SI9300671A - THERAPY COMBINATION CONTAINING HUMAN INTERFERON USED FOR TREATMENT OF VIRAL AND NON-VIRUS INFECTIONS - Google Patents

Abstract

The present invention relates to a therapeutic combination, more particularly to a combination therapy involving the use of human interferon. The therapy involves the treatment of conditions susceptible to treatment with human interferon by a combination of human interferon and a free radical scavenger or a precursor or inducer thereof.

Description

The Wellcome Foundation Limited

Therapeutic combination containing human interferon, useful for the treatment of viral and non-viral infections

The present invention relates to a therapeutic combination, more particularly to a combination therapy involving the use of human interferon.

The first and localized response of animals to viral infection is to produce the lymphokine interferon. Originally thought to be single molecules, interferons are now recognized as a large family of proteins, evolutionary old and widespread in the animal kingdom. Although some limited cross-reactivity may occur, interferons are generally species-specific. Three types of interferon have been characterized, originally known as leukocyte, fibroblast and immune interferon, and are now designated as interferons α, β and -7, respectively.

Human interferon α can be produced by many different cell types, and HPLC separates this type of interferon into over 30 subtypes, each encoded by a different gene. Human interferon β is generally considered to be a single substance made by fibroblasts. Human interferon γ is also a single substance made by T lymphocyte populations induced by helper exposure to antigen or by exposure of human white blood cells, T lymphocytes or T lymphoblastoid cells, to mitogens.

Human interferon-α is produced in commercial quantities by stimulating the Namalwa human lymphoblastoid cell line with Sendai virus to produce a natural mixture of up to 22 subtypes of interferon-α, which is then purified by chromatography to 95% purity and a specific activity of approximately 100 χ 10 ⁶ IU/mg protein. Such a product, identified as human interferon-α-Nl, is commercially available under the name WELLFERON (a registered trademark of The Wellcome Foundation Limited). Natural human interferon-β is derived from human diploid fibroblasts, usually from neonatal tissue, with production induced, e.g., by the addition of synthetic double-stranded RNA. Human interferon-β can be produced from the yellowish-white layer of skin, composed of leukocytes, with a mitogen such as Staphylococcus enterotoxin A as an inducer.

Human interferons α, β and δ can also be produced using recombinant DNA technology, although it is possible that recombinant interferons produced by expression of the relevant gene in bacterial cells do not have the same tertiary structure as the native molecule. Similarly, interferons produced in bacterial cells are not glycosylated and although this does not affect the biological activity of the molecule in vitro, it may alter the conformation and antigenicity and may affect distribution in the body. Such recombinant human interferon preferably has more than 90% amino acid homology to native human interferon; more preferably 95%, even more preferably 97%, even more preferably 98%, even more preferably 99% and most preferably 100% homology. Recombinant human interferons are commercially available and examples are interferon α-2a (ROFERON - Roche) and interferon α-2b (INTRON -Schering). These molecules differ by a single amino acid residue at position 23 (lysine in ROFERON and arginine in INTRON).

Human interferons have been used for several years in the treatment of hepatitis. Initial trials in the mid-1970s, using leukocyte interferon, produced from cells in the yellowish-white layer of the skin, composed of leukocytes, were abandoned after plasma production from donated blood inevitably limited the available supply. In the early 1980s, advances in production techniques led to the use of both natural and recombinant human interferon-α for the treatment of chronic hepatitis B. While treatment can be considered successful in many cases, response rates with human interferon-α alone, as measured by the permanent loss of viral markers, are generally estimated to be less than 50%. Human interferons β and δ have also been investigated for use in chronic hepatitis B, but the treatments have not been established.

All three types of human interferon have also been investigated in the treatment of chronic hepatitis C, although the limited availability of interferon β has limited work with this type of interferon. Quite extensive trials have been conducted using the α interferons referred to above (Interferon α-2a, interferon α-2b and lymphoblastoid interferon) and the results have been encouraging in that 40% of patients have achieved a complete response. However, after 6 months of treatment, it has been estimated that approximately 50% of patients have relapsed, leaving only 20 to 25% of patients with long-term benefit.

For a complete review of the use of interferons in the treatment of hepatitis, see Interferon in the Treatment of Chronic Virus Infection of the Liver; Eddleston and Dixon, Pennine Press, 1990.

In addition, interferons have been proposed for use in the therapy of a number of other conditions, including viral diseases other than hepatitis, disorders involving the immune system, including autoimmune conditions, and various types of cancers, including renal cancer, breast cancer, colon cancer, Kaposi's sarcoma, glioma, and malignant hematological conditions.

Based on evidence, particularly in relation to HIV, we state that chronic viral infections can induce oxidative stress in the infected organism. Induction of oxidative stress by viruses may occur through various mechanisms, including activation of phagocytic cells by immune complexes, promotion of free radical formation by proinflammatory cytokines (TNFα, IL6), and generation of reactive oxygen species by direct interaction between viral surface glycoproteins and cell membranes.

A number of substances are known to act as free radical scavengers at the cellular or whole organism level. For example, reduced glutathione is a widely distributed non-protein thiol present in most mammalian cells that is involved in various metabolic functions, including detoxification reactions for free radicals. Glutathione is a major intracellular defense mechanism against oxidative stress, and factors that increase free radical production lead to the depletion of intracellular glutathione stores. Glutathione also has an immunoregulatory role in modulating lymphocyte activation and proliferation, T cell cytotoxicity, and macrophage-lymphocyte interactions.

N-acetyl cysteine has been known for many years as a mucolytic, corneal agent, and antidote for acetaminophen poisoning. The compound has a relatively mild reducing effect and is thought to act as a mucolytic by cleaving disulfide bonds in mucoproteins. There are several reports that reduced glutathione levels can be reduced in certain chronic viral conditions, particularly HIV infection. N-acetyl cysteine is a precursor and therefore an inducer of glutathione and is intended as a therapeutic agent for use in HIV infection.

EP-A-0 269 017 (Cetus) relates to a combination of a lymphokine or cytotoxin and a free radical scavenger or metabolic inhibitor for the treatment of biological damage in a mammalian host caused by free radical production. Although the description incidentally refers to interferons as lymphokines and also mentions infections as a possible cause of biological damage, it essentially relates to free radicals which may be generated during cancer progression. The biological data given in the description are entirely cancer-related and concern mainly the administration of TNFα to rodents with fibrosarcoma.

The present invention relates to the use of a free radical scavenger or its precursor or inducer as an adjunct to human interferon therapy.

Thus, in one aspect, the present invention provides a method for treating a patient suffering from a condition susceptible to interferon therapy, comprising administering to the patient an effective amount of human interferon and also administering a free radical scavenger or precursor or inducer thereof during all or part of the duration of administration of the human interferon.

According to another aspect, the present invention provides the use of human interferon for the manufacture of a medicament for use in the treatment of a condition susceptible to interferon therapy, by a method wherein a free radical scavenger or a precursor or inducer thereof is also administered during all or part of the duration of administration of the human interferon.

According to another aspect, the present invention also provides the use of a free radical scavenger or a precursor or inducer thereof for the manufacture of a medicament for treating a condition susceptible to interferon therapy, by a method comprising administering human interferon and wherein the free radical scavenger or a precursor or inducer thereof is also administered during all or part of the duration of the administration of human interferon.

A condition susceptible to interferon therapy may be any of the conditions for which human interferons have been established or have been proposed to be effective in treating. Such conditions include viral infections, such as viral hepatitis, infections caused by human papillomavirus, cytomegalovirus, and HIV, non-viral infections, such as tuberculosis, and conditions such as asthma.

The present invention is particularly useful for the use of human interferons in the treatment of hepatitis. As previously mentioned, although such therapy is quite beneficial in many cases, the response rate is relatively low and in the case of hepatitis C there is a significant rate of relapse. Therefore, there is a significant need for improvements in the treatment of hepatitis with human interferon that would overcome these problems.

Examples of humic interferon for use in the invention include those mentioned above.

According to another aspect, the present invention provides a method for treating viral hepatitis infection by administering human interferon, wherein a free radical scavenger or a precursor or inducer thereof is also administered during all or part of the duration of administration of human interferon.

According to a further aspect, the present invention provides the use of human interferon for the manufacture of a medicament for the treatment of viral hepatitis infection by a method wherein a free radical scavenger or a precursor or inducer thereof is also administered during all or part of the duration of administration of the human interferon.

According to a further aspect, the present invention also provides the use of a free radical scavenger or a precursor or inducer thereof for the manufacture of a medicament for the treatment of viral hepatitis infection by a method comprising administering human interferon and wherein the free radical scavenger or a precursor or inducer thereof is also administered during all or part of the duration of administration of human interferon.

According to a further aspect, the present invention provides enhanced combinations, also known as synergistic combinations, of human interferon and a free radical scavenger or a precursor or inducer thereof for use in the treatment of a condition susceptible to interferon therapy. The active ingredients of the enhanced combinations according to the invention may be administered simultaneously or sequentially as separate formulations or as a single combined formulation. If administered sequentially, the lag in the administration of the other active ingredients should not be such as to lose the benefit of the enhanced therapeutic effect of the combination of active ingredients.

The present invention is useful for the treatment of viral hepatitis in all its forms of the five types currently recognized and designated as hepatitis A, B, C, D, and E.

Hepatitis A is an acute viral infection with an incubation period of less than 40 days, transmitted by the fecal-oral route. The virus is a member of the picornavirus family and consists of a 27 nm round, non-enveloped particle. The viral genome has a defined sequence and consists of single-stranded RNA containing approx. 7480 bases.

Hepatitis B is a universal and serial disease with an estimated 200 carriers worldwide. When it was known as serum hepatitis, the disease was diagnosed based on the appearance of symptoms 2 to 3 months after blood transfusion, injection of human plasma fractions, or use of unsterilized injection needles. The identification of serum markers for hepatitis B confirmed the importance of skin penetration and especially blood in the transmission of the virus. After the acute phase of the disease, most adult patients recover spontaneously within a few weeks, but a proportional part of patients do not clear the virus even after many months and become chronic carriers. The hepatitis B virus belongs to the family of hepadnaviruses, whose genome consists of a small incompletely double-stranded circular piece of DNA that replicates by copying its DNA into RNA and then by re-copying the RNA into DNA by reverse transcriptase.

Non-A, non-B hepatitis is recognized as an increasingly serious international health problem. At least 80% of cases of chronic post-transfusion non-A, non-B hepatitis have been shown to be caused by the virus now identified as hepatitis C, and this virus can be considered to be the actual cause of all cases of post-transfusion hepatitis in clinical settings where blood products are screened for hepatitis B.

While approximately half of acute hepatitis C infections resolve spontaneously within a few months, the remainder become chronic, and in many, if not all, cases, the potential outcome of chronic active hepatitis is cirrhosis and hepatocellular carcinoma. The structure of the hepatitis C viral genome has recently been elucidated, and the virus is characterized as a single-stranded RNA virus with similarities to flaviviruses.

Hepatitis D virus was first recognized in 1977 after the identification of a novel antigen in certain hepatitis B carriers. The virus requires hepatitis B (or a closely related hepadna virus) as a helper virus for replication, although replication is sufficiently efficient to achieve higher titers than the helper virus in serum. The hepatitis D genome consists of covalently closed circular DNA and has some structural similarities to certain circular viroids or virus-like agents found in plants. Hepatitis D infection is associated with aggressive liver disease and is more commonly found in patients with severe disease (chronic active hepatitis or cirrhosis) than in those with chronic persistent hepatitis.

Hepatitis E virus is related to the virus that causes hepatitis A (Reyes et al., Science 247, 1335-1339 (1990)) and produces an acute form of hepatitis without a chronic phase. The virus is enteric, waterborne, and normally transmitted by the fecal/oral route. It is particularly prevalent in the Indian subcontinent and causes a high mortality rate in pregnant women.

The present invention is also useful for the treatment of human papillomavirus, which is the agent responsible for non-genital warts, juvenile laryngeal papilloma, capitis and is implicated in cervical cancer. The present invention is also useful for the treatment of other viral infections, such as those caused by human cytomegalovirus and HIV. The present invention is also useful for the treatment of non-viral infections, such as tuberculosis and conditions such as asthma.

The human interferon for use in the invention may be any of the three types listed above, e.g. interferon α, interferon β or interferon γ . In general, the human interferon is interferon α or interferon γ. A preferred human interferon is human interferon α, more preferably human interferon α derived from a human cell line in culture or recombinant human interferon cc. According to one preferred embodiment, the human interferon is recombinant interferon α-2a or interferon α-2b, e.g. one of the products sold under the trademarks ROFERON and INTRON. According to another particularly preferred embodiment, the human interferon is human lymphoblastoid interferon (interferon α-Nl), e.g. a product manufactured by The Wellcome Foundation Ltd. under the trademark WELLFERON.

The term human interferon is intended to include any wild-type interferon whose sequence has been determined for a human, and any allele, variant or mutant thereof which substantially retains the activity of the corresponding wild-type sequence and which has greater than 80% sequence homology to the corresponding wild-type sequence.

Human interferons can be formulated for administration in accordance with the invention in the same manner as for use in the treatment of the condition in question alone, e.g. hepatitis. Thus, interferon is generally administered parenterally, e.g. by injection, preferably subcutaneous injection. Preferably, the interferon is formulated as an aqueous preparation or as a lyophilized product, designed for reconstitution with a suitable carrier, e.g. water for injection. The formulation may also contain a suitable carrier diluent or stabilizer, e.g. another human protein, such as e.g. human serum albumin.

Human interferon is generally administered in accordance with the treatment regimens already established for said product. For example, human interferon a, whether lymphoblastoid or recombinant, may be administered at a dose of 1 to 10 mega units of interferon per day. The dose may be administered on 3 or more days per week, preferably 3 times per week. A preferred dose range is 2 to 6 mega units of interferon per day, on 3 or more days per week, preferably 3 times per week, and specific doses of interferon are 5 mega units or more preferably 3 mega units per day or 3 or more days per week, preferably 3 times per week. In the treatment of hepatitis, the administration of interferon is usually for several weeks, e.g. 12 to 30 weeks, especially about 24 weeks, although in some cases longer periods of treatment of up to 1 year or more may be appropriate.

As used herein, the term free radical scavenger or precursor or inducer thereof means any substance that, when administered to a host, is capable of reducing the level of free radicals (also called oxidative stress) in the host. The substance may cause the reduction of free radicals by directly scavenging free radicals or by inducing, either as a direct biological precursor or by producing in the host a substance that has a scavenging effect on free radicals. Alternatively, the substance may reduce the level of free radicals by exerting an inhibitory effect on the processes that lead to the generation of free radicals.

Preferred free radical scavengers or their precursors or inducers include glutathione and its precursors, such as derivatives of the natural amino acid cysteine. A particularly preferred precursor of glutathione is N-acetyl cysteine. As indicated above, N-acetyl cysteine has well-established pharmaceutical use as a mucolytic and pharmaceutical preparations of the compound are commercially available. Other free radical scavengers or their precursors or inducers include vitamin A, vitamin C (ascorbic acid), vitamin E, uric acid, buthionine sulfoxime, metronidazole diethylmaleate, superoxide dismutase and methionine. Substances that inhibit the generation of free radicals include xanthine oxidase inhibitors, such as allopurinol, and are considered to be free radical scavengers or their precursors or inducers as herein provided.

The free radical scavenger or its precursor or inducer should be administered in a form and dose that is capable of reducing free radical generation and/or mitigating the effects of free radical generation (oxidative stress) in the host. Administration may be by any convenient route, e.g., orally or parenterally, depending on the nature of the substance. Oral administration is preferred where possible.

N-acetyl cysteine is preferably formulated for oral administration in the form of tablets or granules or a liquid preparation, e.g. syrup. A suitable dose of N-acetyl cysteine is in the range of 200 mg to 4 g per dose for administration up to 4 times a day, e.g. 400 to 800 mg for administration 4 times a day, preferably 600 mg for administration 4 times a day.

Although the therapy provided in accordance with the present invention, e.g. for viral hepatitis, consists of the combined administration of human interferon and a free radical scavenger or a precursor or inducer thereof, the two drugs are generally administered as separate preparations. However, in some circumstances it may be preferable to administer the two components as a combined preparation and the invention also encompasses such combined preparations.

Thus, according to a further aspect, the present invention provides a pharmaceutical composition comprising human interferon together with a free radical scavenger or a precursor or inducer thereof.

Generally, any such combination preparation is in a form intended for parenteral administration, e.g. by injection. Such a combination preparation may be in liquid form or in solid form with lyophilized human interferon suitable for reconstitution into a liquid form.

It is also convenient to provide the two drugs together in a convenient form for separate administration. According to another aspect, the invention provides a dual pack containing, for separate administration, human interferon and a free radical scavenger or a precursor or inducer thereof.

The present invention is particularly useful in the treatment of hepatitis B or hepatitis C. As mentioned above, the administration of human interferon, in particular recombinant or lymphoblastoid interferon, is an established therapy for hepatitis B. Furthermore, it has been shown from numerous experiments that the same therapy is also quite useful in the treatment of hepatitis C. According to the present invention, the therapy of patients suffering from hepatitis, and in particular hepatitis B or hepatitis C, with interferon is carried out essentially in accordance with the established regulations, with additional treatment with a free radical scavenger or its precursor or inducer during all or only part of the duration of the therapy with human interferon.

Serum alanine aminotransferase (ALT) levels are highly sensitive markers of liver dysfunction. Both hepatitis B and hepatitis C infections are characterized by elevated ALT levels, and disease progression is generally monitored by serum ALT determination. As noted above, only approximately 50% or fewer of patients with hepatitis B or hepatitis C respond to human interferon-α therapy, as demonstrated by significant clearance of viral markers or reduction in ALT levels.

According to one embodiment of the invention, which is particularly useful for the treatment of hepatitis B or hepatitis C, more particularly hepatitis C, treatment with human interferon, in particular human interferon α, is carried out in a conventional manner for a period of several weeks, e.g. 12 to 30 weeks and in particular about 24 weeks. For patients who fail to respond to an initial course of treatment with human interferon, as evidenced by reduced serum ALT levels, treatment with human interferon is continued and with additional treatment with a free radical scavenger or its precursor or inducer, preferably glutathione or its precursor or inducer, most preferably N-acetyl cysteine. Treatment with human interferon and the free radical scavenger or its precursor or inducer can be continued for a further period of several weeks, e.g. 12 to 30 weeks and in particular about 24 weeks. According to a preferred embodiment of the invention, this treatment regimen is useful for treating hepatitis C with human lymphoblastoid interferon (human interferon α-N1).

As also shown above, certain patients with hepatitis B or hepatitis C may initially respond to treatment with human interferon, particularly human interferon-α, but may later relapse. Such patients may benefit from a combination course of treatment with human interferon and a free radical scavenger or its precursor or inducer as mentioned above.

It is understood that the doses of human interferon and free radical scavenger or its precursor or inducer vary depending on the patient and the precise condition from which the patient suffers. Ultimately, the treatment is controlled and is the responsibility of the attending physician.

The present invention is further illustrated by the following pilot study, which is not intended to limit the scope of the invention in any way.

EXAMPLE

1. Introduction

Hepatitis C virus (HCV) is responsible for most cases of posttransfusion and sporadic non-A, non-B hepatitis. Chronicity of infection is very common and leads to chronic hepatitis, cirrhosis, and ultimately malignant degeneration. Various controlled studies have shown that interferon (IFN) is useful in the treatment of chronic hepatitis C (CHC), but response rates average 50% and the relapse rate after IFN withdrawal can reach 30-40%. Accordingly, the proportion of CHC patients who maintain normal transaminase levels after IFN withdrawal is only about 20-40% of all treated cases.

Reduced glutathione (GSH) is an important antioxidant in mammalian cells that is involved in a wide range of cellular functions, and it is thought that GSH depletion may play a pathogenic role in some chronic viral diseases, such as AIDS. In this study, we measured GSH levels in plasma and peripheral blood mononuclear cells (PBMC) from CHC patients who failed to respond to IFN therapy after at least 4 months of treatment. We also determined the effect of N-acetyl cysteine, a thiol precursor, on GSH levels and on clinical and virological response to IFN therapy.

2. Patients and procedures

2.1 Patients Patients (13 men and 1 woman, mean age 51 years, range 27-71) diagnosed as suffering from CHC by histological and serological criteria, two of them with associated cirrhosis, participated in the study. All patients were treated with α-lymphoblastoid interferon (Wellferon) for a minimum period of 4 months (15 ± 1.6 MU per week, range 9-21 MU per week); all patients showed abnormal ALT levels (above 30 IU/L) at study entry. Most patients with CHC responding to IFN had their transaminase levels normalized within the first 3 months of therapy and those who persisted with high ALT levels after 4 months of treatment could be considered non-responders. Therefore, all patients in this study were considered to be non-responders to IFN. Patients in this study continued essentially the same IFN regimen as before, with the addition of oral N-acetyl cysteine (NAC), 600 mg, every 8 hours daily. No patient had their interferon dose increased after the addition of oral NAC, although in three cases the interferon level decreased slightly (15 ± 1.8 MU/week before NAC vs. 11.5 ± 1.3 MU/week after NAC.

In addition, 10 patients (8 men and 2 women, mean age 32 years, range 24-63), with a recent diagnosis of chronic hepatitis C, who had never received antiviral treatment, received the same amount of oral NAC, but without interferon, for a period of 1 month.

Healthy subjects (14 men and 12 women, mean age 43 years, range 25 to 79) served as the control group. All patients provided written informed consent and the study was approved by the local ethics committee.

2.2 Determination of GSH in PBMC and plasma

Blood samples were obtained from each patient for simultaneous determination of GSH in PBMC (L-GSH) and platelet-poor plasma (P-GSH). PBMC were isolated by centrifugation on a lymphoprep (Nycomed Pharma AS, Oslo, Norway) and washed five times. The isolated cells were killed with 20% perchloric acid (2% final concentration) and after centrifugation (1200 g, 10 min at 4°C) the supernatants were stored at −40°C until use. 20% perchloric acid was added to platelet-poor plasma (2% final concentration) and after centrifugation the supernatants were stored at −40°C until GSH determination.

The stored samples were thawed and GSH was determined by the enzymatic method described by Brigellus et al., Biochem Pharmacol., 32, 2529-2534 (1983) and as modified by Ferrer et al., Biocehm. J. 264, 531-534 (1990). GSH in the presence of GSHS-transferase was conjugated with l-chloro-2,4-dinitrobenzene (CDNB) (SIGMA) and the absorbance of the complex was measured at 340 nm using a Perkin-Elmer Lambda 2 spectrophotometer. Absolute GSH values were obtained using a molar extinction coefficient of 9.6 χ 10 ³ .

2.3 RNA extraction and RT-PCR

Reverse transcription polymerase chain reaction (RT-PCR) for HCV-RNA in serum and for both positive and negative strand HCV-RNA in PBMC was performed essentially as described by Ruiz et al., Hepatology, 16, 637-643 (1992) and Cheng et al., J. Hepatol., in press (1992). We strictly followed the procedures recommended by

Kwoks and Higuchi, Nature, 339, 237-238 (1989), to reduce the risk of contamination. All extractions and reactions were performed simultaneously in positive and negative controls. An aliquot from the last wash of PBMC was also included and PCR was always negative in these samples.

2.4 Statistical analysis

All data are presented as means ± standard error of the mean (SEM). Comparisons for paired and unpaired data were performed using Mann Whitney and Wilcoxon tests.

3. Pictures

The results are described with reference to the accompanying drawings, where:

Figure 1 shows the effect of IFN (-4 to 0 months) and IFN plus NAC (0 to 6 months) on ALT levels for the 14 patients who participated in the study;

Figure 2 shows the detection of positive and negative strands of HCV-RNA in PBMCs during treatment with IFN and with IFN plus NAC;

Figure 3 shows the detection of HCV-RNA in serum at a serum dilution of 1:10 both before and after the addition of NAC to IFN treatment;

Figure 4 shows ALT levels in the patients described in Figure 1 above who responded 11 months after starting therapy with IFN plus NAC.

4. Results

The mean serum ALT levels for the 14 patients who participated in the study are shown in the following table, which also shows L-GSH and P-GSH.

TABLE

Month Mean ALT (IU/L) L-GSH (nmol/l ⁶ cells) P-GSH (μΜ) -4 139 ± 24 - - 0 124 ± 24 1.45 ± 0.27 0.77 ± 0.21 + 1-2 87 ±9 - - +3-4 53 ± 7 3.32 ± 0.18 2.40 ± 0.20 +5-6 37 ± 3

The values for L-GSH and P-GSH for the control group are as follows:

L-GSH 3.43 ± 0.89 nmol/10 ⁶ cells

P-GSH 18.1 ± 4.08 μM

In patients with chronic hepatitis C who had never received antiviral treatment (n = 10), there was a significant decrease in plasma (0.63 ± 0.07 μΜ) and PBMC (1.02 ± 0.09 nmol/10 ⁶ cells) GSH levels compared to healthy controls (18.1 ± 4.08 μΜ and 3.43 ± 0.89 nmol/10 ⁶ cells, respectively, P < 0.01). One month of NAC administration significantly increased PBMC GSH levels (2.22 ± 0.38 nmol/10 ⁶ cells, P < 0.05), but plasma GSH was not significantly modified (0.99 ± 0.22 μΜ, ns). Furthermore, serum ALT levels (128 ± 32 IU/L vs. 110 ± 29 IU/L after one month of NAC therapy) were not significantly altered.

In patients who do not respond to interferon, there is also a significant decrease in GSH levels in PBMC (1.45 ± 0.27 nmol/10 ⁶ cells) and in plasma (0.77 ± 0.21 μΜ) compared to control values (p < 0.01). In these patients, administration of NAC together with interferon, over a period of 3-4 months, results in a significant increase in GSH in mononuclear cells (3.32 ± 0.18 nmol/10 ⁶ cells, p < 0.05) and in plasma (2.40 ± 0.20 μΜ, p < 0.05).

As can be seen from Figure 1 and the table above, in the patients participating in the study (interferon-refractory), ALT levels did not change significantly during the 4 months of IFN therapy (139 ± 24 vs. 124 ± 17 IU/L, n.s.). However, the addition of oral NAC resulted in an immediate and significant decrease in ALT; even after only one month of combined treatment, ALT levels fell significantly (87 ± 9 IU/L, p < 0.05). Furthermore, continuous administration of IFN and NAC over a period of 5-6 months produced a further decrease in ALT levels in all cases (37 ± 4 IU/L), reaching normal values in 41% of cases and near-normal values (maximum 56 IU/L in one case) in the remaining cases. The addition of NAC to the IFN regimen clearly improved the response to IFN in patients previously classified as IFN-refractory. As can be seen in Figure 4, this decrease in ALT levels continued until the most recent patient analyses, 11 months after the start of combination therapy.

The reduction of ALT levels with the combination of IFN and NAC is accompanied by a combined effect on viral replication. In the case of 9 patients classified as IFN-refractory, we tested PBMC for the presence of both the HCV genomic strand (positive strand RNA) and the viral replicative intermediate (negative strand RNA) before and after the addition of NAC to the therapy. As can be seen in Figure 2, when patients were treated with IFN alone, the genomic strand could be detected in 7 cases (77%), while the replicative intermediate was detected in 3 patients (33%). However, after 4-6 months of combined therapy with IFN and NAC, the positive strand was detected in only 2 cases (22%), and the negative HCV-RNA strand could not be detected in any case.

The addition of NAC to the IFN regimen is also accompanied by reduced serum HCV-RNA levels. As can be seen in Figure 3, after the addition of NAC to therapy, an increased serum concentration is required for HCV detection; that is, before the addition of NAC, HCV-RNA can be detected in 100% of cases using a serum dilution of 1:10, while after the addition of NAC to therapy, at the same serum dilution, the virus is detected in only 70% of patients.

For

The Wellcome Foundation Limited:

PATENT OFFICE

Claims (30)

PATENT APPLICATIONS CLAIMS 1. An enhanced combination comprising human interferon and a free radical scavenger or precursor or inducer thereof. The combination of claim 1, wherein the human interferon is human interferon a. Combination according to claim 2, characterized in that human interferon a is recombinant. Combination according to claim 2, characterized in that human interferon a is natural. 5. The combination of claim 4, wherein the natural human interferon is human lymphoblastoid interferon. Combination according to any one of claims 1 to 5, characterized in that the free radical scavenger or its precursor or inducer is glutathione or its precursor or inducer. A combination according to any one of claims 1 to 5, characterized in that the free radical scavenger or its precursor or inducer is N-acetyl cysteine. A combination according to any one of claims 1 to 7, characterized in that it is useful in the treatment of a condition susceptible to interferon therapy. Combination according to claim 8, characterized in that the condition is hepatitis B or hepatitis C infection. Use of human interferon for the manufacture of a medicament for use in the treatment of a patient suffering from a condition susceptible to human interferon therapy, by a method also comprising administering a free radical scavenger or precursor or inducer thereof during human interferon therapy. Use of a free radical scavenger and its precursor or inducer for the manufacture of a medicament for the treatment of a patient suffering from a condition susceptible to human interferon therapy by a method comprising treatment with the human interferon and free radical scavenger or its precursor or inducer. Use according to claim 10 or 11, characterized in that the human interferon is human interferon a. Use according to claim 12, characterized in that human interferon a is recombinant. Use according to claim 12, characterized in that human interferon a is natural. Use according to claim 14, characterized in that the natural human interferon is human lymphoblastoid interferon. Use according to any one of claims 10 to 15, characterized in that the free radical scavenger or its precursor or inducer is glutathione or its precursor or inducer. Use according to any one of claims 10 to 15, characterized in that the free radical scavenger or its precursor or inducer is N-acetyl cysteine. Use according to any one of claims 10 to 17, characterized in that the condition is hepatitis B or hepatitis C infection. 19. The use of human interferon for the manufacture of a medicament for use in the treatment of a patient suffering from a condition susceptible to human interferon therapy, which is unable to respond to human interferon therapy, by a process which also provides a free radical scavenger or precursor thereof, or inducer during human interferon treatment. 20. The use of a free radical scavenger or its precursor or inducer for the manufacture of a medicament for the treatment of a patient suffering from a condition susceptible to human interferon therapy which cannot respond to human interferon therapy by a method comprising human interferon therapy and free radical scavengers or precursors or in19 ductors thereof. Use according to any one of claims 19 and 20, characterized in that the patient has previously responded to treatment with human interferon. Use according to any one of claims 19 to 21, characterized in that the human interferon is human interferon a. Use according to claim 22, characterized in that human interferon a is recombinant. Use according to claim 22, characterized in that human interferon a is natural. Use according to claim 24, characterized in that the natural human interferon is human lymphoblastoid interferon. Use according to any one of claims 19 to 25, characterized in that the free radical scavenger or its precursor or inducer is glutathione or its precursor or inducer. Use according to any one of claims 19 to 25, characterized in that the free radical scavenger or its precursor or inducer is N-acetyl cysteine. Use according to any one of claims 19 to 27, characterized in that the condition is hepatitis B or hepatitis C infection. A pharmaceutical formulation comprising a combination as described in any one of claims 1 to 7 and a pharmaceutically acceptable carrier. 30. A double pack comprising the combined components required for administration to produce the combination as described in any one of claims 1 to 7.