EP0946168A1

EP0946168A1 - Use of ethylene diamine disuccinate for preparing a medicament with antiviral properties

Info

Publication number: EP0946168A1
Application number: EP97921801A
Authority: EP
Inventors: Hans Wilhelm Doerr; Kai Uwe Bindseil; Lutz MÜLLER-KUHRT; Holger Rabenau; Jindrich Cinatl
Original assignee: Doerr Hans-Wilhelm Prof Dr med; Analyticon AG Biotechnologie Pharmazie
Current assignee: Doerr Hans-Wilhelm Prof Dr med; Analyticon AG Biotechnologie Pharmazie
Priority date: 1996-04-27
Filing date: 1997-04-26
Publication date: 1999-10-06
Also published as: US6140367A; CA2252779A1; AU2772997A; DE19616992C1; WO1997040827A1

Abstract

The use is disclosed of (S,S)-N,N'-ethylene diamine disuccinate (EDDS) of formula (I), in which M = H+ and/or stands for any pharmaceutically acceptable cation, for preparing a medicament for treating infections by cytomegaloviruses with immunosuppressive properties.

Description

Use of ethylenediamine disuccinate for the manufacture of a medicament with antiviral properties

The invention relates to the use of ethylenediamine disuccinate including compatible salt-forming ions and the protonated form for the manufacture of a medicament with antiviral properties which also has an immunosuppressive effect.

(S, S) -N, N '-ethylenediamine disuccinate is described in detail in the publication by Takaaki NISHIKIORI et al. (1984), The Journal of Antibiotics, Vol. 37, No. 4: 426-427. (S, S) - N, N'-ethylenediamine disuccinate can be obtained from actinomycetes and can also be prepared synthetically (JA Neal and NJ rose (1968), Inorg. Chem. Vol. 7; 2405-2412). It inhibits phospholipase C and D. If (S, S) - N, N'-ethylenediamine disuccinate is administered intraperitoneally to mice, the antibody production and the DTH reaction (delayed type hypersensitivity) are suppressed. Blastogenesis of B cells and T cells is suppressed in vitro. However, no antimicrobial activity is described in this publication. The toxicity in mice is relatively low. The cytomegaloviruses (CMV) form a group of related viruses that belong to the herpes virus (Lutz Schneider (1990), Pharmaceutical Newspaper, Vol. 135, No. 27, 2396-2400). After an initial infection, the viruses remain latent in the body. The viruses are only reactivated when the immune system is weakened by drug or disease-related immunosuppression. The viruses got the name because they cause histopathologically detectable giant cells with large marginal edges and viruses as inclusion bodies.

The viruses are ubiquitous. The infection of the population varies from 30% to 85%, yes 95%. The infection runs inconspicuously in adults with a functional immune system and shows at most unspecific symptoms, such as fatigue and slightly increased body temperature.

In immunocompromised adults, CMV infections focus on pulmonary diseases, inflammation of the arteries and gastrointestinal disorders. In AIDS patients, CMV infections cause most deaths.

Since the incidence of CMV infections also increases with increasing age, the extent to which the cytomega viruses are involved in arterial plaque formation is discussed immediately. The viruses are able to damage endothelial cells of the vessel walls.

Various substances are being discussed for treatment against the cytomegalovirus.

In the publication by J. Cinatl et al. (1994), Antiviral Research, Vol. 25: 73-77 describes desferrioxamine, which forms iron chelates. This substance can be used successfully in in vitro experiments against herpes simplex viruses, varicella zoster viruses, Epstein-Barr viruses and human cytomegaloviruses. The effect of the substance on chelation with the iron ions is attributed. Furthermore, the cellular ribonucleotide reductase is also to be inhibited. However, some of the previous findings are contradictory in theory. The substance shows low toxicity.

Foscarnet is an antiviral substance with selective activity against human viruses of the herpes group, such as herpes simplex, varicella zoster, Epstein-Barr and cytomegaloviruses as well as hepatitis viruses. The antiviral effect is based on the inhibition of viral enzymes, such as DNA polymerases and reverse transcriptases. Foscarnet has a virostatic effect on cytomegaloviruses, but the viruses cannot be eliminated (Lutz Schneider (1991), Pharmaceutical Newspaper, Vol. 136, No. 46, 33-36). An essential problem of the cytomegalovirus infection is the necessity of the sometimes lifelong long-term treatment of the patients. Another disadvantage is that the cytomegaloviruses have recently become more resistant to this substance (Stanat et al. (1991), Antimicrob. Agents, Chemother. , Vol. 35, No. 11: 2191-2197 and Knox et al. (1991), Lancet, Vol. 337: 1292-1293).

Ganciclovir is described in the publication by Lutz Schneider (1990), Pharmazie, Vol. 135, No. 37, 2396 to 2400. Ganciclovir is one of the nucleoside antimetabolites derived from 2'-deoxiguanosine. It carries an acyclic side chain instead of the 2'-deoxyribose and differs from aciclovir only by an additional hydroxymethyl group in the side chain. Ganciclovir is approved for the treatment of life or eyesight-threatening cytomegalovirus infections in patients with acquired immunodeficiency or medicinal immunosuppression, for example after organ transplants. Although ganciclovir is also effective in other human pathogenic herpes viruses (HSV 1 and 2, Varicella zoster and Epstein-Barr), its use is prohibited because of the high rate of side effects such infections. Ganciclovir leads to neutropenia, and it has also been observed that mice develop tumors during the treatment. Another disadvantage is that the cytomegaloviruses have recently become more resistant to this substance (Stanat et al. (1991), Lancet, Vol. 337: 1292-1293).

WO 94/22438 (DINU, filing date: December 29, 1993) describes diethylenetriaminepentaacetic acid for the treatment of herpes simplex, varicella zoster, encephalomyelitis, polyradiculomiritis, multiple sclerosis, but not of cytomegaloviruses. Immunosuppressants, such as cyclosporin A or tacrolimus, have been used successfully in organ transplants to avoid or mitigate rejection reactions. The disadvantage of this is of course the susceptibility of the treated patients to serious complications caused by viral and bacterial infections. Infection with cytomegaloviruses is particularly worth mentioning in this context.

The technical problem underlying the invention is to provide a compound with an antiviral effect which also has an immunosuppressive effect.

It was assumed that the substance to be used according to the invention, similar to diethylenetriaminepentaacetic acid (Aisen and Listowsky (1980) Annu. Rev. Biochem., Vol. 49: 357-393), did not penetrate the cell because of its strongly hydrophilic character and therefore only free, ie cannot complex metals or ions incorporated into the cell. Desferrioxamine, for example, is known to be able to penetrate the cell. Since the virus multiplication takes place from inside the cell, it had to be assumed that an inhibitory active ingredient must also penetrate the cell in order to be effective. Surprisingly, however, according to in vitro experiments in cell culture, the substance according to the invention is nevertheless effective. Particularly surprising was the effect that EDDS also has an immunosuppressive effect in addition to the antiviral effect. The immunosuppressive effect manifests itself in the inhibition of lymphocyte proliferation stimulated by phytohaemagglutin.

1 shows the concentration-dependent inhibition of lymphocyte proliferation. An almost quantitative inhibition is achieved between 30 μM and 100 μM (corresponding to non-toxic concentrations) EDDS.

Compared to the substance diethylenetriaminepentaacetic acid, the substance to be used according to the invention is significantly more effective in terms of its antiviral activity. Compared to the substance desferrioxamine, the substance according to the invention is more effective by a factor of 30.

Furthermore, it is surprising that only the substance according to the invention (S, S form) can be used as a medicament. However, the other forms, including the modifications (propylene and glutamate) against cytomegaloviruses cannot be used successfully.

The antiviral effect of the substance according to the invention can be modulated by adding iron (II) and iron (III) ions. Antiviral activity is reduced by a factor of 2 to 3, but is not completely eliminated. Thus it becomes obvious that the formation of iron chelate has an observable effect when acting as an antiviral agent, but it is also evident that this effect alone is not sufficient to explain the antiviral effect.

Surprisingly, it has been found that the substance according to the invention can be used very selectively against cytomegaloviruses. Concentrations of the substance according to the invention which have an antiviral effect have no effect on cell growth. The therapeutic index is therefore very high. The substance according to the invention is therefore of great clinical importance.

It is unusual that the substance according to the invention can be used against cytomegaloviruses, but has no influence on certain other viruses, such as adenoviruses (ATTC strain: GB type 3), varicella zoster viruses (ATCC strain: Maclntyre) and herpes simplex viruses (HSV -Vero) has.

The invention further comprises the ethylene diamine succinate according to the invention and its salts and / or acid groups. The salts inevitably result from the environment and the physical state of the substance.

Preferred is the (S, S) -N, N '-ethylenediamine disuccinate, in which the salts comprise cations from the group with the atomic numbers 3-5, 11-13, 19-29, 37-49 or 55-81 or mixtures of the include previously cations.

More preferred is the (S, S) -N, N '-ethylenediamine disuccinate, in which the cations from the group of magnesium (II) -, aluminum (III) -, calcium (II) -, manganese (II) -, iron (II) -, iron (III) -, cobalt (II), nickel (II) -, copper (II) -, zinc (II) - ions and further lithium, potassium and sodium ions are selected or mixtures thereof previously mentioned ions are. Lithium, manganese (II), calcium (II), potassium and sodium ions are also particularly preferred.

In addition, the (S, S) -N, N'-ethylenediamine disuccinate is preferred, in which the salts comprise organic cations.

More preferred is the (S, S) -N, N '-ethylenediamine disuccinate, in which the cations from the group of the primary; secondary or tertiary amines (e.g. ethanolamine, diethanoiamine, triethanolamine, morpholine, glucamine, N, N-dimethylglucamine and N-methylglucamine), lysine, arginine or ornithine are selected or the cations are mixtures of the cations mentioned above.

A mixture of organic and inorganic salts is included in the invention.

The substance (S, S) -N, N'-ethylenediamine disuccinate is preferred as a medicament if it forms a composition with pharmacologically acceptable auxiliaries and carriers. Such auxiliaries and carriers are described in Remington's Pharmaceutical Science, 15th ed. Mack Publishing Company, East Pennsylvania (1980). The compositions can be prepared by known methods.

The (S, S) -N, N'-ethylenediamine disuccinate according to the invention has pharmacological properties and can therefore be used as an active pharmaceutical ingredient. The invention also includes a medicament containing the (S, S) -N, N '-ethylenediamine disuccinate.

In particular, the substance (S, S) -N, N'-ethylenediamine disuccinate according to the invention has an activity against cytomegaloviruses.

The substance according to the invention shows an antiviral inhibition (IC ₅₀ value) at concentrations of 4 μg / ml. Higher concentrations can be used without disturbing the test system. The substance according to the invention can thus be used in a concentration of 0.5 to 100 μg / ml.

The test results of this in vitro test show that the substance according to the invention can be used as a medicament or for medical treatment. These test results can easily be transferred from the in vitro test system to an in vivo system, since the antiviral test system is an established test set-up which serves to detect the antiviral activity (Gerna et al., (1992) Antiviral. Res. Vol. 19: 333-345). EDDS can therefore be used according to the invention for the treatment or prevention of cytomegalovirus infections, the substance also acting as an immunosuppressant. It can be used as an inhibitor in mammals, especially humans, for the treatment of the aforementioned disease.

The invention further provides

(i) the use of a substance according to the invention (for the manufacture of a medicament) as an immun suppressant and / or for the treatment of cytomega-viruses or prevention;

(ii) a method of treating cytomegaloviruses, the method comprising administering an amount of (S, S) - N, N '-ethylenediamine disuccinate, the amount of ethylenediamine disuccinate being given to a patient in need of such a medicament;

(iii) a pharmacological composition for the treatment or prevention of cytomegaloviruses, which comprises a substance according to the invention and at least one pharmaceutical auxiliary and / or carrier.

Different doses are suitable for the therapeutic effect. They depend, for example, on the salts used, on the host, on the type of administration and on the type and severity of the conditions to be treated.

In general, however, satisfactory results are expected in animals when the daily doses range from 1 to 500 mg per kg. Include body weight. In the case of larger mammals, for example humans, a recommended daily dose is in the range from 1 to 50 mg per kg of body weight, a dose of 5 to being most preferred 30 mg per kg body weight. For example, this dose is expediently administered in partial doses up to four times a day. Satisfactory results can be expected if the substance according to the invention is administered subcutaneously or intravenously. Oral administration is also possible.

Examples

The following examples illustrate the invention, in particular the advantageous effects of the substance, without the invention being restricted to these examples.

Antiviral activity: cells and viruses used

Human foreskin fibroblasts (HFF) are grown in a nutrient medium from Eagle's minimal essential medium (EMEM) with 10% addition of fetal calf serum.

The CMV laboratory strain AD169 is used. The viruses are propagated in EMEM culture medium with a 4% addition of fetal calf serum (maintenance medium). The virus titer is determined by determining what are known as immediate early antigen forming units (I.E.F.U.) which are formed in the preservation medium (Gerna et al. (1992), Antiviral. Res., Vol. 19: 333-345).

Antiviral effect

The antiviral effect of the substances on the multiplication of cytomegaloviruses is determined by various parameters:

Production of CMV-late antigens is determined in an ELISA (enzyme-linked immunosorbent assay) test. This result is expressed by the IC ₅₀ value, which represents the concentration of active ingredient which lowers the production rate of the antigen by 50%. The sub- punches have an IC ₅₀ value of 4 μg / ml ± 1 μg / ml.

The cell vitality is measured in an HFF cell culture using an MTT assay. The result is expressed by the TC ₅₀ value, which is the concentration at which 50% of the cells tested are still vital. It is 435 μg / ml in HFF cells for the substances according to the invention.

The quotient TC _S0 / IC _{50 is} formed from both values in order to determine the therapeutic index. In the substances according to the invention, this is 109 (Gerna et al. (1992) Antiviral Res. Vol. 19: 333-345).

Table of measured values:

Highest tested substance concentration X dose not achievable for the patient

Synthesis of all derivatives of the N, N'-diamine disuccinate and glutamate according to JA NEAL and NJ ROSE (1968), Inorg. Chem., Vol. 7: 2405-2412. Immunosuppressive effect

For the proliferation assay, lymphocyte cultures were prepared in mixed lymphocyte cultures (MLC) or with 1% phytohaemaglutinin (PHA) in a total volume of 200 μl culture medium. 18 to 20 hours before the measurement, 0.1 μCi methyl [ ³ H] thymidine (NEN, Germany) was added. Radioactively labeled DNA was harvested on filter membranes (Schleicher & Schüll, Germany). The radioactivity was quantified using a scintillation counter (Zinsser, Germany). The PBL sample affects peripheral blood lymphocytes as a control standard.

Claims

Expectations

Use of (S, S) -N, N '- ethylenediamine di succinate (EDDS) of the formula I.

where M = H ⁴ and / or any pharmaceutically acceptable cation, for the manufacture of a medicament for the treatment of infections against cytomega-viruses with immunosuppressive properties.

Use according to claim 1, wherein EDDS effects an inhibition of phytohaemagglutin-stimulated lymphocyte proliferation.

Use of EDDS according to at least one of claims 1 and / or 2, wherein M comprises the hydrogen ion and / or a cation from the group with the atomic numbers 3-5, 11-13, 19-29, 37-49 or 55-81 or Mixtures thereof included.

Use of EDDS according to claim 3, wherein the cations from the group of magnesium (II) -, aluminum (III) -, calcium (II) -, manganese (II) -, iron (II) -, iron (III) -, Cobalt (II), nickel (II), copper (II), zinc (II) ions and also lithium, potassium and sodium ions are selected or are mixtures of these aforementioned ions.

5. Use of EDDS according to at least one of claims 1 and / or 2, wherein M comprises organic cations.

6. Use of EDDS according to claim 5, wherein the cations are selected from the group of primary, secondary or tertiary amines, lysine, arginine or ornithine or the cations are mixtures of the cations mentioned above.

7. Use of EDDS according to one of the preceding claims, the EDDS forming a composition with pharmacologically compatible auxiliaries and carriers.