WO1998038986A2

WO1998038986A2 - Use of carvedilol or derivatives thereof for the manufacture of a medicament for the prevention and treatment of viral infections

Info

Publication number: WO1998038986A2
Application number: PCT/EP1998/001241
Authority: WO
Inventors: Akira Matsumori
Original assignee: Roche Diagnostics GmbH; Boehringer Mannheim GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1997-03-06
Filing date: 1998-03-05
Publication date: 1998-09-11
Anticipated expiration: 1999-09-06
Also published as: WO1998038986A3; JPH10251148A; JP4126345B2; AU7332498A

Abstract

Medicament for preventive and therapeutic treatment of viral infections which comprises as an active ingredient a substance selected from the group consisting of carvedilol, optically active isomers thereof, its hydroxy carbazole derivatives and pharmaceutically acceptable salts thereof.

Description

MEDICAMENT FOR PREVENTIVE AND THERAPEUTIC TREATMENT OF VIRAL INFECTIONS

BACKGROUND OF THE INVENTION

Technical Field

The present invention relates to a medicament. More specifically, it relates to a medicament useful for preventive and/or therapeutic treatment of viral diseases.

Related Art

Acute myocarditis is a disease accompanied by inflammatory disorders of myocardium. While its prognosis is relatively good when patients are completely recovered from acute stage, it sometimes progresses to chronic state with repetition of increment and arrest of inflammation, and further advances to dilated cardiomyopathy. Dilated cardiomyopathy is an intractable disease, i.e.. approximately a half of the patients die within 5 years after onset, and the patient suffering from this disease accountfor a half of heart transplantation patients in Europe and the United States. Therefore, it is extremely important to treat acute myocarditis for immediately recover from its acute stage, and prevent it from progressing to chronic or intractable state.

Although the cause of acute myocarditis has not yet been fully clarified (noncausal acute myocarditis is sometimes referred to as idiopathic myocarditis), it is considered that most of cases involve viral infection, because presence of viral genes was reported in myocardial biopsy specimens from acute myocarditis and dilated cardiomyopathy patients (cases in which viral infection are directly or indirectly verified are sometimes referred to as viral myocarditis) Persistent infection or repetitive infections of causative virus may advance myocarditis to chronic state through an autoimmune mechanism activated by the infection, and may cause intractable dilated cardiomyopathy Therefore, in the acute stage of acute myocarditis, sufficient treatment of viral infection is required to prevent myocarditis from progressing to chronic state or intractable state of dilated cardiomyopathy, as well as treatment of inflammation of myocarditis (as for dilated cardiomyopathy and myocarditis, see, for example, "Integrated Handbook of Internal Medicine", Vol, 32, "Cardiomyopathy and Myocarditis", published by Nakayama Shoten Co., Ltd. pp. 3 - 9 and pp. 347 - 351).

Although various researches have been made for the treatment of acute myocarditis both from clinical and experimental viewpoints, satisfactory results have not been obtaiend so far to date. For example, steroids are symptomatically applied to inflammation However, steroids may likely advance the infection in the acute stage, and severe adverse effects of steroid, per se, may sometimes be serious clinical problems. Therapeutic effectiveness of antiserum containing specific antibodies and preventive effectiveness of vaccines have been experimentally revealed. However, the methods are considered impractical clinically as it is difficult to identify causative viruses. Development of anti-viral agents exhibiting broad anti-viral spectrums has been desired, however, their practical applications have not yet been achieved.

It is known that cytokines such as TNF-α, interferon-γ, and interleukin- 10 are produced in hearts of viral myocarditis mice [Shioi et al . Research Reports ( 1994), the Ministry of Health and Welfare, Specific diseases, isopathic myocarditis survey and research group, pp. 165 - 167, 1995], In addition, it has experimentally demonstrated that myocardial lesions were recovered and virus amount in myocardia was reduced by administration of an interferon. Therefore, it might be possible that viral myocarditis could be treated by administration of exogenous interferon that has an inhibitory activity against viral proliferation. However, since interferons themselves have inflammatory actions and the like, it is not preferred to administer an exogenous interferon to a patient of myocarditis at acute stage from a viewpoint of an adverse effect. For these reasons, developments of medicaments have been strongly desired which can enhance the effects of symptomatic treatments for inflammation and prevent myocarditis from progressing to intractable or chronic state by reducing or completely eliminating viral infection involved in acute myocarditis. A medicament, which can eliminate viral infection through endogenous mechanism inherent in living bodies, is expected to be particularly useful for the purpose mentioned above.

Carbazolyl-(4)-oxy-propanolamine derivatives having vasodilating and antihypertensive actions are known, and their activities have been clarified to be based on β-blocking action (EP-A-0 004 920). It is also known that their optically active isomers have similar activities (EP-A-0 127 099). Carvedilol, a typical compound among these compounds, i.e., [(±) x 1 (carbazole-4-yloxy)-8-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propanol], has already been widely used clinically for the treatments of hypertension and stenocardia (trade name. Dilatrend, manufactured and sold by Boehringer Mannheim GmbH).

As regards the relation between β-receptors and cytokine production, it has been reported that a β-agonist such as salbutamol inhibits the production of interferon-γ in peripheral blood monocytes stimulated by phytohemagglutinin (Coquert, O . et al . Clin. Exp. Allergy, 25, pp. 304 - 31 1, 1995). However, relation between β-blockers and cytokine production has not yet been clarified. As also to the carbazolyl-(4)-oxy- propanolamine derivatives such as carvedilol mentioned above (EP-A-0 004 920), no publication so far to date teaches or suggests their relations to an interferon production.

An object of the present invention is to provide a medicament useful for preventive and or therapeutic treatment of viral infections. More specifically, the object of the present invention is to provide a medicament useful for preventive and/or therapeutic treatment of viral infections that can eliminate viral infections through an endogenous mechanism inherent in living bodies. Preferably, the object of the present invention is to provide a medicament useful for preventive and/or therapeutic treatment of acute myocarditis involving viral infection, and a medicament that enables treatment of acute myocarditis and can prevent acute myocarditis from progressing to intractable or chronic state. SUMMARY OF THE INVENTION

The inventor of the present invention conducted various researches to achieve the foregoing objects. As a result, he found that, when (±)-l-(carbazoie-4-yloxy)-3-[[2-(o- methoxyphenoxy)ethyl)amino]-2-propanol was administered to myocarditis mice inoculated with Encephalomyocarditis (EMC) virus, amounts of interfereon-γ (sometimes abbreviated as 'TEN x γ" hereinafter in the specification) produced in the hearts were increased, and thereby virus titers in the hearts were lowered The present inventors conducted further researches and found that the above compound had an activity of enhancing IFN-γ production in living bodies and exhibited the activity of eliminating viral infection based on the IFN-γ production-enhancing activity The present invention was achieved on the basis of these findings.

The present invention thus provides a medicament for preventive and/or therapeutic treatment of viral diseases, which comprises, as an active ingredient, a substance selected from the group consisting of l-(carbazole-4-yloxy)-3-[[2-(o-methoxyphenoxy)- ethyl(amino]-2-propanol, optically active isomers thereof, its hydroxycarbazole derivatives and pharmacologically acceptabl salts thereof. According to preferred embodiments of the present invention, there are provided, the aforementioned medicament for preventive and/or therapeutic treatment which is applied to myocarditis involving viral infection; the aforementioned medicament for preventive and/ or therapeu- tic treatment wherein myocarditis is acute myocarditis; the aforementioned medicament for preventive and/or therapeutic treatment which is used for preventing myocarditis from progressing to intractable and/or chronic state; and the aforementioned medicament for preventive and/or therapeutic treatment which has an activity of eliminating viral infections based on the IFN-γ production-enhancing action in living bodies.

According to a further embodiment of the present invention, there is provided an EFN-γ production-enhancing agent comprising, as an active ingredient, a substance selected from the group consisting of l-(carbazole-4-yloxy)-3-[[2-(o-methoxyphenoxy)- ethyl)amino]-2-propanol, optically active isomers thereof, and pharmacologically acceptable salts thereof. According to still further embodiments of the present invention. there are provided a use of the above substance for the manufacture of the aforementioned medicament for preventive and/or therapeutic treatment of viral diseases; use of the above substance for the manufacture of the aforementioned IFN-γ production- enhancing agent; and method for treatment of acute myocarditis involving viral infection, or a method for preventing the advancement of acute myocarditis involving viral infection, which comprises administering to a mammal including a human a substance selected from the group consisting of l-(carbazole-4-yloxy)-3-[[2-(o-methoxyphenoxy)- ethyl]amino]-2-propanol, optically active isomers thereof, its hydroxy carbazole derivatives and pharmacologically acceptable salts thereof.

DETAILED DESCRIPTION OF THE INVENTION

The substances selected from the group consisting of l -(carbazole-4-yloxy)-3-[[2-(o- methoxyphenoxy)ethyl]amino]-2-propanol, optically active isomers thereof, its hydroxy carbazole derivatives and pharmacologically acceptable salts thereof used as the active ingredient of the medicament of the present invention are known, and easily obtained by those skilled in the art. For example, EP-A-0 004 920 specifically discloses, as Example 2, a method for the preparation of the racemate of the compounds mentioned above, and EP-A-0 127 099 specifically discloses optically active isomers thereof. Furthermore, EP-A-0 009 420 and EP-A-0 127 099 also specifically disclose pharmacologically acceptable salts of the compounds. As the active ingredient of the medicament of the present invention, any one or more of racemates, optically active isomers in an optically pure form, any mixtures of the optically active isomers, and physiologically acceptable salts of the compounds can be used. In addition, any hydrates and solvates of these substances may also be used.

The carvedilol derivatives which are hydroxylated at the. carbazole ring are described in WO-A-94/12718. A preferred derivative is l-(3-hydroxycarbazole-4-yloxy)-3-[[2-(o- methoxyphenoxy)ethyl]amino]-2-propanol.

The medicament of the present invention has an activity of enhancing the production of IFN-γ in living bodies, and exhibits an activity of eliminating a viral infections based on the EFN-γ production enhancing action. Therefore, the medicament of the present invention is useful for preventive and/or therapeutic treatment of viral diseases caused by various viral infections. The medicament of the present invention can be used for preventive and/or therapeutic treatment of the above diseases of mammals including humans.

Examples of the viral diseases to which the medicament of the present invention can be applied include viral diseases caused by one or more pathogenic viruses belonging to either of DNA viruses or RNA viruses. Examples of pathogenic virus include DNA viruses such as poxvirus, herpes virus, adenovirus, and parvovirus, and RNA viruses such as revovirus, togavirus, coronavirus, rhabdovirus, paramixovirus, orthomyxovirus, bunvavirus, arenavirus, retrovirus, picornavirus, and calicivirus. However, the viruses to which the medicament of the present invention can be applied are not limited to these viruses

Examples of the viral diseases to which the medicament of the present invention can be applied include viral hepatitis (type A, B, C, E etc.), influenza, virus pneumonia, viral bronchitis, herpes infections (herpes simplex virus, EB virus [glandular fever] or herpes zoster), acute anterior poliomyelitis, AIDS (HIV infection), adult T cell leukemia (ATL), papilloma, measles, German measles, roseola infantum, erythema infectiosum. viral encephalitis, viral meningitis, cytomegalovirus infection, mumps, varicella, hydrophobia, viral enteritis, viral myocarditis, and viral pericarditis. However, the viral diseases to which the medicament of the present invention can be applied are not limited to these viral diseases. Sorts of organs and tissues suffered from viral infection are also not particularly limited. Examples include heart, liver, kidney, pancreas, brain, lung, blood and the like.

Because it is suggested that most cases of myocarditis such as acute myocarditis are involved with viral infection, myocarditis such as acute myocarditis is a preferred disease to which the medicament of the present invention is applied. Although the medicament of the present invention may be applied in a each case of myocarditis, in which infection of pathogenic virus is not verified directly or indirectly, it is preferable that the medicament of the present invention is applied to myocarditis in which involvement of viral infection is verified directly or indirectly Viral infection can be directly verified by, for example, biopsy of cardiac tissue, and indirectly verified by, for example, measuring virus antibody titer in blood. By administering the medicament of the present invention, viral infection of myocardium in acute myocarditis including idiopathic myocarditis can be promptly eliminated, and thereby the cause of acute myocarditis can be eliminated. In addition, by eliminating viral infection, it becomes possible to prevent acute myocarditis from progressing to chronic state or to intractable dilated cardiomyopathy.

Although the substance mentioned above, per se, may be used as the medicament of the present invention, it is usually preferred that pharmaceutical composition containing the above substance as an active ingredient is formulated by using pharmaceutical additives available to those skilled in the art Examples of pharmacologically and pharmacologically acceptable additives include excipients, disintegrating agents or disintegrating aids, binders, lubricants, coating agents, coloring matters, diluents, base material, dissolving agents or dissolving aids, isotonic agents, pH modifiers, stabilizers, propellants, adhesives and the like. Examples of the formulation suitable for oral administration include tablets, capsules, fine granules, granules, liquids, syrups and the like. Examples of the formulations suitable for parenteral administration include injections, drip infusions, suppositories, inhalants, transmucosal preparation, transdermal preparation, nasal drops, ear drops, patches and the like.

Pharmaceutical preparations suitable for oral, transdermal or transmucosal administration may contain, as pharmacologically or pharmacologically acceptable additives, for example, excipients such as glucose; disintegrating agents or disintegrating aids such as carboxymethylcellulose; binders such as hydroxymethylcellulose; lubricants such as magnesium stearate; coating agents such as hydroxypropylmethylcellulose; base materials such as vaseline and the like. In addition, as pharmaceutical additives, propellants such as compressed gases; thickeners such as sodium polyacrylate; base cloths such as cotton cloth and the like can also be used. Pharmaceutical preparations suitable for injections and drip infusions may contain, as pharmaceutical additives, aqueous mediums such as distilled water for injection; dissolving agents or dissolving aids which can be contained in injections dissolved before use; isotonic agents such as glucose; pH modifiers such as inorganic aids, organic aids, inorganic bases, and organic bases

A pharmaceutical preparation containing the particularly preferred active ingredient of the medicament of the present invention, i.e., carvedilol [(±)-l-(carbazole-4-yloxy)-3- [[2-(o-methoxyphenoxy)ethyl]amino]-2-propanol], has already been widely used clinically as ethical medicaments for hypertension and angina pectoris (trade name' Dilatrend, manufactured and sold by Boehringer Mannheim GmbH). Therefore, this pharmaceutical product, per se, may be used as the medicament of the present invention Administration dose of the medicament of the present invention should be varied depending on various factors including the sorts of applicable diseases, conditions and age of patients, purpose of prevention or therapeutic treatment and the like, and can be suitably selected by those skilled in the art by considering these factors It has been already verified that the preferred active ingredient of the medicament of the present invention, carvedilol, is a highly safe substance as used clinically.

EXAMPLES

The present invention will be further explained more specifically by referring to the following examples. However, the scope of the invention is not limited to these examples. In the examples, carvedilol [(±)-l-(carbazole-4-yloxy)-3-[[2-(o-methoxy- phenoxy)ethyl]amino]-2-propanol] was used as the medicament of the present invention, and compared with metoprolol [l-(isopropylamino)-3-[p-(β-methoxyethyl)phenoxy]-2- propanol tartrate], which is also known as a compound which has β-receptor blocking activity

Example 1:

Enhancing activitv of carvedilol on INF-γ production in the heart of viral mvocarditis mouse

Carvedilol (Daiichi Pharmaceutical Co., Ltd.) or metoprolol (Sigma Chemical Co ) was dissolved in phosphate buffered saline (PBS) containing 1% methylcellulose for the use of this experiment. As EMC virus for inoculation, M variant (obtained from American Type Culture Collection) was used, and the concentration was adjusted to 100 pfu/ml (pfu: plaque forming unit) with Eagle MEM culture medium (EMEM: Nissui Pharmaceutical Co., Ltd.) before use. Each of 4-week old DBA/2 male mice in three groups was intraperitoneally inoculated with 0.1 ml of the EMC virus (10 pfu/mouse). From the day of the inoculation, the mice were orally administered with a test compound at the doses set out below every single day. On the seventh day, the hearts were isolated from survived mice, and the hearts were bled and washed with PBS and weighed.

Test group : 10 mg kg body weight of carvedilol

Comparative group: 30 mg/kg body weight of metoprolol

Control group: PBS

The isolated heart was added with 1 ml PBS and then homogenized by using a sonicator (ASTRASON) for 20 seconds. Total volume of the heart was measured, and the whole homogenate was centrifuged at 4°C for 20 minutes (14,000 x rpm, 10,000 g), and the supernatant was separated as samples. The viral myocarditis mice were prepared according to the method of Matsumori et al. (Matsumori, A. and Kawai, C, Circulation, 66. pp. 355 - 360, 1982). Samples were prepared by the methods of Sekido et al. (Sekido, N. et al.. Nature, 365, pp. 654 - 657, 1998) and Torre-Amione et al. (Torre-Amione, G., et al., Circulation, 93, pp. 704 - 711, 1996) with some partial modifications. IFN-γ assay was performed by using INTERTEST™-γ mouse interferon-γ ELISA kit (Genzyme), and the amounts of IFN-γ were indicated as per mg of heart. Statistical analysis was performed by a variance analysis (ANOVA) method according to the multiplex comparison method of Bonferroni, and when p < 0.05. it was judged that there was statistically significant difference. For each group, 9 mice were subjected to the analyses.

While there was no significant difference between the comparison group and the control group as for the amount of IFN-γ per mg of heart, the amounts of LFN-γ per mg of heart of the test group were significantly higher than those of the control group, and in addition, the amounts of IFN-γ was significantly higher compared to the results of comparative group, which revealed the IFN-γ production enhancing activity of carvedilol. The results are shown in Table 1

Table 1

(Mean ± Standard error)

** p < 0 05 vs. Control group and Comparison group

Example 2:

Eliminating activity of carvedilol against viral infection in viral myocarditis mouse

A test compound, comparative compound, and inoculated EMC virus were prepared and used in the same manner as in Example 1. Each of 4-week old DBA/2 male mice, divided in three groups, was intraperitoneally inoculated with 0.1 ml of the EMC virus (10 pfu/mouse). From the day of the inoculation, the mice were orally administered with a test compound at the doses set out below every single day. On the seventh day, the heart rates were isolated from survived mice under sterilized condition, and the hearts were bled and washed with sterilized PBS and weighed.

Test group: 10 mg/kg body weight of carvedilol

Comparative group: 30 mg/kg body weight of metoprolol

Control group PBS The isolated hearts were added with 1 ml of sterilized PBS per weight ( 1 mg) and then homogenized using a sonicator (ASTRASON) for 2 minutes. Total volume of the heart was measured, and the whole homogenate was centrifuged at 4°C for 15 minutes (1,500 x 5,000 rpm), and then the supernatant was separated as samples. Virus titer was determined by EL-plaque assay method (Matsumori, A., et al.. Circulation, 71, pp. 834 - 839, 1985). FL cells (human amnion cells) were cultured to confluent growth as a monolayer in 4 ml of EMEM containing 10% fetal cow serum (FCS) using a 6-well plate (Corning) at 37°C under 5% CO . Then, the wells containing the monolayer of the grown FL cells were washed with PBS three times. One ml of diluted sample was added into these wells, and the mixture was incubated for 1 hour with occasional shaking Four ml of EMEM containing 2% FCS and 1% methylcellulose was added to the mixture, and incubation was further continued for 30 hours at 37°C under 5% C0₂

After the incubation, the cells were fixed with acidic ethyl alcohol and stained with Crystal Violet, and then numbers of plaques were counted. The plaque were twice counted and their average was used as measured value, and virus titer was represented as Log pfu/mg of heart. Statistical analysis was performed by the Kruskal-Wallis test When p < 0 05. it was judged that there was statistically significant difference. 12 mice for the test group, 17 mice for the comparison group, and 18 mice for the control group were finally used While there was no significant difference between the comparison group and the control group as to the virus titer per mg of heart, the test group exhibited a significant decrease of a virus titer compared to the control group, and in addition, the virus titer was significantly decreased compared to the results of comparative group, which revealed the elimination activity of the compound of the present invention on virus infection. The results are shown in Table 2. Table 2

(Mean ± Standard error)

** p < 0.05 vs. Control group and Comparison group

The medicament of the present invention has the activity of enhancing INF-γ production in living bodies, and eliminating activity against viral infection based on the IFN-γ production enhancing activity. Therefore, the medicament of the present invention is useful for preventive and/or therapeutic treatment of viral diseases. For example, the cause of acute myocarditis involving viral infection can be radically eliminated, and thereby effective treatment of acute myocarditis can be achieved.

Claims

WHAT IS CLAIMED IS:

1_. Use of a substance selected from the group consisting of l-(carbazole-4-yloxy)-3- [[2-(o-methoxyphenoxy)ethly]amino]-2-propanol, optically active isomers thereof, its hydroxy-carbazole derivatives and pharmacologically acceptable salts thereof as an active ingredient for the preparation of a medicament for preventive and/or therapeutic treatment of viral infections.

2. The use according to claim 1, wherein the active ingredient is carvedilol.

3. The use according to claim 1, wherein the active ingredient is l-(3-hydroxy- carbazole-4-yloxy)-3-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propanol.

4. The use according to claims 1 to 3, wherein the medicament is applied to myocarditis involving viral infection.

5. The use according to claim 4, wherein myocarditis is acute myocarditis

6. The use according to claim 5, wherein the medicament is used for preventing myocarditis from progressing to intractable and/or chronic state.

7 The use according to any one of claims 1 to 6, wherein the medicament has an activity of eliminating viral infections based on IFN-γ production-enhancing activity in living bodies.

8 Use of a substance selected from the group consisting of l-(carbazole-4-yloxy)-3- [[2-(o-methoxyphenoxy)ethyl]amino]-2-propanol, optically active isomers thereof, its hydroxy-carbazole derivatives and pharmacologically acceptable salts thereof as an active ingredient for the preparation of an IFN-γ production-enhancing agent.