CZ2000788A3 - Use of ribavirin for preparing pharmaceutical preparations, use of alpha interferon for preparing pharmaceutical preparations and the pharmaceutical preparations per se - Google Patents

Abstract

The solution provides a way to treat patients with chronic infection hepatitis C, which is resistant to interferonemal therapy, by using a combination of ribavirin and alpha interferons. Combination therapy is more effective than treatment agents individually.

Description

Use of ribavirin for the manufacture of pharmaceutical compositions, use of interferon alpha for the manufacture of pharmaceutical compositions, and pharmaceutical products

Obiast techniky

Chronic hepatitis C infection is an insidious and slowly progressing disease that has a significant impact on quality of life. It may eventually result in liver cirrhosis, liver decompensation or hepatocellular carcinoma.

BACKGROUND OF THE INVENTION

Interferon alpha monotherapy is commonly used to treat chronic hepatitis C infection. However, this treatment is not always effective and sometimes results in unbearable side effects in proportion to the dose and duration of treatment. Ribavirin monotherapy has been suggested for the treatment of chronic hepatitis C infection (Thomas et al., AASLD Abstracts, Hepatology vol. 20, no. 4, part 2, p. 440, 1994). However, it was found that this monotherapy was usually relatively ineffective and had its own undesirable side effects. Combination therapy with interferon alpha and ribavirin has been proposed (Lai et al., Symposium to the 9 ^th Biennial Scientific Meeting of the Asian Pacific Association for the Study of Liver, 1994). Preliminary results suggest that combination therapy may be more effective than either monotherapy. Hayden FG, Schlepushkin, AN. Interferon2a, rimantadine hydrochloride, and ribavirin together inhibit influenza virus replication in vitro. Antimicrob Agents Chemother. 1984; 25: 53-57. Schvarcz R, Ando Y, S'nnerborg A, Weiland O. Combination therapy of chronic hepatitis C with interferon alfa-2b and ribavirin in patients who did not achieve a sustained response to interferon alone: Swedish experience. J Hepatology, 1995; 232 (suppl. 2): 17-21. Brouwer JT, Nevens F, Michielsen P, et al. What are the options if hepatitis C does not respond to interferon? A placebo-controlled test comparing ribavirin monotherapy with its combination with interferon, performed at a multicenter in Benelux. J Hepatol. 1994; 212 (suppl. 1): S17. Abstract WP2 / 08. Chemello L, Cavalletto L, Bernardinello E et al. Response to ribavirin, interferon and their combination in patients with chronic hepatitis C and its relationship to HCV genotypes. J Hepatol. 1994; 212 (suppl. 1): S12.

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Abstract GS5 / 29. However, no one has described methods that use interferon alpha and ribavirin and are capable of eradicating HCV-RNA efficiently with a long-term effect.

There is a strong need to find a method of treating chronic hepatitis C infection with a combination of interferon alpha and ribavirin, which is capable of eradicating HCV-RNA effectively and in the long term.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of using ribavirin for the manufacture of a medicament for treating patients with chronic hepatitis C infection and eradicating detectable HCV-RNA by a method comprising administering an effective amount of ribavirin in conjunction with an effective amount of interferon alpha in a patient not responding course of treatment with interferon alfa.

In another aspect, the invention provides a method of using interferon alpha for the manufacture of a medicament for treating patients with chronic hepatitis C infection and eradicating detectable HCV-RNA by such a method comprising administering an effective amount of interferon alpha in conjunction with an effective amount of ribavirin in a patient not responding to an earlier course of treatment with interferon aifa.

The invention further provides a method of using both ribavirin and interferon alpha for the manufacture of pharmaceutical compositions for treating patients with chronic hepatitis C infection and eradicating detectable HCV-RNA by such a method comprising administering an effective amount of ribavirin in conjunction with an effective amount of interferon alpha in a non-responder on an earlier course of treatment with interferon alfa.

The pharmaceutical compositions are particularly useful for treating a patient with chronic hepatitis C infection and eradicating detectable HCV-RNA by a method comprising administering an effective amount of ribavirin and an effective amount of interferon alpha for 20 to 30 weeks such that at least 30% of patients detectable HCV-RNA at the end of said 20 to 30 week period, also do not have detectable HCV-RNA at least 24 weeks after the end of administration of said drugs.

Preferably, at least 40% of patients who do not have detectable HCVRNA at the end of said 20-30 week period also do not have detectable HCVRNA at least 24 weeks after the end of administration of said drugs.

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In another embodiment, they can be used to treat a patient with chronic hepatitis C infection and to eradicate detectably HCV-RNA by a method comprising administering an effective amount of ribavirin and an effective amount of interferon alpha for 40 to 50 weeks such that at least 40% of patients who do not have detectable HCVRNA at the end of said 40 to 50 week period, nor does it have detectable HCVRNA at least 24 weeks after the end of administration of said drugs.

Preferably, at least 50% of patients who do not have detectable HCVRNA at the end of said 40 to 50 week period also do not have detectable HCVRNA at least 24 weeks after the end of administration of said drugs.

In another embodiment, they can be used to treat a patient with chronic hepatitis C infection and to eradicate detectably HCV-RNA by a method comprising administering an effective amount of ribavirin and an effective amount of interferon alpha for 60 to 80 weeks such that at least 50% of patients who do not have detectable HCVRNA at the end of said 60-80 week period, nor does it have detectable HCVRNA at least 24 weeks after the end of administration of said drugs.

It is preferred that at least 60% of patients who do not have detectable HCVRNA at the end of said 60-80 week period also do not have detectable HCVRNA at least 24 weeks after the end of administration of said drugs.

In another embodiment, they can be used to treat a patient with chronic hepatitis C infection whose genotype differs from type 1 and has a viral load of less than or equal to 2 million copies per milliliter of serum, as measured by quantitative HCV-RNA PCR, and RNA by such a method comprising administering an effective amount of ribavirin and an effective amount of interferon alpha for 20 to 30 weeks such that at least 60% of patients, and more preferably at least 70% of patients, do not have detectable HCV-RNA at the end of said 20 to 30 week period also has no detectable HCV-RNA at least 24 weeks after the end of administration of said drugs.

It is preferred that at least 80% of patients who do not have detectable HCV-RNA at the end of said 20-30 week period also do not have detectable HCV-RNA at least 24 weeks after the end of administration of said drugs.

In another embodiment, they can be used to treat a patient with chronic hepatitis C infection whose genotype differs from type 1 and has a viral load of more than 2 million copies as measured by HCV-RNA / qPCR, and to detectably detect HCV-RNA by a method that consists of administering an effective amount of ribavirin and an effective amount •

interferon alpha for 20 to 30 weeks such that at least 50% of patients who do not have detectable HCV-RNA at the end of said 20 to 30 week period also do not have detectable HCV-RNA at least 24 weeks after the end of administration of said drugs. It is preferred that at least 60% of patients who do not have detectable HCVRNA at the end of said 20-30 week period also do not have detectable HCVRNA at least 24 weeks after the end of administration of said drugs.

In another embodiment, they can be used to treat a patient with chronic hepatitis C infection of type 1 genotype and having a viral load of less than or equal to 2 million copies as measured by HCV-RNA / qPCR and eradicating detectable HCV-RNA by such a method an effective amount of ribavirin and an effective amount of interferon alpha for 20 to 30 weeks such that at least 30% of patients who do not have detectable HCV-RNA at the end of said 20 to 30 week period also do not have detectable HCV-RNA at least 24 weeks after of medicines. Preferably, at least 40% of patients who do not have detectable HCVRNA at the end of said 20-30 week period also do not have detectable HCVRNA at least 24 weeks after the end of administration of said drugs.

In another embodiment, they can be used to treat a patient with chronic hepatitis C infection of type 1 genotype and having a viral load of more than 2 million copies as measured by HCV-RNA / qPCR, and eradicating detectable HCV-RNA by a method comprising administering an effective amount. ribavirin and an effective amount of interferon alpha for 20 to 30 weeks such that at least 15% of patients who do not have detectable HCV-RNA at the end of said 20 to 30 week period also do not have detectable HCV-RNA at least 24 weeks after the administration of said drugs. Preferably, at least 20% of patients who do not have detectable HCV-RNA at the end of said 20-30 week period also do not have detectable HCV-RNA at least 24 weeks after the end of administration of said drugs.

Preferably, the amount of ribavirin administered is in the range of 400 to 1200 mg per day. Even more preferably, the amount of ribavirin administered is in the range of 800 to 1200 mg per day.

Preferably, the interferon-alpha administered is selected from the group consisting of interferon alpha2a, interferon alpha-2b, consensus interferon, purified interferon alpha product or pegylated (pegyiated) interferon alpha. More preferably, the interferon-alpha to be administered is selected from the group consisting of interferon alpha-2a, interferon alpha-2b or purified. Interferon alpha product and the amount of the product administered, the product of interferon alpha and the amount administered. interferon-alpha is from 2 to 10 million IU per week, administered weekly, TIW, QOD or daily. In a preferred embodiment, the interferon-alpha administered is interferon alpha-2b and the amount of interferon-alpha administered is 3 million IU TIW.

Alternatively, the interferon-alpha administered is a consensus interferon and the amount of interferon-alpha administered is from 1 to 20 µg per week, administered weekly, TIW, QOD, or daily. In another embodiment, the interferon-alpha administered is pegylated interferon alpha-2b and the amount of interferon-alpha administered is from 0.5 to 2.0 pg / kg per week, administered weekly, TIW, QOD, or daily. Alternatively, the interferon-alpha administered is pegylated interferon alpha-2a and the amount of interferon-alpha administered is from 20 to 250 pg / kg per week, administered weekly, TIW, QOD, or daily.

Surprisingly, it has been found in the present invention that, compared to treatment with interferon-alpha alone or ribavirin alone, treatment with a combination of a therapeutically effective dose of ribavirin and a therapeutically effective dose of interferon-alpha for at least 20 to 30 weeks results in 10 more patients. which are not detectable by HCV-RNA in their serum for at least 24 weeks after the end of treatment as compared to either of the two monotherapies.

Detailed description

The term "interferon-alpha" is used herein to refer to a group of highly homologous, species-specific proteins that inhibit viral replication, cell proliferation, and modulate the immune response. Typical suitable interferon-alpha include, but are not limited to, recombinant interferon alpha-2b, such as Intron-A interferon, available from Schering Corporation, Kenilworth, NJ, recombinant interferon alpha-2a, such as Roferon interferon, available from Hoffmann -La Roche, Nutley, NJ, a recombinant interferon alpha-2C, such as interferon Berofor alpha 2, available from Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, CT., Interferon alpha-n1, a purified mixture of natural alpha interferons, such as is Sumiferone, available from Sumitomo, Japan or such as Wellferon interferon alpha-n1 (INS), which is available from Glaxo-Wellcome Ltd., London, UK, or consensus interferon alpha, as described in US Nos. 4,897,471 and 4,695,623 (particularly Examples 7, 8 and 9), and a specific product obtainable from Amgen, Inc, Newbury Park, CA, or interferon alpha-n3, a mixture of natural alpha interferon alpha manufactured by Interferon Sciences and available from Purdue Frederick Co., Norwalk, CT. under the trade name Alferon. The use of interferons alpha-2a or alpha-2b is preferred. Since interferon alfa 2b has the broadest consent for the use of chronic hepatitis C infection of all interferons worldwide, it is most preferred. The preparation of interferon alpha 2b is described in U.S. Pat. No. 4,530,901.

Ribavirin, 1- -D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, obtainable from ICN Pharmaceuticals, Inc., Costa Mesa, California, is described in the ninth edition of Merck Register as Compound No. 5, U.S. Pat. Its production and preparation is described in US No. 4,211,771.

The term "not responding to the previous course of treatment" means that the patient either did not respond completely to the previous course of treatment (commonly referred to in the art as "non-responder") or that the patient responded to the previous course of treatment but subsequently relapsed ( generally referred to as "the person for whom the reaction was not permanent").

The term "difficult to treat patient" refers to a patient who was previously classified as a person who is difficult to respond to treatment; for example because it has a high viral load or because its HCV infection has a genotype that is difficult to treat, such as type 1, for example type 1b. For further details regarding the classification of HCV into individual genotypes, see, for example, "Classification of hepatitis C virus into six major genotypes and senes of subtypes by phylogenetic analysis of the NS-5 region", Simmonds, P. et al., J. Gene. Virol., (1993), 74, 2391-2399 and "A Proposed System for the Nomenclature of Hepatitis C Viral Genotypes", Simmonds, P. et al., Hepatology, 19 (5) (1994), 1321-1323.

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

(a) increased ALT, (b) positive anti-HCV antibody test, (c) presence of HCV demonstrated by positive HCV-RNA test, (d) clinical signs of chronic liver disease, (e) hepatocellular damage.

In a practice of the invention, interferon alpha (here α-IFN) and ribavirin are administered to a patient exhibiting one or more of the above signs or symptoms in amounts sufficient to eliminate or at least alleviate one or more of the above signs or symptoms. . In a preferred embodiment, the combination therapy of the invention is applied to a patient who has not been detectably cleared of HCV-RNA after interferon alpha monotherapy.

Ribavirin is administered to the patient in conjunction with α-IFN, ie, the α-IFN dose is administered during the same period when the patient receives ribavirin doses. Thus, most formulations containing α-IFN are not effective when administered orally, i.e. parenteral administration of α-IFN, preferably by subcutaneous, IV and IM injection, is preferred. Ribavirin may be administered orally in the form of capsules or tablets in conjunction with parenteral administration of α-IFN. Other types of administration of both drugs are contemplated when they become available, such as nasal spray, transdermally, suppositories, sustained release methods, etc. Any form of administration will be effective as long as appropriate dosages are administered without active ingredients would be destroyed.

Detectable HCV-RNA within the scope of the present invention means that less than 100 copies of the patient serum are present in 1 ml of the patient's serum, as measured by reverse transcriptase quantitative PCR. In the present invention, HCV-RNA is preferably measured as described below. This procedure is referred to herein as HCVRNA / qPCR.

RNA is extracted from the patient's serum using guanidium isothiocyanate-phenol-chloroform and then precipitated with ethanol in the presence of ammonium acetate. The precipitated RNA is centrifuged and the resulting sediment dried in a Centrivap apparatus (Labconco, Kansas City, MO). The dry sediment is resuspended in 30 μΙ of a mixture of Rnasin (Promega Corp., Madison, Wl), dithiothreitol and diethylpyrocarbonate treated water. The samples were stored at -20 ° C or lower until reverse RNA (RT) and PCR were performed.

In order to convert the entire RNA sequence into cDNA in the RT reaction, random hexadeoxyribonucleotides (Pharmacia Biotech, Piscataway, NJ) are used as primers to synthesize the first cDNA strand. From the resuspended sample, two 3 μΙ samples were added to 3 μΙ random primers at 100 ng / L, denatured at 70 ° C, then reverse transcribed at 40 ° C for 1 hour using reverse transcriptase M-MLV (USB, Cleveland, OH) in a standard buffer containing 5 mM MgCl ₂ . The final RT reaction volume is 26 L. PCR is started immediately after reverse transcription.

The altered version of the PCR method for amplifying cDNA is performed using a heat stable Taq polymerase. Seventy-five µl of the PCR mixture is added to the entire reaction volume of RT (26 L) to a total final MgCl ₂ concentration of 1.5 mM in a total volume of 101 I. Each 101 L sample is then divided into 50.5 L and a layer is placed mineral oil to prevent evaporation.

The PCR cycle consists of a temperature hybridization for 90 seconds at 55 ° C, an extension phase for 90 seconds at 74 ° C and a denaturation phase for 90 seconds at 94 ° C. For thermocyclic samples, the final extension phase is run at 74 ° C for 7 minutes. Four different sets of cycles are used. By applying the sample in duplicate and halving these samples after RT, four tubes are obtained from each sample. Each of the four tubes underwent a different number of cycles, increasing sensitivity and accuracy in the quantification process. The efficiency of thermocycling will be evaluated by satisfactory amplification of a known number of copies of the standard RNA contained in each set of 60 tubes. Two sets of primers, both from the 5'-untranslated region of the HCV genome, are used for amplification. Both of these two primer sets are highly conserved and detect all known HCV subtypes. Primer Set 1: upstream of 5-GTG GTC TGC GGA ACC GGT GAG T-3 ', downstream of 5-TGC ACG GTC TAC GAG ACC TC-3', which is a 190 base pair product. Primer Set 2: upstream of the 5'-CTG TGA GGA ACT ACT GTC TTC-3 ', downstream of the 5'-CCC TAT CAG GCA GTA CCA CAA-3', which is a 256 base pair product.

The amplified cDNA is then electrophoresed in a 3% agarose gel and transferred to a nylon membrane. Target DNA is detected by Southern blotting and immunostaining using a non-radioactive DNA probe labeled with digoxigenin. These procedures are performed using automatic PCR thermocycling instruments, agerose gel electrophoresis, Southern blot vacuum transfer, hybridization, and immunostaining. Each membrane contains a known number of copies of serially diluted standards, which are used to construct curves for quantitative measurement of zones in samples. The original standard curves are generated from carefully diluted HCV-RNA from transcribed clones. For transcripts, radioactivity incorporation studies, gel electrophoresis, and OD 260 measurements are performed to determine if they are of the expected length. After the production of RNA transcripts from quantified standard clones, "mixed standards" are created that better represent the heterogeneous nature of HCV that can be expected in case of natural infection. These compositions are formed by combining large amounts of serum or plasma from known infected individuals. Serum or plasma mixtures are calibrated by PCR against transcripts from clones and then diluted in a fluid known to be negative in PCR. Finally, those pooled samples that have a high copy number are checked with the Quantiplex nucleic acid detection system cDNA kit from Chiron Inc. (Emeryville, CA). These "double quantified" mixtures were aliquoted and stored at -70 ° C. Dilutions of 5,000,000, 1,000,000, 500,000, 100,000, 10,000, and 1,000 copies / ml are used in each experiment.

Each Southern blot membrane is scanned into a computer using an automatic scanner coupled to a densitometer at intervals during development to determine when the standard curve is most linear. The resulting electronic images are then measured to determine zone areas and average zone density. All readings are standardized to the integrated zone density and compared to a standard curve to obtain a numerical value of the number of viral copies in each zone.

The following clinical protocols were performed:

DETAILED DESCRIPTION OF THE INVENTION

Example 1:

General design and study design

This trial was prospective, performed in many centers, random, double blind and with parallel groups. The study compared treatment with INTRON® A plus ribavirin with treatment with INTRON® A plus placebo for 24 months in patients with compensated chronic hepatitis C infection who responded to previous treatment with one or two cycles of interferon alpha (INTRON® A, Roferon®-A or Wellferon®) (at least 3 MU to a maximum of 6 MU QOD or TIW for a minimum of 20 weeks to a maximum of 18 months) and who experienced a relapse after the last cycle of interferon alpha treatment. Eligible patients had chronic hepatitis C confirmed with HCV-RNA positive serum, liver biopsy and laboratory tests.

Patients were randomly assigned to groups treated with INTRON® A plus ribavirin and INTRON® A plus placebo. The dose of INTRON® A was 3 MU SC TIW; the dose of ribavirin 1000 or 1200 mg PO per day (depending on weight) was divided into two doses. Treatment groups were identified in the same ·

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ratios in the Center for Random Distribution. The random distribution procedure was designed to strike a balance between treatment groups with respect to the presence or absence of cirrhosis in pre-treatment liver biopsies, the level detected by HCV-RNA / qPCR and the HCV genotype.

The treatment method studied was conducted for 24 weeks. The total study period was 48 weeks to determine the long-term effects of the treatment.

Biochemical (ALT), virological (HCV-RNA) and histological (liver biopsy) investigations were performed during and after treatment to assess the nature and duration of response to the treatments studied. The initial efficacy endpoint was a general response, defined as serum HCV-RNA / qPCR loss (<100 copies / ml) measured 24 weeks after the end of treatment, associated with improvement in post-treatment hepatic biopsy as measured by Knodelius histological activity index (HAI). Normalization of ALT was determined as a secondary efficacy variable. The safety of the treatments studied was evaluated by monitoring selected laboratory parameters as well as recording and evaluating the occurrence of any adverse event.

Treatment regimens

The treatment regimens studied were either:

INTRON® A 3 MU SC TIW plus ribavirin 1000 or 1200 mg / day PO divided into two doses for 24 weeks; or INTRON® A 3 MU SC TIW plus placebo instead of ribavirin divided into two doses over 24 weeks.

Study treatment regimens were administered for 24 weeks. The standard INTRON® A (interferon alfa-2b, recombinant) regimen for hepatitis C was administered at a stable dose of 3 MU TIW. Each patient received instructions for the preparation and subcutaneous administration of INTRON® A. Ribavirin was administered twice daily, morning and evening. The dose was determined by the patient's weight at baseline. Patients weighing 75 kg received 1000 mg daily as two 200 mg capsules in the morning and three 200 mg capsules in the evening. Patients weighing> 75 kg received 1200 mg daily as three 200 mg capsules in the morning and evening.

The randomization of the patients into groups was designed to balance the groups with respect to the following basic characteristics:

A. liver histology prior to treatment (cirrhosis or no cirrhosis)

B. serum HCV-RNA / qPCR status (HCV-RNA / qPCR 2,000,000 or HCVRNA / qPCR> 2,000,000 copies / ml); and

C. HCV genotype (1 or other). Patients with mixed genotypes (if they contain type 1) will be classified as type 1 for the purpose of group balance.

Efficiency

Initial for efficacy was a comparison of the two treatment groups with respect to the general extent of response, defined as a decrease in serum HCV-RNA / qPCR within 24 weeks after the end of treatment to undetectable levels or <100 copies / ml, together with improvement in liver status. post-treatment biopsy as defined by Knodeiia's HAI inflammatory index. Secondary signs of efficacy have also been studied:

Secondary signs of efficacy at the end of the test:

D. proportions of patients with normalized ALT after 24 weeks of follow-up;

E. proportions of patients with improved biopsy status (pooled values of categories I + li + III);

F. deviations from baseline values of biopsy indices (pooled values of categories I + II + III);

G. end-of-treatment response ranges based on HCV-RNA / qPCR;

H. proportion of patients with normalized ALT at the end of treatment.

I. Response ranges after 24 weeks of follow-up, based on HCV-RNA / qPCR. Virology: Input state and its changes

Serum testing with HCV-RNA / qPCR was performed in a central laboratory. A positive HCV-RNA test result was required; only HCV-RNA positive patients were eligible. Repeated tests were scheduled for weeks 4, 12, 24, and weeks 12 and 24 after treatment.

The response was rated as defined below:

Respondent: The patient was classified as responding within a given time interval if HCV-RNA / qPCR was negative at this time (<100 copies / ml).

Persistent: The patient was classified as persistent if the patient was a responder within 24 weeks of stopping treatment.

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Note that patients who did not meet these criteria, including those who discontinued participation before the desired HCV-RNA / qPCR score was obtained, were classified as non-responders.

Overall responder: Based on both the serum HCV-RNA / qPCR assay and the change in liver histology evaluated by the Knodelia inflammatory index HAI. A patient was classified as overall responder if he / she was persistent and at the same time his / her Knodelia inflammatory HAI (sum of categories I + II + III) improved by 2 or more units relative to the value determined prior to treatment.

Histology of the liver

Liver biopsies were obtained within six months prior to recruitment and at week 24 following treatment discontinuation. Biopsy assessment was performed by one pathologist using Knodelia histological activity classification. The pathologist was unaware of the patient's identity, treatment group, and biopsy collection time with respect to the treatment period (before and after treatment). The efficacy of the treatments studied was evaluated by comparing the degree of inflammatory activity observed at baseline with the inflammatory activity present at week 24 following treatment discontinuation.

Results.

195 patients were enrolled at the 31st International Center and randomized for treatment with either INTRON® A plus ribavirin (N = 98) or INTRON® A plus placebo (N = 97). Three patients, two randomly assigned to receive INTRON® A plus ribavirin, and one randomly assigned to receive INTRON® A plus placebo, were not treated; thus, all treatment groups contained 192 patients (96 patients for both INTRON® A plus ribavirin and INTRON® A plus placebo). Two out of three patients were not treated because they did not wish to continue, the third because protocol criteria were not met. All discussions on efficacy and safety in this report are based on data from all treatment groups.

Efficiency

The purpose of this study was to compare the combination of INTRON® A plus ribavirin with INTRON® A plus placebo for the general extent of response and 9 9 t 9999 • # 9 9

9 9 9 »virological response range (based on HCV-RNA (qPCR). The primary efficacy aspect for this study is the general response range.

The conclusion on efficacy is as follows:

J. Combination of ribavirin with INTRON® A can dramatically increase the proportion of patients who disappear HCV-RNA and have significantly reduced liver inflammation.

At the end of the post-treatment period, the general extent of response is the sum of serum HCV-RNA (qPCR) loss and changes in liver histology at the end of the period (24 weeks after the end of treatment). The patient was classified as a general responder if HCV-RNA (qPCR) was negative at week 24 of the post-treatment assessment and at the same time the Knodell inflammatory HAI index (sum of categories I + II + III) improved by 2 or more units relative to the value determined prior to treatment. The final virological response values at the end of the post-treatment period, the histological response and the general response ranges are summarized in Tables 1, 2 and 3.

Final HCV-RNA values at the end of the study period: Sustained loss of HCVRNA 24 weeks after the end of treatment.

The proportion of patients with serum HCV-RNA disappeared 24 weeks after treatment was 10 times greater (p <0.001) in the INTRON® A plus ribavirin group compared to those receiving INTRON® A as monotherapy. Table 1 summarizes patient responses at the end of the study period, as indicated by serum HCV-RNA levels.

Table 1 Serum HCV-RNA status at end of study period: Proportion of patients with serum HCV-RNA disappeared 24 weeks after treatment, responses _Number (%) of patients

patients INTRON® A plus ribavirin INTRON® A plus placebo p value all treated 50/95 (52) 5/96 (4) <0.001 patients 95% confidence interval for 1% -10% every way treatments: 4% - 58% for differences between 42% - 62% treatments: Persons responding at the end of treatment 49/80 (60) 5/40 (12)

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Pre- and post-treatment biopsies were available for 81% (78/96) of patients treated with INTRON® A plus ribavirin and for 77% (74/96) of patients treated with INTRON® A plus placebo. Table 2 summarizes the effect of treatment on liver inflammation in patients with liver biopsy results both before and after treatment. As with sustained loss of HCV-RNA replication, the proportion of patients with improved liver inflammation was significantly higher (p <0.001) in patients receiving combination therapy compared to those receiving INTRON® A monotherapy.

Table 2 Liver histology status at the end of the study period: Improvement in liver histology status 24 weeks post-treatment, based on the Knodell HAI index (I + II + III).

state patients Number (%) of patients ” INTRON A plus ribavirin (n = 78) INTRON A plus placebo (n = 74) value ”p Improved biopsy * 49 (52) 30 (31) <0.001

b Patients with liver biopsies both before and after treatment.

c Fischer exact test.

d Changes in the Kodel histological index (HAI) before and after treatment (sum of I + II + U1) included if there was a reduction of 2 or more units relative to the pre-treatment value.

Overall answer

When the study was designed, it was clear that since liver biopsy collection is an invasive procedure, it will be unlikely to obtain liver biopsy from all patients after treatment. Therefore, the protocol and statistical analysis were designed so that the overall response analysis would be based on data for all patients treated and evaluated by the maximum likelihood (MLE) method for patients whose overall response status could not be detected, ie patients with negative HCV- RNA and lack of biopsy evaluation (post-treatment period). The protocol also stipulated that additional analysis should be performed in patients for whom pre-treatment and post-treatment biopsy results are available (ie patients with complete data). The overall response is summarized in Table 3, based on the following analyzes:

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K. maximum likelihood assessment (MLE);

L. patients with complete data (pre-treatment and post-treatment biopsy results);

M. Patients with missing data (missing some or both data for HCV / biopsy), considered as unsuccessful treatment.

Table 3 Share of total responses

analyzed INTRON® A plus INTRON® A plus value * ⁵ d data ribavirin placebo rating maximal 43% 5% <0.001 probability complete patients 38/78 (50%) 4/75 (5%) <0.001 data ^c treatment considered 38/96 (41%) 4/96 (4%) ^e <0.001 as unsuccessful "

1. MLE based on logistic regression.

1. Fischer exact test.

1. Complete data = biopsy results both before and after treatment.

1. Patients who did not have either virological or biopsy data or both were considered unsuccessful.

As could be predicted from individual results regarding the effect of treatment on HCV-RNA clearance at the end of the study period and improvement in liver inflammation, the overall response rate in the INTRON® plus ribavirin group was significantly greater (<0.001) along with 10 to 14 a fold improvement over that observed in the INTRON® plus placebo group for all assessment methods used.

Logistic regression analysis was performed on all input demographic variables and disease characteristics. The only baseline characteristics that were statistically significant for predicting a sustained response at the end of the reporting period were a genotype other than type 1 and a viral load of 2 million.

For viral copy number (2 million,> 2 million), the differences were statistically significant in favor of higher response rates in patients with 2 million copies (Table 4).

When genotype and baseline virus load are combined, it is observed in the hierarchy responses. Patients with a viral genotype other than 1 and a baseline viral genotype

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4 4 4 • 4 4 9 • 4 9 9 With a 2 million copy load, they had the best response at the end of the reporting period; patients with viral genotype 1 and baseline viral load> 2 million copies had the weakest response at the end of the study period.

Table 4 Characteristics of disease versus sustained response: All patients treated

Disease characteristics ^b Number (%) of patients INTRON A plus ribavirin (n = 96) INTRON A plus placebo (n = 96) HCV-RNA / qPCR 2 million 24/36 (68) 5/28 (17) 2 million 26/60 (42) 0/67 (1) Genotype HCV 1 16/53 (29) 2/53 (3) other 34/44 (79) 3/19 (6) Genotype / starting HCV-RNA / qPCR other / 2 million copies 15/16 (92) 3/14 (22) other /> 2 million copies 18/26 (67) 0/29 (0) 1/2 million copies 8/20 (41) 0/16 (0) 1 /> 2 million copies 7/22 (21) 0/37 (0)

At entry, patients were stratified by viral copy number (2 million,> 2 million), genotype (1 or other) and cirrhosis (present or absent).

Example 2.

The second study 2 was performed essentially using the same methodology as described above in study 1. The results are summarized below.

Efficiency

An overall response at the end of the study period occurs when HCV-RNA (qPCR) disappears and changes in liver histology occur at the end of the study period (24 weeks after the end of treatment). A patient was classified as a general responder if HCV-RNA (PCR) was negative at 24 weeks post-treatment and the Knodell HAI inflammatory index (sum of categories I + II + III) improved by 2 or more units relative to the index before treatment. The incidence of endothelial response, histological response and overall response are summarized in Tables 5,6 and 7.

HCV-RNA response at end of study period: Sustained loss of HCV-RNA 24 weeks after treatment discontinuation.

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The proportion of patients who had serum HCV-RNA clearance 24 weeks after treatment was 10-fold (p <0.001) in the INTRON® A plus ribavirin group compared to those receiving INTRON® A as monotherapy. Table 5 summarizes patient responses at the end of the study period, as indicated by serum HCV-RNA levels.

Table 5 Serum HCV-RNA status at end of study period: Proportion of patients with serum HCV-RNA disappeared 24 weeks after treatment discontinuation.

patient responses all patients treated 95% confidence interval for each treatment:

for differences between treatments: End - responders ⁰

INTRON A plus ribavirin

34/78 (44)% - 56%

34/53 (63)

Number (%) of patients_

INTRON A plus placebo

3/76 (4)% - 8%% - 52% p <0.001

3/32 (8)

Pre- and post-treatment biopsies were available for 79% (61/77) of patients treated with INTRON® A plus ribavirin and 84% (64/76) of patients treated with INTRON® A plus placebo. Table 6 summarizes the effect of treatment on liver inflammation in patients with liver biopsy results both before and after treatment. As with sustained loss of HCV-RNA replication, the proportion of patients with improved liver inflammation was significantly higher (p <0.001) in patients receiving combination therapy compared to those receiving INTRON® A monotherapy.

Table 6 Liver histology status at the end of the study period: Improvement in liver histology status 24 weeks post-treatment, based on the Knodell HAI index (I + II + III).

state patients Number (%) of patients ³ INTRON A plus ribavirin (n = 61) INTRON A plus _value b. placebo (n = 64) ^μ Improved Biopsy state ⁰ 38 (48) 27 (36) <0.001

2. Patients with liver biopsies both before and after treatment.

2. Fischer exact test.

2. Changes in the Kodel histological index (HAI) before and after treatment (sum of I + II + III) included if there was a reduction of 2 or more units relative to the pre-treatment value.

Overall answer

The overall response is summarized in Table 7, based on the following analyzes:

N. Maximum Probability Assessment (MLE);

O. patients with complete data (pre-treatment and post-treatment biopsy results);

P. Patients with missing data (some or both of HCV / biopsy data missing), considered unsuccessful.

Table 7 Share of total responses

analyzed INTRON A plus INTRON A plus value “p data ribavirin placebo ML rating ⁸ 36.5% 2.7% <0.001 patients with complete 25/61 (41.0%) 2/64 (3.1%) <0.001 data ⁰ treatment considered 25/77 (32.5%) 2/76 (2.6%) <0.001 as unsuccessful "

1. MLE based on logistic regression.

1. Fischer exact test.

1. Complete data - biopsy results both before and after treatment.

1. Patients who did not have either virological or biopsy data or both were considered unsuccessful.

As predicted from individual outcomes regarding the effect of treatment on HCV-RNA clearance at the end of the study period and improvement in liver inflammation, the overall response rate in the INTRON® plus ribavirin group was significantly greater (<0.001) with 10 to 14 fold improvement of inflammation than observed in the INTRON® plus placebo group in all assessment methods used.

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Logistic regression analysis was performed on all input demographic variables and disease characteristics. The only baseline that was statistically significant for predicting a sustained response at the end of the reporting period was a genotype other than type 1.

For viral copy numbers (2 million,> 2 million), the differences were statistically significant in favor of higher response rates in patients with 2 million copies (Table 8). When genotype and baseline virus load are combined, it is observed in the hierarchy responses. Patients with a viral genotype other than 1 and a baseline viral load of 2 million copies had the best response at the end of the reporting period; patients with viral genotype 1 and baseline viral load> 2 million copies had the weakest response at the end of the study period.

Table 8 Characteristics of disease versus sustained response: All patients treated

Disease characteristics Number (%) of patients INTRON®Aplus ribavirin (n = 77) INTRON® Plus plus placebo (n = 76) HCV-RNA / qPCR 2 million 6/9 (68) 1/12 (7) > 2 million 28/67 (41) 2/64 (2) Genotype HCV 1 12/45 (28) 1/42 (1) other 21/30 (68) 2/34 (5) Genotype / starting HCV-RNA / qPCR other / 2 million copies 4/4 (99) 0/2 (0) other /> 2 million copies 17/26 (62) 2/30 (6) 1/2 million copies 2/5 (41) 1/9 (10) 1 /> 2 million copies 11/40 (28) 0/32 (1)

The described embodiments have been given by way of example only, and the invention is limited only by the terms of the appended claims, along with the full range of equivalents to which these claims justify.

Claims (14)

PATENT CLAIMS Use of ribavirin for the manufacture of pharmaceutical compositions for the treatment of patients with chronic hepatitis C infection to detectably detect HCV-RNA by administering an effective dose of ribavirin together with an effective dose of interferon alpha, a patient who has not responded to a previous course of treatment interferon alpha, characterized in that it is a difficult-to-treat patient with a viral load greater than 2 million copies per milliliter of serum, as measured by HCV-RNA quantitative PCR and difficult to treat type 1 HCV infection. Use of interferons alpha for the manufacture of pharmaceutical compositions for the treatment of patients with chronic hepatitis C infection to remove detectably HCV-RNA, which comprises administering an effective dose of alpha interferons together with an effective dose of ribavirin, the patient not responding to the previous course interferon alpha therapy, characterized in that it is a difficult-to-treat patient with viral load greater than 2 million copies per milliliter of serum, as measured by HCV-RNA quantitative PCR and difficult to treat type 1 HCV infection. 3. Use of both ribavirin and alpha interferons for the manufacture of pharmaceutical compositions for the treatment of patients with chronic hepatitis C infection to remove detectably HCV-RNA by administering an effective dose of ribavirin together with an effective alpha interferon patient that is non-responder not prior treatment with interferon alpha, characterized in that it is a difficult-to-treat patient with viral load greater than 2 million copies per milliliter of serum, as measured by HCV-RNA quantitative PCR and difficult to treat type 1 HCV infection. Use according to any preceding claim, wherein the interferon-alpha is selected from the group consisting of interferon alpha-2a, interferon alpha-2b, consensus interferon, purified manufactured interferon alpha or pegylated ("pegylate) interferon-alpha. • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Use according to any one of the preceding claims, wherein the interferon alpha used is alpha-2b interferon. Use according to any one of the preceding claims, wherein the amount of ribavirin administered is 400 to 1200 mg per day, preferably 800 to 1200 mg per day and the amount of interferon alpha administered is 2 to 10 million IU per week, administered weekly, TIW, QOD or daily , preferably 3 million IU TIW. Use according to any one of the preceding claims, wherein the administration is carried out for a period of 20 to 30 weeks. The use of claim 11, wherein the administration is continued for a total period of 40 to 50 weeks. The use of claim 11, wherein the administration is continued for a total time period of 60 to 80 weeks. Pharmaceutical products comprising ribavirin and interferon alpha in separate but adjacent form. Pharmaceutical products according to claim 10, characterized in that the interferon alpha is in a form suitable for parenteral injection, preferably for subcutaneous injection, and the ribavirin is in a solid dosage form, preferably in a dosage form suitable for oral administration. Pharmaceutical products according to claims 10 or 11, characterized in that they contain an amount of ribavirin sufficient for at least one day of treatment and an amount of interferon alpha sufficient for at least one injection dose. Pharmaceutical products according to claim 12, characterized in that the amount of ribavirin is from 400 to 1200 mg, preferably 800 to 1200 mg, and the amount of interferon alpha is 3 million IU or at least 3 million IU. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 9 9 9 9 9999 9 9 9 99 9 9 99 Pharmaceutical products according to any one of claims 10 to 13, characterized in that they are in kit form with instructions for combined use in the treatment of chronic hepatitis C infections.