Title:  Combination therapy for eradicating detectable HCV-RNA in antiviral treatment naive patients having chronic hepatitis C infection

United States Patent:  6,472,373

Issued:  October 29, 2002

Inventors:  Albrecht; Janice K. (Winter Park, FL)

Assignee:  Schering Corporation (Kenilworth, NJ)

Appl. No.:  311487

Filed:  May 13, 1999

Methods for treating an antiviral treatment naive patient having chronic hepatitis C infection to eradicate detectable HCV-RNA involving administering a therapeutically effective amount of a combination therapy of ribavirin and interferon-alpha for a time period of from 20 up to 50 weeks are disclosed.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, it has been found that, in the case of antiviral treatment naive patients having chronic hepatitis C infection and having HCV genotype 1, or such naive patients having HCV genotype 1 and a viral load of greater than 2 million copies per ml of HCV-RNA by quantitative PCR ("qPCR"), combination therapy with a therapeutically effective amount of ribavirin and a therapeutically effective amount of interferon alpha for a time period of at least 20 to 30 weeks results in ten times more patients having no detectable HCV-RNA in their serum at least 24 weeks after termination of therapy compared to by interferon-alpha monotherapy. When the combination therapy is extended to a time period of 40 to 50 weeks, two to three times more patients have no detectable HCV-RNA in their serum at least 24 weeks after termination of combination therapy compared to those treated with the combination therapy for 24 weeks and eight to nine times more patients have no detectable HCV-RNA in their serum at least 24 weeks after termination of combination therapy compared to those treated with interferon-alpha monotherapy for 48 weeks. See Tables 6, 14, 16 & 17, the rate of sustained virologic response found after using the combination therapy of the present invention depends upon the HCV genotype and the base line viral load as measured by HCV-RNA/qPCR as well as the treatment period of the combination therapy for HCV genotype 1. See Tables 13 & 15. The treatment period of the combination therapy for antiviral treatment naive patients having chronic HCV genotypes 4, 5 and 6 infections is the same as antiviral treatment naive patients having chronic naive patients having chronic HCV genotype 1. The treatment period of the combination therapy for antiviral treatment naive patients having HCV genotypes 2 and/or 3 is shorter, namely 20 to 30 weeks, preferably 24 weeks. See Tables 7, 13 & 15.

The term "interferon alpha" as used herein means the family of highly homologous species-specific proteins that inhibit viral replication and cellular proliferation and modulate immune response. Typical suitable interferon-alphas include, but are not limited to, recombinant interferon alpha-2b such as Intron-A interferon available from Schering Corporation, Kenilworth, N.J., recombinant interferon alpha-2a such as Roferon interferon available from Hoffmann-La Roche, Nutley, N.J., recombinant interferon alpha-2c such as Berofor alpha 2 interferon available from Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, Conn., interferon alpha-n1, a purified blend of natural alpha interferons such as Sumiferon available from Sumitomo, Japan or as Wellferon interferon alpha-n1 (INS) available from the Glaxo-Wellcome Ltd., London, Great Britain, or a consensus alpha interferon such as those described in U.S. Pat. Nos. 4,897,471 and 4,695,623 (especially Examples 7, 8 or 9 thereof) and the specific product available from Amgen, Inc., Newbury Park, Calif., or interferon alpha-n3 a mixture of natural alpha interferons made by Interferon Sciences and available from the Purdue Frederick Co., Norwalk, Conn., under the Alferon Tradename. The use of interferon alpha-2a or alpha 2b is preferred. Since interferon alpha 2b, among all interferons, has the broadest approval throughout the world for treating chronic hepatitis C infection, it is most preferred. The manufacture of interferon alpha 2b is described in U.S. Pat. No. 4,530,901.

The interferon alpha administered is selected from interferon alpha-2a, interferon alpha-2b, a consensus interferon, a purified interferon alpha product or a pegylated interferon-alpha-2a or pegylated interferon alpha-2b.

The therapeutically effective amount of interferon alpha-2a, interferon alpha-2b, or a purified interferon alpha administered in association with ribavirin is from 2 to 10 million IU per week on a weekly, TIW, QOD or daily basis.

The therapeutically effective amount of interferon-alpha-2b administered is 3 million IU TIW.

When the interferon alpha administered in association with ribavirin is consensus interferon, the therapeutically effective amount of interferon-alpha administered is from 1 to 20 micrograms per week on a weekly, TIW, QOD or daily basis.

The term "pegylated interferon alpha" as used herein means polyethylene glycol modified conjugates of interferon alpha, preferably interferon alpha-2a and alpha-2b. The preferred polyethylene-glycol-interferon alpha-2b conjugate is PEG₁₂₀₀₀ -interferon alpha-2b. The phrases "12,000 molecular weight polyethylene glycol conjugated interferon alpha" and "PEG₁₂₀₀₀ -IFN alpha" as used herein mean conjugates such as are prepared according to the methods of International Application No. WO 95/13090 and containing urethane linkages between the interferon alpha-2a or -2b amino groups and polyethylene glycol having an average molecular weight of 12000. The pegylated inteferon alpha, PEG₁₂₀₀₀ -IFN-alpha-2b is available from Schering-Plough Research Institute, Kenilworth, N.J.

The preferred PEG₁₂₀₀₀ -interferon alpha-2b is prepared by attaching a PEG polymer to the epsilon amino group of a lysine residue in the interferon alpha-2b molecule. A single PEG₁₂₀₀₀ molecule is conjugated to free amino groups on an IFN alpha-2b molecule via a urethane linkage. This conjugate is characterized by the molecular weight of PEG₁₂₀₀₀ attached. The PEG₁₂₀₀₀ -IFN alpha-2b conjugate is formulated as a lyophilized powder for injection. The objective of conjugation of interferon alpha with PEG is to improve the delivery of the protein by significantly prolonging its plasma half-life, and thereby provide protracted activity of interferon alpha.

Other interferon alpha conjugates can be prepared by coupling an interferon alpha to a water-soluble polymer. A non-limiting list of such polymers include other polyalkylene oxide homopolymers such as polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof. As an alternative to polyalkylene oxide-based polymers, effectively non-antigenic materials such as dextran, polyvinylpyrrolidones, polyacrylamides, polyvinyl alcohols, carbohydrate-based polymers and the like can be used. Such interferon alpha-polymer conjugates are described in U.S. Pat. No. 4,766,106, U.S. Pat. No. 4,917,888, European Patent Application No. 0 236 987, European Patent Application Nos. 0510 356, 0 593 868 and 0 809 996 (pegylated interferon alpha-2a) and International Publication No. WO 95/13090.

Pharmaceutical compositions of pegylated interferon alphasuitable for parenteral administration may be formulated with a suitable buffer, e.g., Tris-HCl, acetate or phosphate such as dibasic sodium phosphate/monobasic sodium phosphate buffer, and pharmaceutically acceptable excipients (e.g., sucrose), carriers (e.g. human plasma albumin), toxicity agents (e.g. NaCl), preservatives (e.g. thimerosol, cresol or benyl alcohol), and surfactants(e.g. tween or polysorbates) in sterile water for injection. The pegylated interferon alpha-may be stored as lyophilized powders under a refrigeration at 2^o-8^o C. The reconstituted aqueous solutions are stable when stored between 2^o and 8^o C. and used within 24 hours of reconstitution. See for example U.S. Pat. Nos, 4,492,537; 5,762,923 and 5,766,582. The reconstituted aqueous solutions may also be stored in prefilled, multi-dose syringes such as those useful for delivery of drugs such as insulin. Typical suitable syringes include systems comprising a prefilled vial attached to a pen-type syringe such as the NOVOLET Novo Pen available from Novo Nordisk, as well as prefilled, pen-type syringes which allow easy self-injection by the user. Other syringe systems include a pen-type syringe comprising a glass cartridge containing a diluent and lyophilized pegylated interferon alpha powder in a separate compartment.

When the interferon-alpha administered in association with ribavirin is a pegylated interferon alpha-2b and the amount of interferon-alpha administered is from 0.5 to 2.0 micrograms/kilogram per week on a weekly, TIW, QOD or daily basis.

When the interferon-alpha administered in association with ribavirin is a pegylated interferon alpha-2a and the amount of interferon-alpha administered is from 20 to 250 micrograms/kilogram per week on a weekly, TIW, QOD or daily basis.

Ribavirin, 1-.beta.-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, available from ICN Pharmaceuticals, Inc., Costa Mesa, Calif., is described in the Merck Index, compound No. 8199, Eleventh Edition. Its manufacture and formulation is described in U.S. Pat. No. 4,211,771.

A person suffering from chronic hepatitis C infection may exhibit one or more of the following signs or symptoms:

(a) elevated ALT,

(b) positive test for anti-HCV antibodies,

(c) presence of HCV as demonstrated by a positive test for HCV-RNA,

(d) clinical stigmata of chronic liver disease,

(e) hepatocelluar damage.

To practice the invention, the combination therapy of interferon alpha and ribavirin are administered to the patient exhibiting one of more of the above signs or symptoms in amounts sufficient to eliminate or at least alleviate one or more of the signs or symptoms. Interferon alpha formulations, including pegylated interferon alpha formulations, are not effective when administered orally, so the preferred method of administering the interferon alpha or pegylated interferon alpha formulations is parenterally, preferably by subcutaneous, IV, or IM, injection. The ribavirin is administered to the patient in association with the interferon alpha, that is, the interferon alpha dose is administered during the same period of time that the patient receives doses of ribavirin. Ribavirin may be administered orally in capsule, tablet or liquid form in association with the parenteral administration of pegylated interferon-alpha. Of course, other types of administration of both medicaments, as they become available are contemplated, such as by nasal spray, transdermally, by suppository, by sustained release dosage form, and by pulmonary inhalation. Any form of administration will work so long as the proper dosages are delivered without destroying the active ingredient.

The term "antivral treatment naive patients" in the context of the present invention means that the patients have never been treated with ribavirin or any interferon including, but not limited to an interferon-alpha.

The term "no detectable HCV-RNA" in the context of the present invention means that there is less than 100 copies of HCV-RNA per ml of serum of the patient as measured by quantitative, multi-cycle reverse transcriptase PCR methodology. HCV-RNA is preferably measured in the present invention by the methodology described below. This methodology is referred to herein as HCV-RNA/qPCR.

RNA is extracted from patient serum using a guaninidium thiocyanate- phenol-chloroform mister followed by ethanol-ammonium acetate precipitation. The precipitated RNA is centrifuged and the resulting pellet is dried in a Centrivap console (Labconco, Kansas City, Mo.). The dry pellet is then resuspended in 30 microliters of an Rnasin (Promega Corp., Madison, Wis.), dithiothritol, and diethylpyrocarbonate-treated water mixture. Samples are kept at or below -20^o C. (preferably below -70^o C.) until RNA reverse transcription (RT) and PCR.

In order to convert the entire RNA sequence into cDNA in the RT reaction, random hexadeoxyribonucleotides (Pharmacia Biotech, Piscataway, N.J.) are used as primers for the first strand cDNA synthesis. Two aliquots of 3 microliters of resuspended sample is added to 3 microliters of 100 ng/.mu.l random primers and denaturated at 70^o C., then reverse transcribed at 40^o C. for one hour using M-MLV reverse transcriptase (USB, Cleveland, Ohio.) in standard buffer containing 5 mM MgCl₂. The final RT reaction volume is 26 .mu.l. The PCR is started immediately following the reverse transcription.

A modified version of the PCR method is performed using heat-stable Taq polymerase to amplify the cDNA. Seventy-five microliters of PCR mix is added to the entire RT reaction volume (26 .mu.l) to a final MgCl₂ concentration of 1.5 mM in a total volume of 101 .mu.l. Each 101 .mu.l sample is then split into 50.5 .mu.l, and a layer of mineral oil is placed on top to prevent evaporation.

The PCR cycle consists of annealing for 90 sec., extension for 90 sec., and denaturation for 90 sec., at 55^o.times., 74^o C. and 94^o C., respectively. Thermocycling samples is submitted to a final 74^o C. extension for 10 minutes. Four different cycle sets are used. By loading the sample in duplicate, and splitting these samples evenly after RT, there are four tubes from one sample. Each of the four tubes is given a different cycle number, enhancing sensitivity and accuracy in the quantitation process. The thermocycling efficiency will be assessed by satisfactory amplification of known copy number RNA standards included in each set of 60 tubes. Two primer sets are used for the amplification, both from the 5' untranslated region of the HCV genome. Both of these primer sets are highly conserved and detect all known subtypes of HCV. Primer set 1: upstream 5'-GTG GTC TGC GGA ACC GGT GAG T-3' (SEQ ID NO:1, downstream-5'-TGC ACG GTC TAC GAG ACC TC-3' (SEQ ID NO:2) which produced a 190 bp product. Primer set 2: upstream 5'-CTG TGA GGA ACT ACT GTC TTC-3' (SEQ ID NO:3), downstream 5'-CCC TAT CAG GCA GTA CCA CAA-3' (SEQ ID NO:4) which produced a 256 bp product.

The amplified cDNA is then electrophorised in 3% agarose gel and transferred to nylon membrane. The target DNA is detected by Southern blotting and immunostaining using a nonradioactive digoxigenin-labeled DNA probe. These procedures are performed using automated instruments for PCR thermocycling, agarose gel electrophoresis, vacuum-transfer Southern blot, hybridization, and immunostaining. Each membrane contains known copy number serially diluted standards which are used to construct standard curves for quantitative measurement of the specimen bands. Originally standard curves are made from carefully diluted HCV-RNA from transcribed clones. Radioactive incorporation studies, gel electrophoresis, and OD 260 are performed on the transcripts to determine that they are of the expected length. After the production of the RNA transcripts quantitated clone standards "pooled" standards are generated which better represent the heterogeneous nature of HCV, one would encounter in natural infection. These pools are made by combining large amounts of serum or plasma from known infected individuals. The serum/plasma pools are calibrated with PCR, against the clone transcripts and then diluted in the known PCR-negative fluids. Finally, the higher copy number samples of the pools are checked against the cDNA Quantiplex nucleic acid detection system from Chiron Inc. (Emeryville, Calif.). These "double quantitated" pools are aliquoted and saved at -70^o C. Dilutions of 5,000,000, 1,000,000, 500,000, 100,000, 10,000, and 1000 copies/ml are used in each experiment.

Each Southem blot membrane is scanned into a computer using an automated scanner/densitometer, at intervals during development to determine when the standard curve is most linear. The resultant electronic images are then measured for band area and mean band density. All of the reading are standardized to integrated band density and compared to the standard curve to obtain a numerical value of viral copy number for each band.

The term "sustained virologic response" as used in the context of the present invention means that there is no detectable HCV-RNA in the serum of patients treated in accordance with the present invention for at least 24 weeks after the end of the combined therapy treatment. Preferably, the period of sustained virologic response is at least one year--or longer--after the end of treatment.

Claim 1 of 19 Claims

What is claimed is:

1. A method of treating antiviral treatment naive patients having chronic hepatitis C ("HCV") infection comprising identifying antiviral treatment naive patients having HCV genotype 1 and an initial viral load of greater than 2 million copies/ mL of serum HCV-RNA as measured by HCV-RNA/ quantitative Polymerase Chain Reaction ("qPCR") and then administering to said antiviral treatment naive patients a therapeutically effective amount of ribavirin and a therapeutically effective amount of interferon-alpha for a time period of about 40 to about 50 weeks and identifying antiviral treatment naive patients having an HCV genotype 1 and an initial viral load of less than or equal to 2 million copies/ mL of serum HCV-RNA as measured by HCV-RNA/ quantitative Polymerase Chain Reaction ("qPCR") and then administering to said antiviral treatment naive patients a therapeutically effective amount of ribavirin and a therapeutically effective amount of interferon-alpha for a time period of about 20 to about 24 weeks.
 

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