HU199775B - Process for production of formed by fatty acids salts of amin acids and medical compositions containing them - Google Patents

Description

The present invention relates to novel salts having antiviral activity and stimulating the immune system and to pharmaceutical compositions containing them.

The positive antiviral effect of ω-3 polyunsaturated fatty acids (5,8,11,14,17-eicosapentaenoic acid (hereinafter EPA) and 4,7,10,13,16,19-docosahexaenoic acid (hereinafter DHA)) more people were noticing. In his in vitro experiments, Sads (1977, Antimicrobial Agents and Chemotherapy, 72, 523) has shown that polyunsaturated fatty acids such as EPA and DHA are capable of inhibiting viral replication.

The same fact was confirmed by Reinhardt et al. of Virology 25, 479 (1977)], which tested for replication inhibition on bacteriophage PR 4.

No. 4,513,008. U.S. Pat. No. 4,198,125 describes the antiviral activity of polyunsaturated fatty acids, including EPA and DHA, in detail. In animal studies, in mice and guinea pigs, the effects of EPA and DHA were compared with the most commonly used antiviral agent of the period, "Acyclovir" [9- (2-hydroxyethoxymethyl) -guanine]. In particular, DHA-containing formulations have been found to be superior to Acyclovir against herpes virus.

Numerous articles in the literature address the antiviral activity of DHA and EPA, such as Whitaker et al. (1979) Proc. Natl. Acad. Sci. USA 76, No. 11, 5919 and Goodnight et al. 87 (1982)] and Yoshiaki (Biochimia et Biophysica Acta, 793, 80 (1984)).

Prickett et al., Immunology 46: 819 (1982) have shown that eicosapentaenoic acid as an arachidonic acid analogue stimulates the humoral immune response. The specific IgG and IGE production of egg albumin in the inbred Spargue-Dawley rat is increased by 4-8 fold over the control as a result of an EPA-rich diet. It is reported that an EPA-rich diet induces an elevated antibody response. Studies have also shown that EPA acts through inhibition of the suppressive prostaglandin system, ie it can inhibit or correct age-related immunodeficiency and other pathological processes (autoimmune processes, tumorogenesis). These findings are described in Kelley et al., J. Med. of Immunize. 134, 1914 (1985)] and Homey et al., Clin. Exp Immunol. 65, 473 (1986)].

Pearson et al., Proc. Soc Exp Bioi. 79, 409 (1952)], has shown that lysine has an inhibitory effect on encephalomyelitis virus. Later, Tankersley R.W. [J. Bact. 87, 609 (1964)] revealed that lysine in vitro inhibits replication of the herpes simplex virus (HSV) in human cells. According to Kagan's human studies in 1974 (The Chain, 1, 137 (1974)), lysine-induced lesions caused by both oral and genital herpes simplex viruses disappear very quickly.

Griffith et al., Dermatologica, 156, 257 (1978), investigated the therapeutic effect of 1-lysine in 45 patients with HSV I and HSV II at different doses, over different treatment periods. The age of patients (primarily women) ranged from 8 to 60 2 years. The authors found that 1-lysine had only suppressive, but not curative, effects on HSV.

It is an object of the present invention to provide a novel therapeutic salt (s) and a pharmaceutical composition (s) thereof which combine the beneficial therapeutic effects of w-3 unsaturated fatty acids and amino acids, particularly lysine, omitin and histidine, and are more potent than any antiviral agent. show effect. The present invention is based on the discovery that when ω-3 unsaturated fatty acids, especially EPA and DHA, are converted into salts with amino acids or derivatives thereof, especially lysine, the salt formed therefrom has a strong antiviral and immunostimulatory action.

Accordingly, the present invention provides a process for the preparation of novel salts of formula I having antiviral and immunostimulatory activity,

R-COOAH + (I)

- wherein R is a C 1 -C 24 alkyl group containing at least two unsaturated double bonds,

A is a C1-C4 alkyl ester of an amino acid and / or a salt or amide thereof with an amino group or an alkaline cation occurring in living organisms.

According to the present invention, one of the amino acids A of the formula (I) as the base - wherein the substituents are represented by an acid of the formula (H) above

R-COOH (Π)

wherein R is as defined above, in a polar solvent.

The active compounds of the present invention are preferably salts of ω-3 unsaturated fatty acids of the C 18-24 chain length with at least 2 unsaturated double bonds and their basic amino acids or derivatives thereof.

In order to prepare the salt of formula (I) according to the invention, it is convenient to proceed in the following ways:

a) A mixture of ω-3 polyunsaturated fatty acids with an EPA content of 27.6% and a DHA content of 44.6% is added dropwise at room temperature to an aqueous solution of an A '-labelled amino acid such as 1-lysine monohydrate and under nitrogen atmosphere. Stir at C for 3 hours. The solvent was removed in vacuo to give a crystalline salt (m.p. 188-195 C).

b) NaOH is dissolved in a 1: 1 methanol-water mixture and the resulting mixture is dissolved in the same molar amount of 1-tyrosine as the alkali. To the resulting 1-tyrosine alkaline salt solution is added dropwise at room temperature the w-3 polyunsaturated fatty acid mixture of (a). Hereinafter, we will proceed in accordance with point (a). The resulting yellow crystal had an op: 150 'C (partially decomposed)

c) in each case, according to b), except that glutamine is used as the amino acid derivative of formula A. A brownish powdery crystalline salt is obtained, m.p. 181-190 ° C.

d) adding the ω-3 polyunsaturated fatty acid mixture of (a) to a 1: 1 solution of lysine ethyl ester in water-ethyl alcohol at room temperature. Hereinafter, we will proceed in accordance with point (a). A tan solid salt is obtained (m.p. 160-164 'C, dec.)

HU 199775 Β

The efficacy of the compounds of the present invention, 1-lysine salt (ω-3) of a polyunsaturated fatty acid mixture (EPAtart. 27.6%, DHA-tartrate 44.6%) for the proliferation of Hep 2 (human epithelial tumor cell line) morphology and potential toxicity to cell culture. The experiments were performed on a 6x4-well (1.9 cm ² wells) plastic tissue culture plate.

The test compound was prepared in 10 stocks of 10 mg / ml in Eagle MEM medium.

An aliquot of the stock solution was first diluted ten-fold and then diluted twice, and the latter solution was diluted twice more to obtain a solution of the test compound in decreasing concentration (solutions 1-5). Cell treatments were performed with 1 ml of solution containing the active ingredient as shown in the table below:

_ 20

Serial number of the solution The concentration of the active substance is pg / ml First 10,000 Second 1000 Third 500 4th 250 5th 125

The treatment was carried out for 1 hour, then the cultures were washed twice with PBS = Phosphate buffered saline and then medium was added to the cultures and the cell morphology was evaluated by light microscopy. From this it was concluded that the test substance was toxic only at concentrations above 1000 mg / ml.

The viral replication inhibitory activity was assayed on Hep 2 cells as described above. Herpes simplex virus type 1 was used for infection. The virus concentration was approximately 1000 PFU (plague forming units). 1000 of the test compound,

500 and 250 pg / ml solutions were prepared and treated with 0.1 ml (test compound content 500, 250 and 125 pg) by addition of 45 to the undiluted virus suspension for 1 hour at 37 ° C. incubated. The cells were then treated with the virus preincubated with the test compound and observed for 7 days for the development of cytopathological (CP) lesions. 50

The test compound was found to directly inhibit viral growth at doses of 500 and 250 pg / ml, with an infectious titre of cytopathological dose (negative log CPD50) of 0.175 per ml, while untreated control virus cultures Neg. hangs 55 CPO50 value 4.5. The value of viral inhibition is greater than two orders of magnitude relative to the control. Under the same conditions, EPA and DHA did not show appreciable viral inhibition, whereas lysine alone inhibited viral replication, but its magnitude was around the order of magnitude.

In a similar in vitro experiment, vaccine virus was also treated. The test compound has an infectious titer (negative log CPD50) of 1.75 for the vaccinia virus as compared to above and 5.5 for the untreated control virus, which means that the test compound has three orders of magnitude of antiviral activity.

The in vitro activity of the compounds of the invention on the immune system was investigated by the activation of lymphocytes by polyclonal mitogens. Blast transformation of lymphocytes was investigated. During the experiments, the lymphocyte cell population obtained on a Fico Uromiro gradient (Scand. J. Gin. Lab. InvesL 27, 97 (1968)) was pipetted into wells of flat bottom plates and 25 µg / ml Concanavalin A (Con. A, manufacturer) for each parallel culture. Pharmacia Uppsala Sweden), followed by a 0.1, 1.0 and 10 mg / ml solution of the compounds of the invention, as control, culture containing 25 pg / ml Concanavalin A without test compound for 72 hours at 37 ° C in 5% They were kept in an atmosphere containing CO ₂ , but after 64 hours, 0.4 pCi ³ H-thymidine was added to each sample before culture was completed. .

The results are summarized in the following table:

Description Active substance per minute counts (Cpm) effect evaluation conc. (U / a) control 25 8969 ± 2984 (Con. A.) 0.1 12 915 ± 2045 + 1-tyrosine 1.0 12 313 ± 2003 1-Threonine 0.1 + 13 371 2894 ++ + 13 883 1823 + 1-lysine 0.1 11.63 ± 1528 - 1.0 11 833 ± 1823 + ω-3 fatty acids 0.1 + 12 332 2235 - Na salt 1.0 12039 + 1640 - (E.g. composition) control 25 8369 ± 2233 (Con. A) 16th 0.1 + 14 965 2143 +++ of 1.0 12,517 ± 2195 - 1 st of 0.1 + 15 985 2102 +++ product 1.0 14 068 ± 1665 ++ control 25 8816 ± 2630 (Con. A) 13th of 0.1 6506 + 2545 - product 1.0 5115 + 1890

Legend:

- slightly decreasing significance

- not significant + slightly increasing significance ++ significant growth +++ strongly significant growth

Studies have shown that the lysine and tyrosine salts of the compounds of the invention, in particular the polyunsaturated fatty acid mixture, showed significant test results.

HU 199775 Β, that is, it exhibited a strong immunostimulatory effect, whereas the components of the salt according to the invention, alone or not, were found to have low biological significance.

Ci8-24 ω-3, which is one of the components of the salts, is based on unsaturated fatty acids mainly from fish of the North Sea, eg. Salmon, cod, sardine or their liver oils are used, but can be used in freshwater fish such as lingonberries. also oils from the bush. The ω-3 polyunsaturated fatty acids used as starting materials for the salt formation of the present invention are obtained from the above oils by a known method (JACS 59, No. 3, 117 (1982)).

The active compounds of formula (I) may be converted in conventional manner into pharmaceutical compositions which may be encapsulated, tableted, coated, or otherwise formulated using conventional excipients and / or excipients (e. G., Lactose, starch, magnesium stearate, etc.).

The main advantages of the compounds of the present invention and their pharmaceutical composition are:

1. They are useful in the treatment of acute viral infections, and in particular in the suppression of the initial infection in the case of herpes virus infection.

2. Due to their immune stimulating effect, retroviruses, including HTLV ΙΠ and IV, are advantageously used. types of immune deficiency syndromes (such as AIDS).

3. They contain only physiologically essential active ingredients of natural origin, so they are suitable for the prevention of long-term, cure-like treatment in case of viral infection and also in case of immune system diseases.

4. They act internally, thus eliminating the common and inconvenient external treatment in antiviral medicine.

The invention is illustrated by the following non-limiting examples.

Example 1

Dissolve 164 g (1 mol) of 1-lysine monohydrate in 500 ml of water at room temperature and add 320 g (about 1 mol) of ω-3 polyunsaturated fatty acid mixture (EPA 27.6%, DHA 44.6%, E) over 5 minutes. Vitamin 0.1%) is added dropwise. The mixture was stirred for 3 hours under gentle heating (40 ° C) and then concentrated in vacuo (4000-5300 Pa) to give 465 g of crystalline salt having the same composition as the starting material. M.p. 188-195 ° C (decomposed).

Example 2

155 g (1 mol) of 1-histidine were dissolved in 450 ml of water at room temperature and 320 g (about 1 mol) of co-3 polyunsaturated fatty acid (EPA 27.6%, DHA 44.6%) was added dropwise over 5 minutes. Hereinafter, the procedure of Example 1 is followed. 471 g of crystalline material are obtained; op: 192-202 (decomposed).

Example 3

All proceed as in Example 2, but using 133 g of l (+) - omitin instead of 1-histidine. 440 g of crystalline product are obtained; op. 189-195 (dec.).

Example 4

1-Lysine monohydrate (1.64 g, 0.01 mol) was dissolved in water (5 ml) at room temperature and then 3.02 g (0.01 mol) of eicosapentaenoic acid (EPA manufactured by Sigma, St. Louis USA E-7006) was added in small portions. (1987). After stirring for 3 hours at 40 ° C under N ₂ , the mixture is concentrated under a pressure of 4000 to 5300 Pa to give 4.9 g of a tan crystalline salt. 194-196 ° C (dec.).

Example 5

In each case, Example 4 was repeated except that 3.28 g (0.01 mol) of docosahexaenoic acid (DHA from Sigma, Cat. No. D-6508 (1987)) were used instead of eicosapentaenoic acid.

4.85 g of crystalline salt are obtained; 195-198 'C (dec.).

Example 6

All proceed as in Example 4, but using 1.55 g (0.01 mol) of 1-histidine instead of 1-lysine monohydrate. 4.8 g of crystalline salt are obtained; Mp: 196-200 'C (decomposed).

Example 7

All proceed as in Example 4 but using 1.55 g (0.01 mol) of 1-histidine instead of 1-lysine monohydrate and 3.28 g (0.01 mol) of DHA instead of EPA. The resulting crystalline salt weighed 3.78 g; Mp: 196-199 ° C (decomposed).

Example 8

In any event, the procedure of Example 4 was followed except that 1.32 g (0.01 mol) of l (+) - omitin was used instead of 1-lysine monohydrate. 4.6 g of crystalline salt are obtained; M.p. 190-193 C (dec.).

Example 9

All proceed as in Example 4, except using 1.33 g (0.01 mole) of l-lysine monohydrate, 3.28 g (0.01 mole) of DHA instead of EPA. The resulting crystalline salt weighed 4.55 g; M.p. 191-195 'C (dec.).

Example 70 1-Alanine g (22 mmol) was dissolved in a solution of water (0.88 g, 22 mmol) in NaOH (20 mL) at room temperature. 7.5 g (22 mmol) of a mixture of ω-3 polyunsaturated fatty acids (EPA 27.6%; DHA 44.6%, vitamin E 0.1%) are added dropwise at 40 'C.

The mixture was then stirred at 40 ° C for 2 hours under N ₂ and the solvent was evaporated in vacuo (4000-5300 Pa). 10.4 g of a light brown pasty solid are obtained, m.p. 210-220 ° C.

11-14. In each of Examples 1 to 4, the same amino acid was used as in Example 10 instead of 1-alanine.

HU 199775 Β

Example 11 10.0 mL of methanol 1.5 g of l-proline (13.0 mmol) 15.0 ml of water 40.0 ml of water 0.38 g NaOH (9.5 mmol) 0.52 g NaOH (13.0 mmol) 3.17 g of ω-3 fatty acid mixture (Example 10) (9.5) 4.35 g of ω-3 fatty acid (Example 10) (13.0 mmol) 5 mmol). After evaporation, 4.5 g light brown The resulting brown product weighed 6.3 g. crystalline product, Melting point: 175 ° C. Example 12 1.0 g of 1-leucine (7.6 mmol) Example 18 5.0 ml of water 10 2.0 g of glutamine (13.77 mmol) 0.3 g NaOH (7.6 mmol) 20.0 mL of methanol 2.54 g ω-3 fatty acid (7.6 mmol) 105.0 ml of water Obtained as a brown crystalline product: 3.70 g 0.55 g NaOH (13.77 mmol) liquefies in air. 4.60 g of a mixture of ω-3 fatty acids (13.77 mmol) 15 Brownish powdery crystal weight: 7.11 g; Example 13 Mp: 181-190 ° C. 0.5 g of 1-threonine (4.2 mmol) 10.0 ml of water Example 79 0.16 g NaOH (4.2 mmol) Preparation of an encapsulated pharmaceutical composition 1.4 g ω-3 fatty acid (4.2 mmol) 20 Prepared according to Example 1 salt mixture - Weight of yellow crystalline products: 1.95 g; Op: 204- 500 mg of active ingredient are administered by known encapsulation procedure. 213 'C (dec.). hard gelatin capsules suitable for receiving Juk. Example 14 1.0 g of 1-aspartic acid (7.5 mmol) 25 Example 20 40.0 ml of water Preparation of tablets 0.6 g NaOH (15.0 mmol) Prepared according to Example 1 salt as follows 2.5 g ω-3 fatty acid (7.5 mmol) tablets are prepared as follows: Yellow crystalline salt: 3.7 'g: Op: 200' C Example 1 Salt 500 mg (Dec) 30 lactose 120 mg starch 63 mg Example 75 polyvinylpyrrolidone 3.5 mg 1.5 g (7.1 mmol) of 1-arginine HCl in 20 mL of 0-10 ° C magnesium stearate 3.5 mg between chilled anhydrous alcohol dissolve in which If desired, the tablets can be coated with 7.1 mmol of metal sodium was also dissolved. The mixture 35 cover.

after stirring at room temperature for 20 minutes, the filtrate is concentrated in vacuo. The residue was dissolved in a solution of 0.28 g of NaOH (7.1 mmol) in 15 mL of water.

To the resulting solution was added 2.37 g (7.1 mmol) of a mixture of ω-3 polyunsaturated fatty acids (composition EPA 27.6%, DHA 44.6%, vitamin E 0.1%). The mixture was then stirred at 40 ° C for 2 hours and the solvent was evaporated in vacuo (4000-5300 Pa). 4.05 g of yellow crystalline salt are obtained. M.p. 207-211 ° C (dec.).

Example 16

1-Tyrosine (2.0 g, 11.0 mmol) was dissolved in a solution of NaOH (0.44 g, 11.0 mmol) in water (20 mL) and methanol (20 mL) at room temperature. To the resulting solution was added dropwise a mixture of 3.68 g (11.0 mmol) of ω-3 polyunsaturated fatty acid (same composition as in Example 10) at 40 ° C.

The reaction mixture was stirred under N _{2 for} 2 hours at 40 ° C and then the solvents were distilled off at 4000-5300 Pa. A yellowish powdery solid crystalline salt remains, weighing 6.18 g, m.p. 150 DEG C. (part decomposition).

17-18. In each of Examples 1 to 4, Example 16 is followed by salt formation with the appropriate amino acid instead of 1-tyrosine.

Claims (5)

A process for the preparation of salts of the formula I with antiviral and immunostimulatory properties R-COOAH + (D Example 17 1.0 g 1-serine (9.5 mmol) - in which R represents a C18-24 alpha group containing at least two unsaturated double bonds, "A" represents an amino acid occurring in living organisms and / or a C 1 -C 4 alkyl ester or an alkaline cation salt or amide thereof. Characterized in that an amino acid of formula A, wherein A is as defined above, with one or more acids of formula (Π) R-COOH (Π) wherein R is as defined above in a polar solvent 55 reaction. A process for preparing the 1-lysine salt of eicosapentaenoic acid according to claim 1, wherein the eicosapentaenoic acid is reacted with 1-lysine. The process for preparing the 1-lysine salt of docosahexaenoic acid 60 according to claim 1, wherein the docosahexaenoic acid is reacted with 1-lysine. 4. A process for the preparation of a 1-lysine salt of docosahexaenoic acid and eicosapentaenoic acid according to claim 1, wherein the mixture of docosahexaenoic acid and eicosapenic taenoic acid is reacted with 1-lysine. HU 199775 Β A process for the preparation of a pharmaceutical composition having an antiviral and an immunostimulatory effect, characterized in that a compound of the formula I according to claim 1, wherein the substituents are as defined in claim 1, is optionally used in the preparation of a medicament and / or together with excipients, in a manner known per se.