the HCV RNA values, the median maximal decline in HCV RNA was 3.9 log 10 IU/mL for the dual regimen and 5.0 log 10 IU/mL for the triple regimen. The median time to maximal decline in HCV RNA was 7 days for the dual regimen and 14 days for the triple regimen, with the difference attributed to delayed incidence and onset of viral breakthrough in the ribavirin containing arm. Three of 15 (20%) subjects receiving the dual regimen and 10 of 13 (77%) subjects receiving the triple regimen had nadir HCV RNA values 5 ⁇ 30 IU/mL (excluding non-GT1 subjects).
• Compound 2+Compound 1 alone and in combination with RIBA were well-tolerated for up to 28 days by HCV subjects in this study, both before and following the addition of PEG or PEG/Ribavirin. Both regimens yielded potent suppression of HCV RNA, with greater and more sustained activity in the three drug regimen.
• Subjects 1011, 1012, and 1043 at one French study center were excluded; Subject 1004 was not excluded **Breakthrough defined as >1 log increase in HCV RNA above nadir value or HCV RNA >25 IU/mL following a nadir of ⁇ 25 IU/mL
• the mean time to HCV breakthrough which is a measure of the eventual increase in HCV viral load as the virus mutates and becomes less susceptible to the antiviral drugs, is greater in the presence of ribavirin than in the absence of ribavirin. Further, the number of subjects showing viral breakthrough is substantially less in the presence of ribavirin than in the absence of ribavirin.

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