The present invention relates to the use of recombinant IFN-beta for the production of a medicament for the treatment of HCV infection by subcutaneous administration to patients of Asian race, which failed to respond to a previous treatment with interferon-alpha, is herein reported. According to a preferred embodiment of the invention, this treatment can be better and further focused to those patients which after at least 4 weeks of initial treatment with IFN-beta show HCV RNA clearance.

Description of the Invention

FIELD OF THE INVENTION

This invention relates to the use of recombinant IFN-beta for the production of a medicament for the treatment of HCV infection by subcutaneous administration to patients of Asian race, which failed to respond to a previous treatment with interferon alpha.

BACKGROUND OF THE INVENTION

The hepatitis C virus (HCV) produces a state of chronic infection in nearly all acutely infected individuals. Approximately 20% of patients with chronic HCV infection (CHC) develop cirrhosis with subsequent liver failure, portal hypertension, ascites, encephalopathy, and bleeding disorders (Alter M., 1992). Long-term follow-up suggests that these estimates may be conservative (Davis G L, 1990); moreover, chronic HCV infection is strongly associated with hepatocellular carcinoma (Tabor E. et al., 1992).

Interferons (IFNs) are glycoproteins produced by the body in response to a viral infection. They inhibit the multiplication of viruses in protected cells. Consisting of a lower molecular weight protein, IFNs are remarkably non specific in their action, i.e. IFN induced by one virus is effective against a broad range of other viruses. They are however species-specific, i.e. IFN produced by one species will only stimulate antiviral activity in cells of the same or a closely related species. IFNs were the first group of cytokines to be exploited for their potential antitumour and antiviral activities.

The three major IFNs are referred to as IFN-alpha, IFN-beta and IFN-gamma. Such main kinds of IFNs were initially classified according to their cells of origin (leukocyte, fibroblast or T cell). However, it became clear that several types might be produced by one cell. Hence leukocyte IFN is now called IFN-alpha, fibroblast IFN is IFN-beta and T cell IFN is IFN-gamma. There is also a fourth type of IFN, lymphoblastoid IFN, produced in the "Namalwa" cell line (derived from Burkitt's lymphoma), which seems to produce a mixture of both leukocyte and fibroblast IFN.

In particular, human fibroblast interferon (IFN-beta) has antiviral activity and can also stimulate natural killer cells against neoplastic cells. It is a polypeptide of about 20,000 Da induced by viruses and double-stranded RNAs. From the nucleotide sequence of the gene for fibroblast interferon, cloned by recombinant DNA technology, Derynk et al. (Derynk R. et al., Nature 285, 542-547, 1980) deduced the complete amino acid sequence of the protein. It is 166 amino acid long.

Shepard et al. (Shepard H. M. et al., Nature, 294, 563-565, 1981) described a mutation at base 842 (Cys.fwdarw.Tyr at position 141) that abolished its anti-viral activity, and a variant clone with a deletion of nucleotides 1119-1121.

Mark et al. (Mark D. F. et al., Proc. Natl. Acad. Sci. U.S.A., 81 (18) 5662-5666, 1984) inserted an artificial mutation by replacing base 469 (T) with (A) causing an amino acid switch from Cys.fwdarw.Ser at position 17. The resulting IFN-beta was reported to be as active as the `native` IFN-.beta. and stable during long-term storage (-70.degree. C.).

Rebif.RTM. (recombinant human Interferon-beta-1a) is the latest development in interferon therapy for multiple sclerosis (MS) and represents a significant advance in treatment. Rebif.RTM. is Interferon (IFN)-beta-1a, produced from mammalian cell lines.

The mechanisms by which IFNs exert their effects are not completely understood. However, in most cases they act by affecting the induction or transcription of certain genes, thus affecting the immune system. In vitro studies have shown that IFNs are capable of inducing or suppressing about 20 gene products.

There is no completely effective therapy for CHC. The best results have been obtained with interferon-alpha, although this is not a universally-recommended therapy. Many clinicians only observe patients with CHC because of the uncertain natural history of HCV infection and the toxicity associated with interferon-alpha.

Most patients with CHC do not achieve complete responses to treatment with interferon-alpha. Controlled trials of interferon-alpha administered for six months resulted in normalisation of serum ALT in 40 to 50% of patients at the end of treatment, but this response was sustained in only 15 to 25% (Hoofnagle J H et al., 1997).

Dose escalations and increased duration of therapy have resulted in small increases in sustained response, but at the cost of increased expense and toxicity (Poynard T. et al., 1996). In addition, the benefit of higher doses is often transient and relapses are common after therapy has been discontinued (Lindsay K L et al., 1996).

A study of 35 non-responders to interferon-alpha reported no benefit from prolongation of therapy from six to 12 months, increasing the dose of interferon-alpha, switching therapy from recombinant to lymphoblastoid interferon or using steroids (Piccinino F et al., 1993).

The natural history of HCV infection following lack of response to interferon-alpha has not been adequately studied, but in one study follow-up of 28 patients for at least 2 years after therapy found only one case of eventual remission at 16 months (normalisation of ALT and disappearance of HCV RNA) (Takeda T et al., 1993).

Several factors have been found to be associated with greater probability of long-term sustained response to interferon-alpha: non-type 1 genotype, low serum HCV RNA concentration, shorter duration of infection, lower body weight, mild activity on liver biopsy, absence of cirrhosis and low levels of serum ferritin, iron, transferrin saturation and hepatic iron concentration (Schvarcz R et al., 1989, Bacon B R et al., 1995, Conjeevaram H S et al., 1995, Bonkovsky H L et al., 1997).

Patients with CHC who fail to achieve a sustained response after interferon-alpha therapy are thought to have a more aggressive disease course, possibly due to the selection of resistant genotypes, but the development of neutralising antibodies to interferon-alpha may also be a contributing factor. There appears to be a strong correlation between development of neutralising antibodies to interferon-alpha-2a and lack of clinical benefit, in both CHC and hepatitis B virus (HBV) infections (Douglas D D et al., 1993, Milella M M et al., 1993, Lok A S F et al., 1990). In fact, the development of antibodies to a single recombinant type of interferon-alpha may neutralise other Interferon-alpha subtypes (Brand C M et al., 1993).

There is relatively little experience with interferon-beta in HCV infection. Very promising results have been reported for interferon-beta therapy of acute HCV infection, with 7 of 11 patients achieving sustained normalisation of ALT at one year compared to only one of 14 controls (Omata M et al., 1991). The eleven patients were treated for an average of 30 days with a mean IV dose of 52 MU of fibroblast-derived, "native", and interferon-beta. Notably, no significant toxicity was reported.

Today, in Japan natural IFN-beta is commonly used for the treatment of chronic hepatitis C and the recommended regimen is a daily dose of 3-6 MIU administered i.v. for 6-8 weeks (see Habersetzer et al., Liver, 2000, 20, 438, 4th line).

Very poor clinical efficacy of intramuscular administration of IFN-beta (3 MU t.i.w) in HCV patients of non-Asian race has been shown (Perez R. et al., J. Virol. Hepat. 1995, 2(2), 103-6).

Always in non-Asian (Caucasian) HCV patients subcutaneous administration (9 or 12 MU) of recombinant IFN-beta has shown efficacy at least in a group of patients (Habersetzer et al., Liver, 2000, 20 437-441).

Kishiara et al. (Fukukoka Acta Med., 86(4), 113-20, 1995) disclose a treatment with natural IFN-beta administered i.v. at a dose of 6MIU to HCV patients not responding to IFN-alpha.

In a preliminary comparative study of interferon-alpha vs. interferon-beta in HBV and HCV, response rates were 81% for interferon-alpha and 86% for interferon-beta, with similar response rate maintenance at 6 months (72% for interferon-alpha and 79% for interferon-beta) (Tundo L, 1993). Notably, side effects led to interruption of therapy for 24% of the interferon-alpha group compared to 0% of the interferon-beta group.

The encouraging initial results of some previous studies carried out by the Applicant, along with the good safety and tolerability profile of IFN-beta-1a, led to the design of the study, which explored higher and more intense dose regimens for a longer treatment period in patients with chronic hepatitis C who had failed treatment with IFN-alpha.

DESCRIPTION OF THE INVENTION

Because of a spontaneous report of good efficacy results by the Investigator in the Taiwanese centre, exploratory analyses by centre and by demographic characteristics were performed, which led to identification of differences between patients of Asian and non-Asian origin. The study's analysis plan was therefore amended to include complete evaluation of these two populations.

The main object of the present invention is the use of recombinant IFN-beta for the production of a medicament for the treatment of HCV infection by subcutaneous administration to patients of Asian race, which failed to respond to a previous treatment with interferon alpha.

Another object of the present invention is, therefore, the method for treating HCV infection comprising administering subcutaneously an effective amount of IFN-beta, together with a pharmaceutically acceptable excipient, to patients of Asian race, who failed to respond to a previous treatment with IFN-alpha.

An "effective amount" refers to an amount of the active ingredients that is sufficient to affect the course and the severity of the disease, leading to the reduction or remission of such pathology. The effective amount will depend on the route of administration and the condition of the patient.

"Pharmaceutically acceptable" is meant to encompass any carrier, which does not interfere with the effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which is administered. For example, for parenteral administration, the above active ingredients may be formulated in unit dosage form for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution.

Besides the pharmaceutically acceptable carrier, the compositions of the invention can also comprise minor amounts of additives, such as stabilizers, excipients, buffers and preservatives.

The term "recombinant interferon-beta (IFN-beta)", as used in the present invention, is intended to include human fibroblast interferon, as obtained by DNA recombinant techniques from prokaryotic or eukaryotic host cells as well as its salts, functional derivatives, variants, analogs and fragments.

"Functional derivatives" as used herein covers derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they do not destroy the biological activity of the proteins as described above, i.e., the ability to bind the corresponding receptor and initiate receptor signaling, and do not confer toxic properties on compositions containing it. Derivatives may have chemical moieties, such as carbohydrate or phosphate residues, provided such a derivative retains the biological activity of the protein and remains pharmaceutically acceptable.

For example, derivatives may include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives or free amino groups of the amino acid residues formed with acyl moieties (e.g., alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl group (e.g., that of seryl or threonyl residues) formed with acyl moieties. Such derivatives may also include for example, polyethylene glycol side-chains, which may mask antigenic sites and extend the residence of the molecule in body fluids.

Of particular importance is a protein that has been derivatized or combined with a complexing agent to be long lasting. For example, pegylated versions, or proteins genetically engineered to exhibit long lasting activity in the body, can be used according to the present invention. A pegylated version of interferon-beta-1a has been described in WO 99/55377 and is considered as included in the definition of interferon-beta according to the present application.

The term "derivatives" is intended to include only those derivatives that do not change one amino add to another of the twenty commonly occurring natural amino acids.

The term "salts" herein refers to both salts of carboxyl groups and to acid addition salts of amino groups of the proteins described above or analogs thereof. Salts of a carboxyl group may be formed by means known in the art and include Inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with mineral acids, such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids, such as, for example, acetic acid or oxalic acid. Of course, any such salts must retain the biological activity of the protein relevant to the present invention, i.e., the ability to bind to the corresponding receptor and initiate receptor signaling.

A "fragment" according to the present invention refers to any subset of the molecules, that is, a shorter peptide, which retains the desired biological activity. Fragments may readily be prepared by removing amino acids from either end of the molecule and testing the resultant for its properties as a receptor agonist. Proteases for removing one amino acid at a time from either the N-terminal or the C-terminal of a polypeptide are known, and so determining fragments, which retain the desired biological activity, involves only routine experimentation.

A "variant" according to the present invention refers to a molecule, which is substantially similar to either the entire proteins defined above or a fragment thereof. Variant peptides may be conveniently prepared by direct chemical synthesis of the variant peptide, using methods well known in the art. Of course, such variant would have similar receptor binding and signal initiating activity as the corresponding naturally occurring protein.

Amino acid sequence variants of the protein defined above can be prepared by mutations in the DNAs, which encode the synthesized derivatives. Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity. Obviously, the mutations that will be made in the DNA encoding the variant peptide must not alter the reading frame and preferably will not create complementary regions that could produce secondary mRNA structure.

At the genetic level, these variants ordinarily are prepared by site-directed mutagenesis of nucleotides in the DNA encoding the peptide molecule, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. The variants typically exhibit the same qualitative biological activity as the non-variant peptide.

An "analog" of the protein defined above, according to the present invention, refers to a non-natural molecule, which is substantially similar to either, the entire molecules or to an active fragment thereof. Such analog would exhibit the same activity as the corresponding naturally occurring protein.

The types of substitutions, which may be made to interferon-beta, according to the present invention, may be based on analysis of the frequencies of amino acid changes between homologous proteins of different species. Based upon such analysis, conservative substitutions may be defined herein as exchanges within one of the following five groups:

SmaII, aliphatic, non-polar or slightly polar residues:

Ala, Ser, Thr, Pro, Gly II. Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gln

III. Polar, positively charged residues: His, Arg, Lys

IV. Large, aliphatic non-polar residues: Met, Leu, Ile, Val, Cys

V. Large aromatic residues: Phe, Tyr, Trp

Within the foregoing groups, the following substitutions are considered to be "highly conservative": Asp/Glu His/Arg/Lys Phe/Tyr/Trp Met/Leu/Ile/Val

Semi-conservative substitutions are defined to be exchanges between two of groups (I)-(IV) above which are limited to supergroup (A), comprising (I), (II), and (III) above, or to supergroup (B), comprising (IV) and (V) above. Substitutions are not limited to the genetically encoded or even the naturally-occurring amino acids. When the epitope is prepared by peptide synthesis, the desired amino acid may be used directly. Alternatively, a genetically encoded amino acid may be modified by reacting it with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.

Cysteinyl residues most commonly are reacted with alpha-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxylmethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, alpha-bromo-beta-(5-imidazoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl-2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Parabromophenacyl bromide is also useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino acid-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methyliosurea; 2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal; 2,3-butanedione; and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine, as well as the arginine epsilon-amino group.

The specific modification of tyrosyl residues per se has been studied extensively, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyl tyrosyl species and .epsilon.-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'N--C--N--R') such as 1-cyclohexyl-3-[2-morpholinyl-(4-ethyl)]carbodiimide or 1-ethyl-3-4-azonia-4,4-dimethylpentyl)carbodimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.

Examples of production of amino acid substitutions in proteins which can be used for obtaining analogs for use in the present invention include any known method steps, such as presented in U.S. Pat. Nos. RE 33,653; 4,959,314; 4,588,585 and 4,737,462, to Mark et al; U.S. Pat. No. 5,116,943 to Koths et al; U.S. Pat. No. 4,965,195 to Namen et al; and U.S. Pat. No. 5,017,691 to Lee, et al, and lysine substituted proteins presented in U.S. Pat. No. 4,904,584 (Shaw et al).

Preferably, the variant or analog, as defined above, will have a core sequence, which is the same as that of the "native" sequence or biologically active fragment thereof, which has an amino add sequence having at least 70% identity to the native amino add sequence and retains the biological activity thereof. More preferably, such a sequence has at least 80% identity, at least 90% identity, or most preferably at least 95% identity to the native sequence.

The term "sequence identity" as used herein means that the sequences are compared as follows. The sequences are aligned using Version 9 of the Genetic Computing Group's GAP (global alignment program), using the default (BLOSUM62) matrix (values -4 to +11) with a gap open penalty of -12 (for the first null of a gap) and a gap extension penalty of -4 (per each additional consecutive null in the gap). After alignment, percentage identity is calculated by expressing the number of matches as a percentage of the number of amino acids in the claimed sequence.

Analogs or variants in accordance with the present invention may also be determined in accordance with the following procedure. The DNA of the native sequence is known to the prior art and is found in the literature. Polypeptides encoded by any nucleic acid, such as DNA or RNA, which hybridizes to the complement of the native DNA or RNA under highly stringent or moderately stringent conditions, as long as that polypeptide maintains the biological activity of the native sequence, are also considered to be within the scope of the present invention.

Stringency conditions are a function of the temperature used in the hybridization experiment, the molarity of the monovalent cations and the percentage of formamide in the hybridization solution. To determine the degree of stringency involved with any given set of conditions, one first uses the equation of Meinkoth et al. (1984) for determining the stability of hybrids of 100% identity expressed as melting temperature Tm of the DNA--DNA hybrid: Tm=81.5.degree. C.+16.6 (.sub.LogM)+0.41 (% GC)-0.61 (% form)-500/L, where M is the molarity of monovalent cations, % GC is the percentage of G and C nucleotides in the DNA, % form is the percentage of formami de in the hybridization solution, and L is the length of the hybrid in base pairs. For each 1.degree. C. that the Tm is reduced from that calculated for a 100% identity hybrid, the amount of mismatch permitted is increased by about 1%. Thus, if the Tm used for any given hybridization experiment at the specified salt and formamide concentrations is 10.degree. C. below the Tm calculated for a 100% hybrid according to equation of Meinkoth, hybridization will occur even if there is up to about 10% mismatch.

As used herein, highly stringent conditions are those, which are tolerant of up to about 15% sequence divergence, while moderately stringent conditions are those, which are tolerant of up to about 20% sequence divergence. Without limitation, examples of highly stringent (12-15.degree. C. below the calculated Tm of the hybrid) and moderately (15-20.degree. C. below the calculated Tm of the hybrid) conditions use a wash solution of 2.times.SSC (standard saline citrate) and 0.5% SDS at the appropriate temperature below the calculated Tm of the hybrid. The ultimate stringency of the conditions is primarily due to the washing conditions, particularly if the hybridization conditions used are those, which allow less stable hybrids to form along with stable hybrids. The wash conditions at higher stringency then remove the less stable hybrids. A common hybridization condition that can be used with the highly stringent to moderately stringent wash conditions described above is hybridization in a solution of 6.times.SSC (or 6.times.SSPE), 5.times. Denhardt's reagent, 0.5% SDS, 100 .mu.g/ml denatured, fragmented salmon sperm DNA at a temperature approximately 20.degree. to 25.degree. C. below the Tm. If mixed probes are used, it is preferable to use tetramethyl ammonium chloride (TMAC) instead of SSC (Ausubel, 1987-1998).

While the present invention provides recombinant methods for making the above-defined derivatives, these derivatives may also be made by conventional protein synthesis methods, which are well known to those skilled in the art.

According to the present invention "a race" is a population that can be distinguished as a distinct subgroup within a species (e.g. the human species). A race possesses a unique and distinct ensemble of genes, and is identified by the traits (both mental and physical) produced by the genetic ensemble. Members of the same race share distinguishing genetic characteristics, because they share a common genetic ancestry and a consequently similar genetic ensemble.

Based on the nuclear DNA studies of Luigi Cavalli Sforza and his colleagues at least 6 human races/populations can be defined: the Caucasoid (which include the European and Indian populations), the African, the Asian, the Arctic, the American Indian, and the Pacific one (L. Cavalli-Sforza, Scientific American, 72-78, November 1991).

According to the present invention "Asian" means any person having origins in any of the original peoples of China, Mongolia, Taiwan, Singapore, Korea, Japan, Vietnam, Cambodia, Laos, Burma, Thailand, Malaysia, Indonesia and Philippines.

"Non-Asian" is herein intended to refer to all the other human races/populations, which do not fall under the above-definition of "Asian".

Patients normally are requested to self-identify by "race" or the doctor on the basis of their somatic traits and/or the country of origin assigns the race.

According to the present invention, "patients who failed to respond to a previous treatment with IFN-alpha" are those HCV patents who underwent a previous treatment with any type (or types) of interferon-alpha (at least 12 weeks of treatment at a dose of at least 3 MIU 3 times a week), with one of the following outcomes: (a) failure to normalise serum ALT, or (b) normalisation of ALT followed by breakthrough (ALT elevation) before the end of therapy. The dosages and the regimens can be selected by the doctor depending on the severity if the disease, the age and the sex of the patient. According to the present applications the following four regimens and dosages have been used. Regimen A: 12 MIU (44 mcg) recombinant IFN-beta-1a three times a week, Regimen B: 12 MIU (44 mcg) recombinant IFN-beta-1a daily, Regimen C: 24 MIU (88 mcg) recombinant IFN-beta-1a three times a week, or Regimen D: 24 MIU (88 mcg) recombinant IFN-beta-1a daily.

According to a preferred embodiment of the invention, the treatment with IFN-beta is to be carried out only on the subgroup of patients who show HCV RNA clearance after 4 weeks of treatment In fact it has been noted that for this subgroup of patients the probability that the treatment will be successful after the 48 weeks of treatment is very high, close to 100%. "HCV RNA clearance" means absence of detectable HCV RNA in the serum of the treated patients.

In other words the treatment of the present invention may be advantageously preceded by a "test-phase", in which the patients undergo the same treatment with IFN-beta for 4 weeks and at the end of this "test-phase" preferentially the patients who show HCV RNA clearance are encouraged to carry out the treatment for more weeks.

According to a further preferred embodiment of the present invention the treatment with IFN-beta can be coupled with a concomitant treatment with another antiviral drug. The most commonly used antiviral drug in the treatment of HCV is ribavirin (a nucleoside analog), but other drugs show some potential in this treatment and are listed in a recent review (T Wilkinson, Curr. Op. Invst. Drug, 2(11), 1516-22, 2002) and include serine protease inhibitors, inhibitors of the RNA-dependent RNA polymerase (RdRp) and helicase inhibitors. These drugs can be administered simultaneously, separately or sequentially when combined with recombinant IFN-beta.
 

Claim 1 of 7 Claims

1. A method of treating hepatitis C virus infections comprising the subcutaneous administration of an effective amount of a composition comprising interferon-beta (IFN-.beta.) to Asian patients that had failed to respond to a previous treatment with interferon-.alpha..

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