DE19860802A1 - Production of an agent against hepatitis B, HI, Paramyxo and Orthomyxo viruses - Google Patents

Abstract

A substance of the general formula I DOLLAR F1 in which DOLLAR A (A) R¶1¶ and R¶2¶ are each hydrogen and R¶3¶ is a halogen, -CF¶3¶, alkoxy with 1 or 2 carbon atoms or halogen-substituted alkoxy with Is 1 or 2 carbon atoms, DOLLAR A (B) R¶2¶ is hydrogen and R¶2¶ and R¶3¶, which are the same or different, are halogen or -CF¶3¶, DOLLAR A (C) R ¶1¶ is hydrogen, R¶2¶ is alkyl with 1 or 2 carbon atoms and R¶3¶ is halogen, or DOLLAR A (D) R¶1¶ is hydrogen and R¶2¶ and R¶3¶ 3 ' , 4'-methylenedioxy is used to produce an agent for the treatment of hepatitis B viruses, HI viruses, viruses of the paramyxo group and / or the orthomyxo group, or for treating an infection of such viruses, or for detecting such viruses.

Description

The invention relates to the use of a substance of the general Formula I for the preparation of an agent for the treatment of hepatitis B viruses (HBV), HI viruses, viruses of the paramyxo group and / or the orthomyxo group, a method of reducing or inhibiting growth, or Propagation of such viruses, and a method for the detection of such Viruses.

Hepatitis B virus infection leads to acute and in certain cases chronic liver disease. According to estimates, worldwide WHO persistently infected about 350 million people with HPB. Chronic Infected people are at high risk of chronic active hepatitis, Liver cirrhosis or hepatocellular carcinoma. Even though Highly effective HV vaccines that have been available since 1982 can already infected patients are not treated with it. The available antiviral Therapy with interferon alpha is associated with side effects and only with some of the patients can be used and are effective. Newer treatment possibilities with nucleoside analogues such as 3'thiacytidine appear promising and tolerable, but the long-term effectiveness is so far unknown, especially considering the development of resistance already observed. The provision of agents with minor side effects that are not intended to Leading resistance is therefore desirable. The increasing Importance of combination therapies requires the development of additional ones Means that are associated with other stages of the infection cycle or disease ver interfering.  

In most cases, an HIV infection leads to different lengths of time Latency phases for the development of AIDS (acquired immune deficiency syndrome), whose clinical picture and course in addition to the effects of Primary infection by the HI virus to a considerable extent from secondary infections is determined. Combination therapy with multiple effects substances (nucleoside analogs, protease inhibitors) also play a role in therapy HIV-infected patients play the crucial role, but that's just it a delay and relief of the course of the disease possible, none AIDS cure. A vaccine is currently not available.

Viruses of the Orthomxyviridae family include group A influenza viruses, B and C. Influenza A and B cause viruses in infected patients Range of clinical symptoms, from asymptomatic to viral Pneumonia with fatal outcome is enough. These viruses can, conditionally through reassortment of genetic information between different Tribes, trigger global epidemics with excessive mortality. Vaccination of risk groups with the annual recommended by the WHO Inactivated vaccine composition is routinely performed. Amantadine and Rimantadine are available for prophylaxis and therapy Antivirals are available, but are only effective against influenza A, not against influenza B, which in a quarter of cases for hospitalization is responsible, or against resistant strains. Paramyxoviruses include mumps, measles, dog flu, bovine and respiratory Syncytial virus.

Therefore, there has long been a need for effective means against Hepatitis B viruses, HI viruses, viruses of the paramyxo and orthomyxo groups, the have minor side effects and inexpensive in suitable Dosage forms can be provided.

An object of the present invention is therefore to provide means for Treatment of hepatitis B viruses, HI viruses, viruses of the paramyxo and / or  to provide the orthomyxo group, which has a good activity against these viruses have minor side effects.

According to the invention, this object is achieved by using a substance of the general formula I.

wherein

• A) R ₁ and R _{2 are} each hydrogen and R _{3 is} a halogen, -CF ₃ , alkoxy with 1 or 2 carbon atoms or a halogen-substituted alkoxy with 1 or 2 carbon atoms,
• B) R _{2 is} hydrogen and R ₂ and R ₃ , which are the same or different, are halogen or -CF ₃ ,
• C) R _{1 is} hydrogen, R _{2 is} alkyl with 1 or 2 carbon atoms and R _{3 is} a halogen, or
• D) R _{1 is} hydrogen and R ₂ and R _{3 are} 3 ', 4'-methylenedioxy, for the preparation of an agent for the treatment of hepatitis viruses, HI viruses, viruses of the paramyxo group and / or the orthomyxo group.

The compounds of the formula I are classified under the leflunomides. Leflunomide denotes a group of substances of N- (4-trifluoromethyl phenyl) -5-methylisoxazole-4-carboxamiden, which was originally used as a herbicide in agriculture. In the meantime, she has also become one anti-inflammatory and immunosuppressive effect, so that their use, especially in transplant patients, for suppression  a transplant rejection seems possible. An antiviral activity for However, leflunomide or its derivatives have not been reported to date.

It was surprising that the substances used according to the invention in addition to herbicide activity and anti-inflammatory and one immunosuppressive effect also good effectiveness in particular against hepatitis B viruses, HI viruses, viruses of the paramyxo group and the Show orthomyxo group.

The agents obtained according to the invention with substances according to formula I as Active ingredients are characterized by their high effectiveness, so already small amounts are sufficient for successful treatment.

On the other hand, substances according to formula I are well tolerated, so that the in the dose obtained according to the invention is not used adverse side effects is limited. In addition, the Substances used according to the invention are unproblematic in their Manufacture and thus easily and inexpensively available in large quantities bar.

In the substance according to formula I, the oxazole ring is closed or open in front. After taking an agent obtained according to the invention, the Includes substance according to formula I with a closed oxazole ring this metabolizes in the body and the oxazole ring opens, causing a more physiologically effective form of the substance (basic structure: N- (4- Trifluoromethylphenyl) -2-cyano-3-hydroxycrotonamide (A771726)) is formed.

The agent obtained according to the invention can furthermore be conventional carrier, auxiliary or / and contain additives which are known to the person skilled in the art and therefore do not have to be mentioned explicitly. Are particularly preferred Carriers, auxiliaries and / or additives that increase the shelf life, absorption, Increase kinetics and / or the tolerance of the agent according to the invention.  

The agent preferably contains at least one pyrimidine, preferably one Uridine, cytidine and / or thymidine. By using pyrimidine in the compatibility of the agent according to the invention is used Substance according to formula I further increased and possible side effects can be reduced. It also makes it possible to Increase dose of the substance used according to the invention. So can a serious virus infection can also be effectively combated. The The pyrimidine content can be up to 90% by weight, preferably 5 to 70% by weight, more preferably 10 to 60% by weight and most preferably 30 up to 40% by weight.

In preferred embodiments, the substance of the formula I is used Production of anti-virus products from the hepatitis group B virus, HIV-1 or HIV-2 virus, avian paramyxovirus, parainfluenza virus, preferably type 1, mumps virus, measles virus, canine distemper virus, Rinderpest virus, pneumovirus and influenza virus, preferably influenza A Virus.

As a substance according to formula I for the agent, (A) N- (4-trifluoro methylphenyl) -5-methylisoxazole-4-carboxamide, (B) the open form Oxazole ring of (A) N- (4-trifluoromethylphenyl) -2-cyano-3-hydroxycroton amide, (C) a sodium salt of N- (4-trifluoromethylphenyl) -2-cyano-3- hydroxy-crotonamide, (D), (E), (F) and / or a mixture thereof.

The content of the substance according to formula I in the agent according to the invention can, depending on the type of virus to be treated and, if necessary, the The severity of the virus infection can be selected and adjusted.

The agent produced according to the invention is generally in such Amount administered, the 0.1 to 100 mg / day of substance of formula I. corresponds, depending on the protein binding of the respective substance.  

In a further embodiment, the agent can be further antiviral contain effective substances. The selection of Sub is advantageous punch the same virus type specificity as that according to the invention have used substance. This has the advantage of being effective against a certain virus type, possibly through synergistic Effects of the substances can be increased. If applicable, this shows Substance a virus type specificity other than the substance according to formula I, so that, on the other hand, in addition to the primary virus, secondary virus Infections can be fought.

The agent produced according to the invention can be in any suitable form be prepared. The agent is preferably in the form of a Tablet, a prolonged-release tablet, a coated tablet, a capsule, a granulate, an ampoule, solution for infusion, solution for injection or one Concentrate for solution for infusion or injection prepared solution.

In a further embodiment, the invention relates to a method for Reduce or inhibit the growth of hepatitis B viruses, HI viruses, Viruses of the paramyxo group and / or the orthomyxo group in with these Virus infected cells, comprising contacting the cells with a sufficient effective amount of a substance according to formula I to the Slow down or inhibit virus replication.

In a preferred embodiment of this method, the Reduce or inhibit the growth or multiplication of the viruses achieved by slowing down or inhibiting virus assembly.

The effective amount of a substance according to formula I is the adapted the respective virus type and the infected cells in a suitable manner. In general, the effective amount of the substance of formula I is in a concentration of 1 to 1000 µM.  

In yet another embodiment, the invention relates to a method for the detection of a hepatitis B virus, a HI virus or a virus of Paramyxo group or the Orthomyxo group, characterized in that is that 1. to patient cells in culture to a substance according to formula I. is added to the culture medium and 2. cytopathic or cell ver changing effects compared to a control culture without adding this Substance to be measured. This procedure has the advantage that simple visual inspection makes it possible to detect the viruses mentioned above is.

In a further embodiment, the invention relates to a method for prophylactic or therapeutic treatment of infections through Hepatitis B viruses, HI viruses, viruses of the paramyxo group and / or Orthomyxo group by administering an agent to a patient, which contains a substance of general formula I. The amount of substance according to formula I, the virus type present and the Adjusted the severity of the virus infection. The patient becomes useful a daily dose of 0.1 to 100 mg of the substance according to formula I. administered. The administration of the agent obtained according to the invention can be done in any suitable manner, preferably a systemic Application by e.g. B. oral, intravenous, intramuscular or subcutaneous Application takes place. The agent with the substance according to formula I can Patients can also be administered in the form of an aerosol. Another possible type of application is topical, e.g. B. in the form of drops, creams, Plasters or suppositories.

As a substance according to formula I, (A) N- (4-trifluoromethylphenyl) -5- methylisoxazole-4-carboxamide, (B) the form with an open oxazole ring of (A) N- (4-trifluoromethylphenyl) -2-cyano-3-hydroxycrotonamide, (C) a sodium salt of N- (4-trifluoromethylphenyl) -2-cyano-3-hydroxy-crotonamide or a Mixture thereof preferred.  

According to the effectiveness or / and the tolerance of the substance The agent according to the invention can further increase Formula I at least one pyrimidine, preferably a uridine, cytidine or / and Contain thymidine.

The substance according to formula I can be antiviral together with other effective agents are administered, this simultaneously or can be done in succession. Examples of other antivirals Substances include acyclovir, gancyclovir, vidaravidin, foscarnet, Cidovir, amantadine, ribavirin, trifluorothymidine, interferon-α, cidovudine, Didanosine or calcitabine.

The invention is further illustrated by the following figures and examples explained.

The figures show:

Fig. 1, the reduction of the cytopathic effect (CPE) of HIV-1 in C8166 cells by the substances N- (4-trifluoromethylphenyl) -5-methylisoxazole-4-carboxamide (A), the metabolized, active open ring form of (A ) N- (4-trifluoromethylphenyl) -2-cyano-3-hydroxycrotonamide (B) and its sodium salt N- (4-trifluoromethylphenyl) -2-cyano-3-hydroxy crotonamide (C) in various dilutions,

Fig. 2 shows the influence of the substances A, B and C at various dilutions on the formation of CPE (Syuncytien formation),

Fig. 3, Fig. 4 and Fig. 5, the effect of N- (4-trifluoromethyl phenyl) -5-methylisoxazole-4-carboxamide in various dilutions (at different time points Day 2-5, 5-9 and 9-12 ) on duck hepatitis B virus production in primary duck hepatocytes.

example 1

Principle of the test: Carrying out a series of infections of C8166 cells with HIV-1 and subsequent evaluation of the cytopathic effect (CPE = giant cell formation due to virus-related cell fusion tongues (syncytia)), each with a virus dilution series without substance was used as a control. With these The infections with HIV were experiments without Substance carried out in a falcon tube after 1 Hour adsorption was centrifuged and washed to then apply the cells to the plate. The washing reduces the number of unbound viruses in the supernatant approx. 100 / ml. There are further indicator cells and on the plate Submitted medium with appropriate substance.

Cell line: C8166, human T lymphocytes, indicator cell line for HIV-1

Virus: HIV-1 was derived from the culture supernatant from infected H9 Cells (isolate from a male homosexual Munich; Provided by Prof. L. Gürtler, Munich).

Virus titer: approx. 3 × 10 ³ -1.6 × 10 ⁴ / ml (determined via CPE after 3-4 days)

The cytopathic effect was evaluated after 3-4 days.

Test substances

A = N- (4-trifluoromethylphenyl) -5-methylisoxazole-4-carboxamide

B = metabolized, active open ring form of (A): N- (4-trifluoro methylphenyl) -2-cyano-3-hydroxycrotonamide

C = the sodium salt of B: N- (4-trifluoromethylphenyl) -2-cyano-3- hydroxy crotonamide  

The test was carried out for substance A on 24-well plates, all further tests were carried out on 48-well plates. The number of C8166 cells used was approximately 10 ³ -10 ⁴ / well. The first column of the plate remained virus-free; the remaining columns contain HIV-1 supernatant in increasing dilution from left to right (1: 5, 1: 25, 1: 125, 1: 625, 1: 3125, 1: 15 625; 1: 78 125). The first line of the plate was usually reserved for the control dilution series, in the further series the corresponding substances were added to the medium (EK in the medium: 100 µM, 50 µM and 20 µM). After three to four days, when the cytopathic effect in the control series was fully developed, the syncytia in each well were evaluated.

Results

Substance A: CPE decreased by a factor of 5 to 25; it is not to recognize clear concentration dependence.

Substance B: CPE decreased by a factor of 25 to 125; it is only one to recognize weak concentration dependency.

Substance C: CPE decreased by a factor of 5 to 125; it is only one to recognize weak concentration dependency.

These results are shown in Fig. 1.

The inventive use of substances A, B and C was able to achieve a clearly reproducible reduction in cytophati effect of HIV-1 viruses on C8166 indicator cells become.

Example 2 Decrease in the cytopathic effect of HIV-1 (IIIb) on C8166 cells through substances A, B and C.

Principle of the test: Carrying out a series of infections of C8166 cells with HIV-1 and subsequent evaluation of the cytopathic effect. A virus dilution series was used as a control value  uses one virus dilution series per tested substance was used.

Cell line: C8166, human T lymphocytes, indicator cell line for HIV-1

Virus: HIV-1 (HIV IIIb) was from the culture supernatant of infected HUT78 cells;

Virus titer: approx. 5 × 10 ² -1 × 10 ³ / ml

Test substances: A, B, C (50 mM), were in a 1: 500 dilution used.

A = N- (4-trifluoromethylphenyl) -5-methylisoxazole-4-carboxamide

B = metabolized, active open ring form of (A) N- (4-trifluoro methylphenyl) -2-cyano-3-hydroxycrotonamide

C = the sodium salt of (B) N- (4-trifluoromethylphenyl) -2-cyano-3- hydroxy crotonamide

The test was carried out on 24-well plates. The number of C8166 cells used was approximately 10 ³ -10 ⁴ / well. The first column of the plate remained virus-free; the other columns contain HIV IIIb supernatant in increasing dilution from left to right (1: 5, 1: 25, 1: 125, 1: 625; 1: 3125). The first row of the plate was reserved for the control dilution series, in the other three rows substances A, B and C were added to the medium. After three days, when the control CPE was fully developed, the syncytia in each well were counted. The experiment was repeated three times. Fig. 2 shows the averaged values.

The result clearly shows that the syncytia formation of HIV IIIb C8166 cells by applying the substances according to the invention a factor of approximately 100 is suppressed. Because of the low Virus titers, however, are the values obtained at the resolution limit of the Testing.  

Example 3 Reductions in the multiplication of ortho- and paramyxoviruses by the Substances A, B, C

Test principle: carrying out infections of cells in the presence of the individual substances and quantification of the released Viruses by HA test (orthomyxoviruses) or TCID test (Paramoxy viruses). Additional microscopic assessment of the by the substances alone or by viruses in the presence of the Substances triggered by the cytopathic effect (CPE).

Viruses: Influenza A virus, 2560 HA / ml; Parainfluenza Type 1 (Sendai) virus, 6.4 x 10 ⁵ TCID ₅₀ / ml;

Cell lines: MDCK cells for influenza A viruses; MCF-10A cells and HeLa- Sendai virus cells;

Experiment: cells in 6-well plates were infected with 128 HA influenza A virus, 1.6 × 10 ⁴ TCID ₅₀ Sendai virus for two hours; Viruses which had not been taken up were removed by washing and the cells were incubated in serum-free medium in the presence of the test substance for 24 or 48 hours; the number of viruses released into the cell culture supernatant was then determined in the HA or TCID ₅₀ test.

Results: In several independent test series, a reduction in the replication of ortho- and paramyxoviruses depending on the concentration of the substances used (e.g. substance B) was found:

Substance B showed a 2-fold better inhibition of Virus replication compared to substances A and C.

In the presence of substance B, the virus infection reduced it conditional CPE to 90% -75% compared to the positive control, in particular the change in cell shape in the direction of "spindle-shaped" was reduced.

Example 4 Reduction of pressure hepatitis B virus (DHBV) secretion from primary Duck hepatocytes by N- (4-trifluoromethylphenyl) -5-methylisoxazole-4- carboxamide (substance A)

METHODS: One day after hatching, a duckling was injected intravenously with 200 µl duck serum containing 10 ¹⁰ DHBV DNA genome equivalents / ml. Two weeks after infection, primary duck hepatocytes (PDH) were prepared and cultured as described (JV (1986) 58, 17-251; JV (1992) 66, 2829-2836): hepatocytes were isolated by two-step collagenase perfusion and sown in six-well plates with approx. 10 ⁶ cells / well. Cells were kept at 37 ° C in 5% CO ₂ in William's medium E (Gibco BRL) containing gentamycin (50 µg / ml), L-glutamine (2.25 mM), glucose (0.06%), HEPES pH 7.4 (23 mM), hydrocortisone (4.8 µg / ml), inosine (1 µg / ml), penicillin (50 IU / ml), streptomycin (50 µg / ml) and dimethyl sulfoxide (1.7%) had been supplemented. Medium change took place on days 2, 5, 9 and 12 after plating.

Substance A was added on day 5 and on day 9 after plating. Culture supernatants were collected on day 5, 9 and 12 after plating. A quarter of the culture medium (corresponds to 2.5 × 10 ⁵ cells) per well was analyzed with the aid of dot blot hybridization relative to a DNA standard. A linearized plasmid carrying a copy of the DHBV genome was labeled with "random priming" to a specific activity of about 10 ⁸ cpm / µg and used as a probe for hybridization. The signals were quantified using a Molecular Dynamics Phosphoimager.

Explanation of the figures

Fig. 3 shows the DNA dot-blot of PDH supernatants. PDH were treated with substance A (comp. A, comp. A rev) in the concentrations indicated 5 days after plating. "Control" shows the DMSO control (the substance was dissolved in DMSO). On day 9 after plating, fresh medium with (comp. A) or without substance A (comp. A rev) was added to the cells. The DNA standard that was used to convert the data into the diagram of FIG. 4 is also shown. The numbers in FIG. 4 represent the median values from 3 (A rev), 6 (untreated d9, untreated d12) or 12 individual wells (untreated d5, A), of which the non-specific background (7.7 × 10 ⁶ DNA equivalents / ml) has already been withdrawn. "d2-5" stands for supernatants collected on day 5, "d5-9" on day 9 and "d9-12" on day 12.

The diagram of a dot blot with supernatants of PDH which have been treated with substance A in concentrations of 5 to 200 μM is shown in FIG. 5. Numbers here represent median values of two (treated with A) or three (untreated) individual wells; the non-specific background corresponded to 4.3 × 10 ⁶ DNA equivalents / ml.

Results

The viral release of the untreated PDH was low in the first days after plating, but increased to 3.8 × 10 ⁸ DNA equivalents / ml on day 12 ( FIG. 4 (-), cf. d2-5, d5-9, d9 -12). In contrast, the production of progeny viruses in PDH that had been treated with 20 or 50 μM substance A remained at the low initial level of 5 or 7 × 10 ⁷ DNA equivalents / ml even on day 12 ( FIG. 4 20 μM A , 50 µM A). Comparable results were obtained in a concentration range from 5 µM to 200 µM A ( Fig. 5). Removal of substance A on day 9 ( FIG. 4A rev) resulted in higher virus release between days 9 and 12 compared to cells treated further ( FIG. 4A), indicating that the effect of substance A is reversible. In summary, it was shown that substance A prevented the increase in virus secretion which is observed in untreated cells about 1.5 weeks after plating.

Claims (13)

1. Use of a substance of the general formula I
wherein

• A) R ₁ and R _{2 are} each hydrogen and R _{3 is} a halogen, -CF ₃ , alkoxy with 1 or 2 carbon atoms or a halogen-substituted alkoxy with 1 or 2 carbon atoms,
• B) R _{2 is} hydrogen and R ₂ and R ₃ , which are the same or different, are halogen or -CF ₃ ,
• C) R _{1 is} hydrogen, R _{2 is} alkyl with 1 or 2 carbon atoms and R _{3 is} a halogen, or
• D) R _{1 is} hydrogen and R ₂ and R _{3 are} 3 ', 4'-methylenedioxy, for the preparation of an agent for the treatment of hepatitis B viruses, HI viruses, viruses of the paramyxo group and / or the orthomyxo group,

2. Use according to claim 1, characterized, that the agent continues to be conventional carriers, auxiliaries and / or additives includes. 3. Use according to one of the preceding claims, characterized, that the agent comprises at least one pyrimidine.   4. Use according to claim 3, characterized, that the pyrimidine is a uridine, cytidine and / or thymidine. 5. Use according to claim 1, characterized, that the HI virus is an HIV-1 or HIV-2 virus. 6. Use according to claim 1, characterized, that the virus of the paramyxo group is an avian paramyxovirus, a Parainfluenza virus, a mumps virus, a measles virus, a dog staupe virus, a swine fever virus or a pneumovirus. 7. Use according to claim 1, characterized, that the orthomyxo group virus is an influenza virus. 8. Use according to one of the preceding claims, characterized, that the substance of formula I is selected from (A) N- (4- Trifluoromethylphenyl) -5-methylisoxazole-4-carboxamide, (B) the form with an open oxazole ring of (A) N- (4-trifluoromethylphenyl) -2-cyano- 3-hydroxycrotonamide, (C) the sodium salt of N- (4-trifluoromethyl phenyl) -2-cyano-3-hydroxy-crotonamide or a mixture thereof. 9. Use according to one of the preceding claims, characterized, that the agent has at least one other antiviral substance contains. 10. Use according to one of the preceding claims,  characterized, that the agent is in the form of a tablet, a prolonged-release tablet, one Dragees, a capsule, a granulate, an ampoule, an infu solution, a solution for injection or a concentrate Prepared solution for infusion or solution for injection becomes. 11. A method of reducing or inhibiting the growth of Hepatitis B viruses, HI viruses, viruses of the paramyxo group and / or Orthomyxo group in cells infected with these viruses, comprising Contacting the cells with a sufficiently effective amount a substance of general formula I of claim 1 to the Slow down or inhibit virus replication. 12. Method of treating infection with hepatitis B virus, HI Viruses, viruses of the paramyxo group and / or the orthomyxo group by administering a sufficiently effective amount of Substance of the general formula I of claim 1. 13. A method for the detection of a hepatitis virus, a HI virus or a virus of the paramyxo group or the orthomyxo group, characterized in that

• 1. a substance according to formula I of claim 1 is added to the culture medium to patient cells in culture, and
• 2. cytopathic or cell-altering effects can be measured in comparison with a control culture without adding this substance.