LU87471A1 - ANTIVIRAL AND IMMUNOSTIMULANT PHARMACEUTICAL COMPOSITIONS AND PROCESS FOR PREPARING THE SAME - Google Patents

Description

Antiviral and immunostimulatory pharmaceutical compositions and process for preparing them.

The present invention relates to antiviral and immunostimulatory pharmaceutical compositions.

Another aspect of the present invention relates to providing a process for the preparation of these compositions. The beneficial antiviral action of gras-3 polyunsaturated fatty acids [5,8,11,14,17-eicosapentaenoic acid (hereafter: EPA) and 4,7,10,13,16 acid , 19-docosahexaènoïque (thereafter: the □ HA)] attracted a lot of attention. Szads (Antimicrobial Agents and Chemoterapy 12, 523 (1977)] has shown by in vitro experiments that polyunsaturated fatty acids, for example both EPA and DHA, are capable of inhibiting virus replication. fact has been reported by Reinhardt et al. (J, of Virology 25, 479 (1977)] studying the inhibition of replication on a bacteriophage PR 4. The antiviral effect of polyunsaturated fatty acids and among others, that of l EPA and DHA is described in detail in the description of US Patent No. 4,513,008. The effect of EPA and DHA was compared in animal experiments performed on mice and guinea pigs, to that of Acyclovir [9- (2-hydroxyethoxymethylj-guanine), which is the most frequently used antiviral agent currently in use. 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 of Whitaher et al. [Proc. Natl. Acad. Sci. USA ZS. 5919 (1979) 1 as well as from Goodnight et al. [Arteriosclerosis, 2, 87, (1982) 1 and de Yoshiaki [Biochimica and Biophysica Acta 793, 80 (1984)].

Prichett and coil. [Immunology 46, 819 (1982) 1 have shown that the humoral immune response is stimulated by eicosapentaenolic acid used as an analogue of arachidonic acid. With a diet rich in EPA, the specific production of Ig6 and IgE in response to albumin from the egg was increased by 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 ΓΕΡΑ acts by inhibiting the prostaglandin suppressor system and can thus inhibit or correct, respectively, the accompanying immunodeficiency 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 de Homey et al, [Clin. Exp. Immunol. 65, 473 (1986)].

It has long been known from in vivo studies by Pearson et al. [Proc. Soc. Exp. Biol. Med. 7g, 409 (1952)] that L-lysine has an inhibitory effect on the encephalomyelitis virus. Later, Tankerslay [J. Bact. 87, 609 (1964)] drew attention to the fact that replication of the herpes simplex virus (subsequently; HSV) is inhibited by lysine in human cells under in vitro conditions. From human examinations, Kagan reported [The Lancet, 1, 137 (1974)] that both oral and genital lesions induced by HSV disappeared very quickly following treatment with L-lysine.

Griffith et al. [Dermatologica, 156, 257 (1978)] studied the therapeutic action of L-lysine at various doses on 45 patients infected with HSV 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 a new pharmaceutical composition combining the advantageous therapeutic effects of unsaturated ω-3 fatty acids with those of amino acids and certain essential amino acids, in particular lysine, omithine and histidine, and having a stronger action than that of any antiviral agent known until now. The invention is based on the observation that the advantageous antiviral effects known separately in se of the unsaturated ω-3 fatty acids, in particular of ΓΕΡΑ and DHA, as well as amino acids, respectively, in particular of lysine, are increased so synergistic in a mixture formed from these and in addition, the mixture also has an immunostimulatory action.

Thus, the present invention relates to an antiviral and / or immunostimulatory pharmaceutical composition, which comprises as active components, amino acids appearing in living organisms (such as taurine, cycloserine, homoserine, homocysteine, canavanine, sancosine, hydroxyproline, hydroxylysine, 4-hydroxyphenylalanine, essential amino acids) and / or their derivatives in which the carooxy group is substituted by a C1-4 alkyl group or an amino group or by an alkali metal, alkaiino metal cation - earthy or ammonium, preferably a sodium or potassium cation; or the hydrates and / or salts of these compounds, with one or more C] g-24 fatty acids containing an alkyl group carrying at least two double bonds, optionally in admixture with carriers and / or additives and antioxidants commonly used in the pharmaceutical industry, where the molar ratio of amino acid and fatty acid (s) is 1.4-4: 1.

Cω-24 ω-3 fatty acids containing at least two double bonds and amino acids, of basic character, are preferably used as components of the compositions according to the present invention, it is suitable to use lysine, histidine or omithine, or their derivatives as essential amino acids having a basic character. □ he substances usually used in the pharmaceutical industry such as lactose, starch or magnesium stearate, can be used as carriers and additives in the compositions according to the invention.

To inhibit the oxidation of the composition, c-tocopherol (vitamin E), glutathione or traditional antioxidants such as butylhydroxytoluene should be used.

Among the compositions according to the invention, the activity and the possible toxicity of mixtures containing a mixture of polyunsaturated ω-3 fatty acids (containing 27.6% of EPA and 44.6% of DHA) and of L- lysine in molar ratios of 1: 1, 1: 4 and 4: 1, respectively (test compositions) were studied through the effect that these mixtures exert 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 3 each).

A stock solution containing the test composition at a concentration of 10 mg / ml, was prepared in an Eagle MEM medium (manufactured by Serva 6mbH Co., Heidelberg, RFA). An aliquot of the stock solution was diluted 10 times, the solution obtained was diluted twice and then the last solution was subjected to double dilution twice, providing successively decreasing concentrations of the test composition (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 Biemsa solution (manufactured by Reanal, Budapest, Hungary) and the cell morphology was evaluated under an illuminating microscope. It has been established that the test compound does not turn out to be toxic except at a concentration of 1GOÛ Jig / ml. The inhibition effect of viral replication was examined on Hep 2 cells as described above. Type I of HSV was used for infection. The virus concentration was approximately 1000 PFU (plaque forming unit). Solutions containing the test composition at a concentration of 1000, 500 and 250 Ug / ml, respectively, were used and for 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 the test composition and the development of cytopathic alterations (CP) was observed for 7 days. establish that viral replication was directly inhibited by a dose of 500 μg / ml or 250 μg / ml of the test composition: the infectious titre value of the cytopathological dose (neg. log. CPD50) was 1 , 75 to 2.04 calculated for 0.1 ml, compared to the value of log neg CPD50 of 4.5 presented by the culture of untreated control virus. The value of viral inhibition exceeds by two orders of 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 compared to the control.

The vaccine virus was also treated in an in vitro experiment carried out as described above. The value of neg. log. CPD50 of the test composition measured on the vaccine virus was 1.75 to 2.15 relative to the value of 5.5 of the untreated control virus, that is to say that the test composition had a virus inhibition effect which was three orders of magnitude greater than that of the control. The in vitro action on the immune system of the compositions according to the invention was 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 gradient. Fico Uromiro [Scand. J. Clin. Lab. Invest. 21, 97 (1968)] in the hollow holes of flat-bottomed leaves, then adding 25 Jig / ml of Concanavalin A (called Con A) (manufactured by Pharmacia, Uppsala, Sweden) and then a solution containing the compositions according to l invention at a concentration of 0.1, 1.0 or 10 Jig / mi respectively, in each of the parallel cultures. A culture containing 25 Ug / ml Con A without test composition was used as a control. The leaves were kept in an air atmosphere containing 5% carbon dioxide at 37 ° C for 72 hours and 0.4 μΟϊ of ^ H-thymidine was added to each sample 64 hours later, before the culture stopped. 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 collated in the following table.

It is evident from these examinations that the compositions according to the invention, in particular the mixture of polyunsaturated fatty acids used with lysine and tyrosine, gave a significant result in the tests, that is to say a strong immunostimulating effect, while the separate components of the test materials according to the invention in themselves had only little or no biological action.

As the starting material, 8-3 unsaturated Ci8-24 fatty acids representing a component of the composition should be used, mainly oils which can be obtained from fish from the northern seas such as salmon, cod, sardines. or from their liver, but oils from freshwater fish can also be used. Polyunsaturated fatty acids (Ö-3 can be obtained from the above oils by a known method [J. Am. Chem. Soc. 59, 117 (1982)]. These fatty acids are liable to undergo oxidation. inhibit oxidation, antioxidants such as vitamin E should be used

The active components mentioned above can be put in the form of capsules, tablets, dragees or other pharmaceutical compositions formulated according to a method known in itself.

The main advantages of the pharmaceutical composition according to the present invention can be summarized as follows: a) The antiviral therapeutic effect of the active components of the composition is increased synergistically. b) It can be used against acute viral infections, especially infections with a herpes virus, to suppress the infection in an initial stage. c) Thanks to the immunostimulating effect, it can preferably be used against retroviruses, in particular against immunodeficiency syndromes (for example AIDS) induced by viruses of types HTLV III and IV. d) It contains exclusively natural active compounds which are essential from the biological point of view; thus, it is useful for a 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. e) It also acts internally; the inconvenient external treatment commonly used in antiviral treatment can thus be avoided.

The compositions according to the present invention are illustrated in detail in the following nonlimiting examples. EXAMPLE 1 164 g (1 mole) of L-lysine monohydrate and 320 g (about 1 mole) of a mixture of ω-3 polyunsaturated fatty acids (containing 27.6% EPA and 44.6% DHA) are mixed at room temperature. The homogeneous mixture thus obtained is introduced by a known encapsulation process, into hard gelatin capsules capable of receiving 500 mg of active component. EXAMPLE 2 155 g (1 mole) of L-histidine are mixed with 320 g (about 1 mole) of a mixture of polyunsaturated to-3 fatty acids (containing 27.6% EPA and 44.6% DHA). The procedure described in Example 1 is then followed. EXAMPLE 3 41.0 g (0.25 mole) of L-iysine monohydrate are mixed with 320 g (about 1 mole) of a mixture of ω-3 polyunsaturated fatty acids (containing 90.0% of EPA and 0 , 1% vitamin E), then the procedure described in Example 1 is followed. EXAMPLE 4

The procedure of Example 3 is followed, except that 164 g (1 mole) of L-lysine monohydrate and, as poinsaturated fatty acid, 328 g (I mole) of docosahexaenoic acid (DHA) (manufactured by Sigma, St Louis, USA, under catalog number D- 6508 (1987) 1 are used EXAMPLE 5

The procedure of Example 1 is followed, except that the L-lysine is replaced by 181 g (1.0 mole) of L-tyrosine. EXAMPLE 6

The procedure of Example 1 is followed, except that the L-lysine is replaced by 120 g (1.0 mole) of L-threonine. EXAMPLE 7

The procedure of Example 3 is followed, except that 164 g (1 mole) of L-lysine monohydrate and 80 g (about 0.25 mole) of a mixture of ω-3 polyunsaturated fatty acids (containing about 90, 0% DHA and 0.1% vitamin E) are used. EXAMPLE 8

The procedure of Example 3 is followed, except that 164 g (1 mole) of L-iysine monohydrate and that instead of a mixture of polyunsaturated fatty acids, 302 g (1 mole) of eicosapentaenoic acid (EPA) ) [manufactured by Sigma, St Louis, USA, under catalog number E-7006 (1987)] are used. EXAMPLE 9

The procedure of Example 1 is followed, except that the L-lysine is replaced by 132 g (1.0 mole) of L-omithine. EXAMPLE 10

The procedure of Example 3 is followed, except that the L-lysine is replaced by 33 g (0.25 mole) of L-omithine. EXAMPLE 11

The procedure of Example 4 is followed, except that the L-lysine is replaced by 132 g (1.0 mole) of L-omithine. EXAMPLE 12

The procedure of Example 1 is followed, except that the L-lysine is replaced by 133 g (1.0 mole) of L-aspartic acid. EXAMPLE 13

The procedure of Example 1 is followed, except that the L-lysine is replaced by 211 g (1.0 mole) of L-arginine hydrochloride. EXAMPLE 14

The procedure of Example 1 is followed, except that the L-lysine is replaced by 105 g (1.0 mole) of L-serine. EXAMPLE 15

The procedure of Example 1 is followed, except that the L-lysine is replaced by 145 g (1.0 mole) of L-glutamine. EXAMPLE 16

Preparation of tablets □ the tablets are prepared from the composition described in Example 1; they each contain the following components: mg

Composition of Example 1 5Q0

Lactose 120

Starch 63

Polyvinylpyrrolidone 3.5

Magnesium stearate 3.5

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

Claims (10)

1. Antiviral and / or immunostimulatory pharmaceutical composition, characterized in that it comprises, as active components: amino acids appearing in living organisms and / or their derivatives in which the carboxy group is substituted by a Ci-4 alkyl group or an amino group or by an alkali metal, alkaline earth metal or ammonium cation; or the hydrates and / or salts of these compounds, with one or more C18-24 fatty acids containing an alkyl group carrying at least two double bonds, optionally in admixture with carriers and / or additives and antioxidants commonly used in I pharmaceutical industry, in which the molar ratio of amino acid and fatty acid (s) is 1: 4 to 4: 1. 2. Composition according to claim 1, characterized in that it comprises fatty acids obtained from fish oils as unsaturated fatty acids. 3. Composition according to claim 1, characterized in that it comprises eicosapentaenoic acid (EPA) and L-lysine as active components. 4. Composition according to claim 1, characterized in that it comprises docosahexaenoic acid (DHA) and L-lysine as active components. 5. Composition according to claim 1, characterized in that it comprises docosahexaenoic acid, eicosapentaenoic acid and L-lysine as active components, 6. Process for the preparation of an antiviral and / or immunostimulatory pharmaceutical composition, characterized in that as active components are mixed: amino acids appearing in living organisms and / or their derivatives in which the carboxy group is substituted by a C1-4 alkyl group or amino group or by an alkali metal, alkaline earth metal or ammonium cation; or the hydrates and / or salts of these compounds, with one or more C18-24 fatty acids containing an alkyl group carrying at least two double bonds, optionally with carriers and / or additives and antioxidants commonly used in the pharmaceutical industry , and that they are put into the form of a pharmaceutical composition, in which the molar ratio of the amino acid and of the fatty acid (s) is from 1: 4 to 4: 1. 7. Method according to claim 6, characterized in that fatty acids obtained from fish oils are used as unsaturated fatty acids. 8 · Process according to claim 6, characterized in that eicosapentaenoic acid (EPA) and L-lysiqe are used as active components. 9. Method according to claim 6, characterized in that docosahexaenoic acid (DHA) and L-lysine are used as active components. 10. Method according to claim 6, characterized in that docosahexaenoic acid, eicosapentaenoic acid and L-iysine are used as active components.