LU87470A1 - NOVEL ANTIVIRAL SALTS, PHARMACEUTICAL COMPOSITIONS CONTAINING THEM AND PROCESS FOR PREPARING THE SAME - Google Patents

Description

New antiviral salts, pharmaceutical compositions containing them and process for their preparation.

The present invention relates to new antiviral and immunostimulating salts and to pharmaceutical compositions containing these salts.

Another aspect of the present invention relates to providing a process for the preparation of the new salts. The advantageous antiviral action of ω-3 polyunsaturated fatty acids [5,8,1 1,14,17-eicosapentaenoic acid (hereafter: EPA) and acid 4,7,10,13, 16,19-docosahexaènoïque (thereafter: the □ HA)] attracted much attention. Szads [Antimicrobial Agents and Chemoterapy] 2, 523 (1977)] has shown by in vitro experiments that polyunsaturated fatty acids, for example both EPA and DHA, are capable of inhibiting virus replication. This fact has been pointed out by Reinhardt et al. [J. of Virology 25, 479 (1978)] studying the inhibition of replication on a bacteriophage PP 4. The antiviral effect of polyunsaturated fatty acids and inter alia that of EPA and DHA is described in detail in the Description of US Patent No. 4,513,008. The effect of EPA and DHA has been compared in animal experiments performed on mice and guinea pigs, with that of Acyclovir [9- (2-hydroxyethoxymethylj-guanine), which is the most antiviral agent. often used today It has been established that compositions containing in particular DHA were more active against the herpes virus than Acyclovir.

A number of articles have been devoted to the antiviral effect of DHA and EPA, such as those by WhitaHer et al. [Proc. Nat !. Acad. Sci. USA 76, 5919 (1979)] as well as Boodnight et al. [Arteriosclerosis, 2, 87, (1982)] and Yoshiahi [Biochimica and Biophysica Acta 793, 80 (1984)}.

PricHett and coil. [Immunology 4g, 819 (1982)] have shown that the humoral immune response is stimulated by eicosapentaenoic acid used as an analogue of arachidonic acid. With a diet rich in EPA, the specific production of IgS and IgE in response to the albumin of the egg, was increased from 4 to 8 times compared to the control, in inbred Sprague-Dawley rats. According to this article, an increased antibody response was induced by the diet rich in EPA In addition, it has been shown by studies that EPA acts by inhibiting the prostaglandin suppressor system and can thus inhibit or correct, respectively, the immunodeficiency accompanying aging or other pathological processes (autoimmune processes, tumorogenesis). These findings were reported in the articles by Kelley et al. (J. of Immunol. 134, 1914 (1985)] as well as Homey et al. [Clin. Exp. Immunol. 65, 473 (1986)].

It has been known for a long time thanks to in vivo studies by Pearson and coil. [Proc. Soc. Exp. Biol. Med. 79, 409 (1952)] that L-lysine has an effect of inhibiting the encephalomyelitis virus. Later, Tanherslay [J. Bact. 87, 609 (1964)] drew attention to the fact that replication of the herpes simplex virus (hereinafter: HSV) is inhibited by lysine in human cells under in vitro conditions. From human examinations, Kagan reported [The Lancet, J., 137 (1974)] that both oral and genital lesions induced by HSV disappeared very quickly following treatment with L-lysine .

Griffith and coil. [Dermatologica, 156, 257 (1978)] studied the therapeutic faction of L-lysine at various doses in 45 patients infected with H5V I and HSV II, during various periods of treatment. The age of the patients (especially women) was between 8 and 60 years. It has been established that L-lysine exerts only a suppressant effect but no curative effect on HSV.

The aim of the present invention is to provide new therapeutically effective salts and to use these salts to prepare pharmaceutical compositions combining the advantageous therapeutic effects of fatty acids (0-3 unsaturated with those of amino acids, in particular of ta lysine, I ornithine and histidine, and having a stronger action than that of any antiviral agent known until now The invention is based on ia observation that the salts obtained by the salification of fatty acids to- 3 unsaturated, in particular EPA and DHA, with amino acids or their derivatives, respectively, in particular with lysine, have powerful antiviral and immunostimulatory effects

Thus, the present invention relates to new antiviral and immunostimulatory salts represented by the general formula (I) R-COCTAH + (I) in which R is a Cig-24 alkyl group containing at least two double bonds; and "A" represents an amino acid appearing in living organisms and / or a derivative thereof containing a carboxy group substituted by a C 1-4 alkyl group or an amino group or by an alkali metal cation.

According to the present invention, these new salts are prepared by reacting, as basic component an amino acid "A" as defined with respect to the general formula (I), in which the substituents are as defined above, with a acid of general formula (II), R-COOH (II) in which R is as defined above, in a polar solvent.

The compounds of general formula (I) are preferably salts formed from unsaturated ω-3 fatty acids containing at least two double bonds with basic amino acids or their derivatives, respectively

The possible toxicity vis-à-vis a cell culture of the salts according to the invention was studied with a salt formed from a mixture of ω-3 polyunsaturated fatty acids (containing 27.6% of EPA and 44.6% DHA) and L-lysine, through the effect that this salt exerts on the propagation and morphology of Hel 2 cells (human epithelial tumor cell line). These experiments were carried out on a plastic tissue culture sheet, containing 6 × 4 hollow spaces (holes having a floor area of 1.9 cm 2 each).

A stock solution containing the test compound at a concentration of 10 mg / ml was prepared in an Eagle MEM medium (manufactured by Serva BmbH Co., Heidelberg, RFA). An aliquot of the stock solution was diluted 10 times, the resulting solution was diluted twice and then the last solution was subjected to double dilution twice, providing successively decreasing concentrations of the test compound (solutions No . 1 to 5). The cells were treated with 1 ml of each of the solutions containing the active component at the following concentrations:

The treatment was carried out for 1 hour, then the cultures were washed twice with a buffered sodium chloride solution (phosphate buffered saline, hereinafter referred to as PBS), then a nutrient medium was added to the cultures. 24 hours later and after fixing with methanol, the cultures were stained with an ethanolic Giemsa solution (manufactured by Reanal, Budapest, Hungary] and the morphology of the cells was evaluated under an illuminating microscope. It was established that the compound d the test was found to be toxic only above a concentration of 1000 Mg / ml. The effect of inhibiting viral replication was examined on Hep 2 cells as described above. Type I of HSV a was used for infection. The virus concentration was approximately 1000 PFU (plaque forming unit). Solutions containing the test compound at the concentration of 1000, 500 and 250 Mg / ml, respectively, were used and for the treatment, 0.1 ml of solution was added to the undiluted virus suspension, the mixture was incubated at 37 ° C. for 1 hour, then the cells were treated with the preincubated virus with compound d and the development pement of cytopathological alterations (thereafter; CP) was observed for 7 days.

It was established that viral replication was directly inhibited by a dose of 500 jJg / ml or 250 Mg / ml of the test compound and the value of the infectious titre of the cytopathological dose (neg. Log. CPDso) was 1 , 75 calculated for 0.1 ml, relative to the neg. log. CPDso of 4.5 presented by the untreated control virus culture. The value of viral inhibition exceeds that of the control by two orders of magnitude. Under the same conditions, EPA and DHA did not show any significant viral inhibition and lysine itself only inhibited viral replication by about an order of magnitude more than the control.

The vaccine virus was also treated in an in vitro experiment carried out as described above. The value of neg. log. CPDso of the test compound measured on the vaccine virus was 1.75 compared to the value of 5.5 of the untreated control virus, that is to say that the test compound exhibited an inhibiting effect of the virus which was three orders of magnitude greater than that of the control. The in vitro action on the immune system of the compounds according to the invention has been studied on the activation of lymphocytes by polyclonal mitogens. The blastic transformation of lymphocytes was studied by pipetting a cell population of lymphocytes obtained on a Fico Uromiro gradient [Scand. J. Clin. Lab. Invest. 2J [, 97 [1968)] in the hollow holes of flat-bottomed leaves, then adding 25 j-ig / ml of Concanavaiin A (called Con A) (manufactured by Pharmacia, Uppsala, Sweden] and then a solution containing the new compound according to the invention at a concentration of 0.1, 1.0 or 10 JJg / mi respectively, in each of the parallel cultures. A culture containing 25 Mg / mi of Con A without test compound was used as a control The sheets were kept in an air atmosphere containing 5% carbon dioxide at 37 "C for 72 hours and 0.4 J-iCi ^ H-thymidine was added to each sample 64 hours later, before stopping the culture 72 hours later, the cultures were filtered through a glass filter, the filters were placed in a scintillation cuvette and the radioactivity was measured each time in 5 ml of toluene solution with a beta radiation counter device. The results are collected in the following table

It is evident from these examinations that the compounds according to the invention and in particular the lysine or tyrosine salt formed with a mixture of polyunsaturated fatty acids provides significant results in the tests, that is to say a strong immunostimulating effect, while the separate components forming the salt had in themselves only little or no biological action.

As starting material, ω-3 unsaturated C] g-24 fatty acids representing a salt component should be used, mainly oils which can be obtained from fish from the northern seas such as salmon, cod, sardines or their liver, but oils from freshwater fish can also be used. Polyunsaturated ω-3 fatty acids can be obtained from the above oils by a known method [J. Am. Chem. Soc. 59, 1 17 [1982}].

The active compounds of general formula [1} can be put in the form of capsules, tablets, dragees or other pharmaceutical compositions formulated according to a method known in itself with the supports and / or additives such as lactose, starch, stearate magnesium and the like, commonly used in the pharmaceutical industry.

To inhibit the oxidation of the composition, it is advisable to use Γα-tocopherol [vitamin EL of glutathione or traditional antioxidants such as, for example, butylhydroxytoluene.

The main advantages of the compounds according to the present invention and of the pharmaceutical compositions prepared from them, can be summarized as follows: 1) They can be used against acute viral infections; they are in particular preferable in the infection by a virus of herpes to suppress the infection during an initial stage. 2} Due to their immunostimulating effect, they can preferably be used against retroviruses, in particular against immunodeficiency syndromes (for example AIDS) induced by viruses of types HTLV III and IV. 3) They contain exclusively compounds natural active ingredients that are essential from a biological point of view; thus, they are useful for long-term prophylactic treatment, of the cure type in the event of risk of viral infection as well as in diseases of the immune system. 4) They also act internally; the inconvenient external treatment commonly used in antiviral treatment can thus be avoided.

The compounds and compositions according to the present invention, as well as their preparation process, are illustrated in detail in the following nonlimiting examples. EXAMPLE 1 164 g (1 mole] of L-lysine monohydrate dissolved in 500 ml of water at room temperature, are added dropwise 320 g (about 1 mole) of a mixture of fatty acids ω- 3 polyunsaturates (containing 27.6% of EPA, 44.6% of DHA and 0.1% of vitamin E.) The mixture is stirred under slight heating (at 40 * 0 and under nitrogen for 3 hours and then evaporated under reduced pressure (4 to 5.3 HPa) to obtain 465 g of a crystalline salt, mp 188-195 ° C (with decomposition), the composition of unsaturated fatty acids of which is identical to that of the starting mixture. 155 g (1 mole) of L-histidine are dissolved in 450 ml of water at room temperature and dropwise added with 320 g (about 1 mole) of a mixture of ω-3 polyunsaturated fatty acids ( containing 27.6% of EPA and 44.6% of DHA) in 5 minutes The procedure described in Example 1 is then followed and provides 471 g of a crystalline substance, mp: 192-200 ° C (with decomposition) .EX EMPLE 3

The procedure described in Example 2 is repeated, except that L-histidine is replaced by 133 g of L (+) - omithine and provides 449 g of a crystalline product, mp: 189-195 * C (with decomposition} EXAMPLE 4

After dissolving 1.64 g (0.01 mole) of L-iysine monohydrate in 5 ml of water at room temperature, 3.02 g (0.01 mole) of eicosapentaenoic acid [manufactured by Sigma, St Louis , USA, under catalog number D-7006 (1987)] are added to the solution in small portions. The mixture is maintained at 40 ° C. under nitrogen for 3 hours, then evaporated under a pressure of 4 to 5.3 kPa to give 4.9 g of yellowish-brown crystalline salt, mp: 194-196 "C (with decomposition) EXAMPLE 5

The procedure of Example 4 is followed, except that 3.28 g (0.01 mole) of docosahexaenoic acid (DHA) (manufactured by Sigma, St Louis, USA, under catalog number D-65Q8 (1987) ] are used in place of eicosapentaenoic acid to give 4.85 g of a crystalline salt, mp: 195-198 * C (with decomposition).

The procedure of Example 4 is followed, except that 1.55 g (0.01 mole) of L-histidine is used in place of L-lysine monohydrate to give 4.8 g of a crystalline salt, mp , 196-200 "C (with decomposition). EXAMPLE 7

The procedure of Example 4 is followed, except that 1.55 g (0.01 mole) of L-histidine is used in place of L-lysine monohydrate and that 3.28 g (0.01 mole) of DHA is used in place of EPA to obtain 3.78 g of a crystalline salt, mp, 196-199'C (with decomposition). EXAMPLE 8

The procedure of Example 4 is followed, except that the L-lysine monohydrate is replaced by 1.32 g (0.01 mole) of L (+) - omithine to obtain 4.6 g of a crystalline salt, mp , 190-193'C (with decomposition). EXAMPLE 9

The procedure of Example 4 is followed, except that the L-lysine monohydrate is replaced by 1.32 g (0.01 mole) of L (+) - omithine and the EPA by 3.28 g (0.01 mole) of DHA to obtain 4.5 g of a crystalline salt, mp, 191-195 "C (with decomposition). EXAMPLE 10 2 g (22 mmol) of L-alanine are dissolved in the solution of 0.88 g ( 22 mmol) of sodium hydroxide in 20 ml of water with stirring at room temperature, then 7.5 g (22 mmol) of a mixture of ω-3 polyunsaturated fatty acids (containing 27.6% of EPA, 44.6% DHA and 0.1% vitamin E) are added dropwise at 40 ° C. to the above solution. The mixture is stirred under nitrogen at 40 ° C. for 2 hours, then the solvent is distilled off under a reduced pressure of 4 to 5.3 hPa to give 10.4 g of a light brown pasty solid product, mp: 210-220 "C.

In the following examples 11 to 14, the process described in example 10 is repeated, except that the L-alanine is replaced by the corresponding amino acid. EXAMPLE 11 L-Proline 1.5 g (13.0 mmol)

Water 40.0 ml

Sodium hydroxide 0.52 g (13.0 mmol) Mixture of fatty acids ω-3 (according to example 10) 4.35 g ί 13.0 mmol)

An oily brown product is obtained with a yield of 6.3 g. EXAMPLE 12 L-Leucine 1.0 g (7.6 mmol)

Water 5.0 ml

Sodium hydroxide 0.3 g (7.6 mmol) Mixture of fatty acids ω-3 2.54 g (7.6 mmol)

A crystalline brown product is obtained with a yield of 3.70 g. It liquefies in air, EXAMPLE 13 L-Threonine 0.5 g (4.2 mmol)

Water 10.0 ml

Sodium hydroxide 0.16 g (4.2 mmol) Mixture of co-3 fatty acids 1.4 g (4.2 mmol)

A yellow crystalline product is obtained with a yield of 1.98 g; mp: 204-213 "C (with decomposition). EXAMPLE 14

L-aspartic acid 1.0 g (7.5 mmol)

Water 40.0 ml

Sodium hydroxide 0.6 g (15.0 mmol) Mixture of fatty acids ω-3 2.5 g (7.5 mmol)

A yellow crystalline salt is obtained with a yield of 3.97 g, m.p .: 200 "C (with decomposition). EXAMPLE 15 7.1 mmol of metallic sodium are dissolved in 20 ml of anhydrous ethanol, then the solution is cooled to between 0 * and 10 ° C. and 1.5 g (7.1 mmol) of L-arginine hydrochloride is added, after 20 minutes of stirring at room temperature, the mixture is filtered and the filtrate is evaporated under reduced pressure The evaporation residue is dissolved in the solution of 0.28 g (7.1 mmol) of sodium hydroxide in 15 ml of water, then 2.37 g (7.1 mmol) of a mixture of polyunsaturated ω-3 fatty acids (containing 27.6% of EPA, 44.6% of DHA and 0.1% of vitamin E) are added. The mixture is then stirred at 4 ° C. under nitrogen for 2 hours, then the solvent is distilled off under a reduced pressure of 4 to 5.3 kPa to give 4.05 g of a yellow crystalline salt, mp: 207-21 l'C (with decomposition). EXAMPLE 16 2.0 g (11.0 mmol) of L-tyrosine are dissolved in a solution containing 0.44 g (11.0 mmol) of sodium hydroxide in 20 ml of water and 20 ml of methanol to the room temperature, then 3.68 g (11.0 mmol) of a mixture of ω-3 polyunsaturated fatty acids (containing 27.6% of EPA, 44.6% of DHA and 0.1% of vitamin E ) are added dropwise to the above solution at 4 ° C. After stirring the reaction mixture under nitrogen at 40 ° C. for 2 hours, the solvents are evaporated under a reduced pressure of 4 to 5.3 kPa to give a saline, crystalline, solid, yellowish powdery residue with a yield of 6.18 g, mp: 150 "C (with partial decomposition). In Examples 17 and 18 below, the process described in Example 16 is repeated using the amino acid corresponding to the place of L-tyrosine EXAMPLE 17 L-Serine 1.0 g (9.5 mmol) Methanol 10.0 mi

Water 15.0 ml

Sodium hydroxide o, 38 g (9.5 mmol) Mixture of fatty acids ω-3 (according to example 1) 3.17 g (9.5 mmol)

After evaporation, a light brown crystalline product is obtained with a yield of 4.5 g, which decomposes above 175 "C. EXAMPLE 18

Glutamine 2.0 g (13.77 mmol)

Methanol 20.0 ml

Water 105.0 ml

Sodium hydroxide o, 55 g (13.77 mmol) Mixture of fatty acids ω-3 4.60 g [13.77 mmol)

A brownish powdery crystalline salt is obtained with a yield of 7.11 g, m.p .: 181-190 ° C. EXAMPLE 19

Preparation of a pharmaceutical composition in the form of capsules

A salt mixture prepared as described in Example 1 is introduced by a known encapsulation method into hard gelatin capsules capable of receiving 500 mg of active component, EXAMPLE 20

Preparation of tablets

Tablets are prepared from the salt mixture obtained according to the procedure of Example 1; they each contain the following components: mg Salt mixture of example 1,500

Lactose 120

Starch 03

Polyvinylpyrrolidone 3.5

Magnesium stearate 3.5

The tablets can optionally be coated with a sugar coating with a suitable machine.

Claims (11)

1. New antivipal and immunostimulating salts, characterized by the general formula (I) i R-COO "AH + fl) in which R is a C) alkyl group 8-24 containing at least two double bonds; and" A "represents a amino acid appearing in living organisms and / or a derivative thereof containing a carboxy group substituted by a C 1-4 alkyl group or an amino group or by an alkali metal cation. 2. Salt according to claim 1, characterized in that it comprises I eicosapentaenoic acid (EPA) as the acid component and L-lysine as the basic component. 3. Salt according to claim 1, characterized in that it comprises I docosahexaenoic acid (DHA) as the acid component and fa L-iysine as the basic component. 4. Salt according to claim 1, characterized in that it comprises a mixture of docosahexaenoic acid and eicosapentaenoic acid as the acid component and L-lysine as the basic component. 5. Antiviral and immunostimulatory pharmaceutical composition, characterized in that it comprises a salt of general formula OL in which the substituents are as defined in claim 1, in admixture with carriers and / or additives and optionally antioxidants commonly used in the pharmaceutical industry. 6. Process for the preparation of the new salts of general formula fl) R-COQ-AH + f |) in which R and A are as defined in claim 1, characterized in that a basic component is reacted with a amino acid, wherein the substituents are as defined in claim 1, with an acid of general formula (II], R-COOH (II) in which R is as defined in claim 1, in a polar solvent. 7. Method according to claim 6, characterized in that eicosapentaenoic acid is reacted with L-lysine, 8. Method according to claim 6, characterized in that docosahexaenoic acid is reacted with L-lysine. 9. Method according to claim 6, characterized in that a mixture of docosahexaenoic acid and eicosapentaenoic acid is reacted with L-lysine. 10. Process for preparing antiviral and immunostimulatory pharmaceutical compositions, characterized in that a salt of general formula (I) prepared by the process according to claim 6 is mixed with supports and / or auxiliary agents and optionally antioxidants used commonly in the pharmaceutical industry and that the mixture thus obtained is transformed into pharmaceutical preparations in the form of tablets, dragees, capsules, suppositories, etc. 11. Use of the salts of general formula (I) according to claim 1, characterized in that it relates to the preparation of pharmaceutical compositions having antiviral and immunostimulating activities.