FI94591C - Method for preparing antiviral and immunostimulatory pharmaceutical compositions - Google Patents

Description

94591

The present invention relates to a process for the preparation of antiviral and immunostimulatory pharmaceutical compositions.

The beneficial antiviral activity of ω-3-polyunsaturated fatty acids [5,8,11,14,17-eicosa-pentaenoic acid (hereinafter EPA) and 4,7,10,13,16,19-docosahexaenoic acid (hereinafter DHA)] has been considerable attention to their part. Szads [Antimicrobial Agents and Chemotherapy 12, 523 (1977)] has shown in in vitro experiments that polyunsaturated fatty acids, e.g. both EPA and DHA, are able to inhibit viral replication. This has been supported by Reinhardt et al. [J. of Virology 25, 479 (1978)] by studying replication inhibition in PR 4 bacteriophage.

The antiviral activity of polyunsaturated fatty acids, including EPA and DHA, is detailed in U.S. Patent No. 4,513,008. The effect of EPA and DHA in animal studies in mice and guinea pigs was compared with acyclovir (9- (2-hydroxyethoxymethyl) guanine), which is currently the most widely used antiviral agent. It has been found that compositions containing especially DHA had a more beneficial effect on herpes virus than acyclovir.

Several articles on the antiviral effect of DHA and EPÄ have been reported in the literature, such as: Whitaker et al. [Proc. Natl. Acad. Sci. USA 76, 5919 (1979)] as well as Goodnight et al. [Arteriosclerosis, 2, 87 (1982)] and Yoshia-ki [Biochimica et Biophysica Acta 793, 80 (1984)].

Prickett et al. [Immunology 46, 819 (1982)] have shown that the humoral immune response is stimulated by eicosapentaenoic acid such as an arachidonic acid analog. In an EPA-rich diet, specific IgG and IgE production in response to 2,94591 protein increases in inbred Sprague-Dawley rats by 4- to 8-fold compared to controls. According to this publication, an improved antibody reaction is achieved with an EPA-rich diet. Further, studies show that EPA acts by inhibiting the suppressive prostaglandin system, which can inhibit or respond to immune deficiency due to aging and other pathological processes [autoimmune processes, tumorogenesis (tumor development)]. These statements are supported by Kelley et al. [J. of Immunol. 134, 1914 (1985)] as well as Homey et al. [Clin. Exp. Immunol.

65, 473 (1986)].

Pearson et al. [Proc. Soc. Exp. Biol. Med. TS_, 409 (1952)], it has long been known from L vivo lysine that it has an inhibitory effect on encephalomyelitis virus. Later, Tankersley [J. Bact. 87, 609 (1964)] drew attention to the fact that herpes simplex virus (hereinafter HSV) replication is inhibited by lysine in human cells in vitro. Based on human studies, Kagan reported [The Lancet 1, 137 (1974)] that both orally and genitally HSV-induced injuries rapidly disappeared upon treatment with L-lysine.

Griffith et al. [Dermatologies 156, 257 (1978)] studied the therapeutic effect of L- .. lysine in 45 patients with HSV I and HSV II infection at different doses during varying treatment periods. The age of the patients (mainly women) was 8-60 years. It was found that L-lysine had only a suppressive but no curative effect on HSV.

·. It is an object of the present invention to provide novel pharmaceutical compositions which combine the beneficial therapeutic effects of ω-3-unsaturated fatty acids with the effects of amino acids and certain vital amino acids, in particular lysine, ornithine and histidine, and which have a stronger effect than any known antiviral agent.

3,94591

The invention is based on the finding that the antiviral effects of β-3-unsaturated fatty acids, in particular EPA and DHA, and the corresponding amino acids, in particular lysine, which are known per se, are synergistically enhanced in the mixture formed therefrom and also have an immunostimulatory effect.

Thus, the present invention relates to a process for the preparation of an antiviral and / or immunostimulatory pharmaceutical composition comprising as active ingredients amino acids present in living organisms (such as taurine, cycloserine, homoserine, homocysteine, channelanine, sarcosine, hydroxyproline, hydroxyproline, hydroxyproline, hydroxyproline). Basic amino acids) and / or derivatives thereof, the carboxyl group of which is substituted by a C 1-4 alkyl group or an amino group or an alkali metal, alkaline earth metal or ammonium cation, preferably a sodium or potassium cation, or hydrates and / or salts of these compounds together with one or more Cxe-24 ~ with a fat containing an alkyl group containing at least two double bonds, optionally mixed with carriers and / or additives and antioxidants commonly used in the pharmaceutical industry, wherein the amino acid and the fatty wax the molar ratio of pon (s) is 1: 4 to 4: 1.

The components of the compositions according to the invention are preferably C18-24 "®" 3 "fatty waxPP ° and / or containing at least two double bonds, and amino acids of a basic nature. It is preferred to use lysine, histidine or ornithine or their derivatives as basic amino acids of a basic nature.

Materials commonly used in the pharmaceutical industry, such as lactose, starch or magnesium stearate, can be used as carriers and additives in the compositions of the invention.

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To inhibit the oxidation of the composition, it is preferable to use α-tocopherol (vitamin E), glutathione or conventional antioxidants such as butylated hydroxytoluene.

The compositions of the invention were tested for activity and possible toxicity of mixtures containing a mixture of ω-3 polyunsaturated fatty acids (27.6% EPA and 44.6% DHA) and lysine in molar ratios of 1: 1, 1: 4 and 4: 1, respectively. (test compositions), based on their effect on the proliferation and morphology of Hep 2 cells (human epithelial tumor cell). These experiments were performed on plastic tissue culture plates containing 6x4 hollow space (wells with a bottom area of 1.9 cm 2).

A stock solution containing 10 mg / ml of test compound was prepared in Eagle MEM medium (manufactured by Serva GmbH Co., Heidelberg, Federal Republic of Germany). An aliquot of the stock solution was diluted 10-fold, the resulting solution was diluted 2-fold and then diluted twice, the latter solution being prepared 2-fold solutions of test compounds with gradually lower concentrations (solutions Nos. 1 to 5). The cells were treated with 1 ml of solution, each containing the active ingredient in the following concentrations:

Solution No. concentration of active ingredient uq / ml 1 10,000 2 1,000 3,500 4,250,525 The treatment lasted for one hour, then the cultures were washed twice with buffered sodium chloride solution (phosphate-buffered saline, hereinafter PBS), then the cultures were supplemented with 94591 medium. After 24 hours and after fixation with methanol, the cultures were stained with ethanolic Giemsa solution (manufactured by Reanal, Budapest, Hungary) and the cell morphology was assessed by light microscopy. It was found that the test compound was only toxic at concentrations above 1000 ug / ml.

The viral replication inhibitory effect was studied in Hep 2 cells as described above. HSV I virus was used for infection. The virus concentration was about 1000 PFU (plaque forming unit). Solutions containing test compound 1000, 500 and resp. 250 μg / ml and 0.1 ml of solution was added to the undiluted virus suspension for treatment. The mixture was incubated at 37 ° C for one hour. The cells were then treated with virus preincubated with the test compound, and changes in cytopathological changes (hereinafter CP) were monitored for 7 days. It was found that viral replication was directly inhibited by a dose of 500 μg / ml or 250 μg / ml of the test composition: the cytopathological dose (neg. Log. CPD50) had an effective titer of 1.75 to 2.04 calculated from 0.1 ml compared to the untreated control virus culture neg. log. To a CPD 50 value of 4.5. The value of viral inhibition exceeds the value of the control by two orders of magnitude. Under the same conditions, EPÄ and DHA had no valuable viral inhibition, and lysine itself inhibited viral replication to an amount only about one order of magnitude higher than the control.

Vaccinia virus was also treated in an in vitro experiment performed as described above. Infectious titer value (neg. Log.) Measured in the vaccinia virus of the test composition.

CPD50) ranged from 1.75 to 2.15 compared to 5.5 for the untreated control virus, i.e. the test composition had a viral inhibitory effect that was three orders of magnitude stronger than the control.

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The in vitro effect of the compositions of the invention on the immune system was studied by activating lymphocytes with polyclonal mitogens. The blast transformation of lymphocytes was studied so that a lymphocyte cell population prepared in a Fico Uromiro gradient [Scand. J. Clin. Lab. Invest. 21, 97 (1968)], was pipetted into the wells of flat-bottom plates and then 25 pg / ml Concanavaline A (hereinafter: Con A) (manufactured by Pharmacia, Uppsala, Sweden) was added to each co-culture and then a solution containing new compositions 0.1, 1.0 or resp. 10 ug / ml. A culture containing 25 pg / ml Con A without test composition was used as a control. The plates were maintained in an air atmosphere containing 5% carbon dioxide at 37 ° C for 72 hours and then 0.4 μCi of 1H-thymidine was added to each sample 64 hours before the end of the culture. After 72 hours, the cultures were filtered through a glass filter, the filtrates were placed in a scintillation cuvette and the radioactivity was measured in 5 ml of toluene solution using a beta counter. The results are shown in the following table.

l: 7 94591 No. Compound Active substance- Calculations / min Concentration of the effect component cpm evaluation _thio ^ g / g_ 1. Control 25 896912984 (Con A) 2. L-tyrosine 0.1 1291512045 slightly significant increase 1.0 1231312003 3 L-lysine 0,1 1106311528 not significant 1.0 1183311823 slightly significant increase 4. ω-3-fatty acid 0,1 1233212235 not relevant mixture (eg 1 composition)

Na salt 1.0 1203911640 not significant 5. Control 25 839612233 (Con A) 6. Example 5 0.1 1460511747 very product significant increase 1.0 1201012285 not significant 7. Example 1 0.1 1608512063 very product significant • increase 1.0 1468111769 significant increase 8. Example 3 0.1 1266111548 significant product increase β 94591 «· 1.0 1242011452 significant increase 9. Example 4 0.1 1192012005 significant product increase 1.0 1224011950 significant increase These studies show that the compositions of the invention, especially the mixture of polyunsaturated fatty acids , used in combination with lysine and tyrosine, produce a significant test result, i.e. they have a strong immunostimulatory effect, whereas the individual components of the test substances according to the invention themselves did not appear to have any biological effect or had only a weak biological effect.

It is preferable to use as starting material the β-3-unsaturated Cia-24 fatty acids which form one component of the composition, in particular oils obtained from North Sea fish such as salmon, cod, sardines or their livers, but oils obtained from freshwater fish can also be used. -3-polyunsaturated fatty acids are obtained from the above oils using a known method [J. Am. Chem. Soc. 59, 117 (1982)]. These: · fatty acids tend to oxidize. Antioxidants such as E-Vitamin are preferably used to prevent oxidation.

The above-mentioned active ingredients can be converted into capsules, tablets, granules or other known pharmaceutical compositions prepared in a manner known per se.

The main advantages of the pharmaceutical composition according to the invention are the following: 9 94591 a) The antiviral therapeutic effect of the active ingredients of the composition is increased synergistically.

b) It can be used against acute viral infections, especially in herpes virus infection to suppress the infection in the early stages.

c) Due to its immunostimulatory effect, it can be advantageously used against retro viruses, in particular immunodeficiency syndrome (e.g. AIDS), induced by HTLV

III and HTLV IV viruses.

(d) It contains exclusively natural active substances which are biologically relevant. Thus, it can be used for prophylactic, long-term, curative treatment in the presence of a risk of viral infection as well as in diseases of the immune system.

e) It also has an internal effect. Thus, the uncomfortable external treatment commonly used in antiviral therapy can be avoided.

The compositions of the invention are further illustrated by the following non-limiting examples.

Example 1 164 g (1 mole) of L-lysine monohydrate and 320 g (ca. 1 mole) of a ω-3-monounsaturated fatty acid mixture (containing 27.6% EPA and 44.6% DHA) are stirred at room temperature . The homogeneous mixture thus obtained is filled using known encapsulation methods into hard gelatine capsules containing 500 mg of active ingredient.

Example 2 155 g (1 mole) of L-histidine and 320 g (ca. 1 mole) of a ω-3-polyunsaturated fatty acid mixture (containing 27.6% EPA and 44.6% DHA) are mixed together. Otherwise, the method of Example 1 is followed.

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Example 3 41.0 g (0.25 moles) of L-lysine monohydrate and 320 g (ca. 1 mole) of a ω-3 polyunsaturated fatty acid mixture (containing ca.

90.0% EPA and 0.1% vitamin E) are mixed together, then the procedure of Example 1 is followed.

Example 4

The procedure of Example 3 is followed except that 164 g (1 mole) of L-lysine monohydrate and 328 g (1 mole) of docosahexaenoic acid (DHA) are used as the polyunsaturated fatty acid [manufactured by Sigma Co., St. Louis, USA, catalog number D-6508 (1987)].

Example 5

The procedure of Example 1 is followed, except that 181 g (1.0 mol) of L-tyrosine are used instead of L-lysine.

Example 6

The procedure of Example 1 is followed, except that 120 g (1.0 mol) of L-threonine are used instead of L-lysine.

Example 7 • ·

The procedure of Example 3 is followed, except that 1.64 g (1 mole) of L-lysine monohydrate and 80 g (about 0.25 moles) of a mixture of ω-3 polyunsaturated fatty acids (containing about 90.0% DHA) are used. and 0.1% vitamin E).

Example 8

The procedure of Example 3 is followed except that 164 g (1 mole) of L-lysine monohydrate and 302 g (1 mole) of • 11,94591 eicosapentaenoic acid (EPA) are used instead of the polyunsaturated fatty acid mixture [manufactured by Sigma, St. Louis, USA, catalog number E-7006 (1987)].

Example 9

The procedure of Example 1 is followed except that 132 g (1.0 mol) of L-ornithine are used instead of L-lysine.

Example 10

The procedure of Example 3 is followed except that 33 g (0.25 moles) of L-ornithine are used instead of L-lysine.

Example 11

The procedure of Example 4 is followed except that 132 g (1.0 mol) of L-ornithine are used instead of L-lysine.

Example 12

The procedure of Example 1 is followed except that 133 g (1.0 moles) of L-aspartic acid are used instead of L-lysine.

Example 13 »

The procedure of Example 1 is followed, except that 211 g (1.0 mol) of L-arginine hydrochloride are used instead of L-lysine.

Example 14

The procedure of Example 1 is followed, except that 105 g (1.0 mol) of L-serine are used instead of L-lysine.

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Example 15

The procedure of Example 1 is followed, except that 145 g (1.0 mol) of L-glutamine are used instead of L-lysine.

Example 16 Preparation of tablets

The composition prepared in Example 1 is prepared into tablets containing the following components: e.g. composition according to 1 500 mg lactose 120 mg starch 63 mg polyvinylpyrrolidone 3.5 mg magnesium stearate 3.5 mg

If desired, tablets may be sugar coated, using a panning machine.

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Claims (4)

A process for preparing an antiviral and immunostimulatory pharmaceutical composition, characterized in that amino acids and / or derivatives thereof present in living organisms which contain a carboxyl group substituted by a C 1-4 alkyl group or an amino group or an alkali metal or alkali metal, alkaline earth metal mixture are mixed together as active ingredients. , or hydrates and / or salts of these compounds, and one or more C18-24 omega-3 fatty acids prepared from fish oil, optionally together with carriers and / or additives and antioxidants commonly used in the pharmaceutical industry, wherein the amino acid and fatty acid ( fatty acids) is 1: 4 to 4: 1. Process according to Claim 1, characterized in that eicosapentaenoic acid (EPA) and L-lysine are used as active ingredients. Process according to Claim 1, characterized in that docosahexaenoic acid (DHA) and L-lysine are used as active ingredients. Process according to Claim 1, characterized in that docosahexaenoic acid, eicosapentaenoic acid and L-lysine are used as active ingredients. m 94591 14