FR2628324A1 - ANTIVIRAL AND IMMUNOSTIMULANT PHARMACEUTICAL COMPOSITIONS AND PROCESS FOR PREPARING THE SAME - Google Patents

Abstract

The present invention relates to novel antiviral and / or immunostimulatory pharmaceutical compositions, which comprise as active components: amino acids occurring in living organisms and / or their derivatives in which the carboxy group is substituted by a C1 - 4 alkyl group or a group amino or with 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 supports and / or additives and antioxidants commonly used in the pharmaceutical industry, in which the molar ratio of the amino acid and the fatty acid (s) is from 1: 4 to 4: 1. The invention further relates to a process for the preparation of these compositions.

Description

The present invention relates to compositions

  antiviral and immunostimulatory drugs.

  Another aspect of the present invention relates to the

  providing a process for the preparation of these compositions.

  ) Advantageous antiviral action of w-3 polyunsaturated fatty acids [5,8,11,14,17-eicosapentaenic acid [hereinafter: EPA]) and 4,7,10,13 acid , 16,19-docosahexaenoic (subsequently: DHA)] has attracted much attention. Szads [Antimicrobial Agents and Chemotherapy 12, 523 (1977)] has shown by in vitro experiments that polyunsaturated fatty acids, e.g. both EPA and OHA, are capable of inhibiting virus replication. This fact has been reported by Reinhardt et al. [J. of Virology 25, 479 [1977]] student

  inhibition of replication on bacteriophage PR 4.

  The antiviral effect of polyunsaturated fatty acids and between, inter alia, that of EPA and DHA, is described in detail in the

  US Patent Specification No. 4,513,008. The effect of EPA and ODHA has

  have been compared in animal experiments carried out on

  mice and guinea pigs, to that of Acyclovir [9- (2-

  hydroxyethoxyjmethyl) -guanine], which is the antiviral agent most often used today It has been established that the compositions containing especially DHA, were more active against the virus

of herpes as Acyclovir.

  A number of articles have been devoted to the effect

  antiviral agents of DHA and EPA, such as those of Whitaker et al. [Proc.

  Natl. Acad. Sci. USA 76, 5919 (1979)] as well as Goodnight et al.

  [Arteriosclerosis, 2, 87, (19821) and Yoshiaki [Biochimica and

Biophysica Acta 793, 80 [1984] 1.

  Prichett et al. [Immunol. 46, 819 (1982) 1] have shown that the humoral immune response is stimulated by the eicosapentaenoic acid used as an analogue of arachidonic acid. With an EPA-rich diet, the specific production of Ig and IgE in response to egg albumin was increased 4- to 8-fold relative to the control in inbred Sprague-Dawley rats. According to this article, an increased antibody response was induced by the EPA-rich diet. In addition, studies have shown that EPA acts by inhibiting the suppressor prostaglandin system and thus can inhibit or correct, respectively, the immunodeficiency accompanying aging or other pathological processes (autoimmune processes, tumorigenesis). These findings have been reported in articles by Kelley et al. [J. of Immunol. 134, 1914 (1985)] as well as

  Homey et al. [Clin. Exp. Immunol; 65, 473 (19861).

  We have known for a long time thanks to in-depth studies of

  Pearson et al. [Proc. Soc. Exp. Biol. Med. 79, 409 [1952]) that the L-

  lysine has an inhibitory effect on encephalomyelitis virus. Later, Tankerslay [J. Bact. 87, 609 (1964)] has drawn attention to the fact that replication of herpes simplex virus (subsequently: HSV) is inhibited by lysine in human cells under in vitro conditions, from In human studies, Kagan reported [The Lancet, 1, 137 (1974)] that both HSV-induced oral and genital lesions disappeared very rapidly as a result of treatment with

L-lysine.

  Griffith and coil. [Dermatologica, 156, 257 (1978)] investigated the therapeutic action of L-lysine at various doses in 45 patients infected with HSV I and HSV II for various treatment periods. The age of the sick (especially women) was between 8 and 60 years old. It has been established that L-lysine only exerts a suppressive but not curative effect on HSV. The object of the present invention is to provide a novel pharmaceutical composition combining the advantageous therapeutic effects of w-3 unsaturated fatty acids with those of amino acids and certain essential amino acids, in particular uysin, ornithine and histidine, and having a stronger action

  than any known antiviral agent to date.

  The invention is based on the finding that the advantageous antiviral effects known separately from unsaturated fatty acids, in particular EPA and DHA, as well as amino acids, in particular lysine, are synergistically increased in a mixture formed from these and more, 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 occurring in living organisms (such as taurine, cycloserine, homoserine, homocysteine, canavanine, sarcosine, Hydroxyproline, hydroxylysine, 4-hydroxyphenylalanine, essential amino acids) and / or their derivatives in which the carboxy group is substituted with a C 1-4 alkyl group or an amino group or with an alkali metal cation, metal alkaline earth or ammonium, preferably a sodium or potassium cation; or the hydrates and / or salts of these compounds, with one or more Cl 8-24 fatty acids containing an alkyl group bearing at least two double bonds, optionally in admixture with carriers and / or additives and antioxidants used in the pharmaceutical industry, in which the molar ratio of the amino acid and

  of the fatty acid (s) is from 1: 4 to 4: 1.

  C1-8-24 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 appropriate to use lysine, histidine or ornithine, or their derivatives as essential amino acids having a

basic character.

  Normally used substances 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, it is advisable to use tetocopherol [vitamin E1, glutathione or

  traditional antioxidants such as butylhydroxytoluene.

  Among the compositions according to the invention, the activity and the possible toxicity of mixtures containing a mixture of polyunsaturated fatty acids (containing 27.6% of EPA and 44.6% of OHA) and L-lysine at molar ratios of 1: 1, 1: 4 and 4: 1, respectively (test compositions) were studied via the effect these mixtures exert on the propagation and morphology of Hel 2 cells ( human epithelial tumor cell line). These experiments were performed on a plastic tissue culture sheet, containing 6x4

  hollow spaces (holes having a floor area of 1.9 cm 2 each).

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

1 10000

2,000

3,500

4,250

5,125

  The treatment was carried out for 1 hour, then the cultures were washed twice with buffered sodium chloride solution (phosphate buffered saline, hereinafter referred to as PBS), and nutrient medium was added to the cultures. 24 hours later and after fixation with methanol, the cultures were tinted with an ethanolic solution (manufactured by Peanal, Budapest, Hungary) and the morphology of the cells was evaluated under a light microscope. It was found that the test compound was toxic only

concentration of 1000 Jig / ml.

  The inhibitory effect of viral replication was examined on IHep 2 cells as described above. Type I HSV was used for infection. The concentration of the virus was approximately 1000 PFU (platelet-forming unit.) Solutions containing the test composition at the concentration of 1000, 500 and 250 μg / 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 virus preincubated with the test composition and the development of cytopathological alterations ( CP) was observed for 7 days. It could be established that the viral replication was directly inhibited by a dose of 500 μg / ml or 250 μg / ml of the test composition: the value of the infectious titre of the cytopathological dose. (Neg log CPOso) was 1.75 to 2.04 calculated for 0.1 mi, relative to the value of n / a CPDOO of 4.5 presented by the untreated control virus culture. viral inhibition is two orders of magnitude higher Under the same conditions, EPA and DI-HA showed no significant viral inhibition, and lysine itself inhibited viral replication only.

  about an order of magnitude more than the control.

  The vaccine virus has also been treated in an experiment

  vitro performed as described above. The value of neg. log.

  CPOSO of the test composition measured on the vaccine virus was 1.75 to 2.15 compared to the value of 5.5 of the untreated control virus, i.e., the test composition showed an effect of inhibition of the virus that was three orders of magnitude greater than

that of the witness.

  The in vitro action on the immune system of the compositions according to the invention has been studied on the activation of lymphocytes by polyclonal mitogens. The blast transformation of lymphocytes has been studied by pipetting a lymphocyte cell population obtained on a gradient. Fico Uromiro [Scand. J. Clin. Lab. Invest. 21, 97 (19681) in the hollow holes of flat-bottomed leaves, and then adding 25 μl / ml Concanavalin A (referred to as Con A) (manufactured by Pharmacia, Uppsala, Sweden) and then a solution containing the compositions according to US Pat. at a concentration of 0.1, 1.0 or 10 μg / ml respectively, in each of the parallel cultures A culture containing 25 μg / ml Con A without test composition was used as a control. were maintained in an air atmosphere containing 5% carbon dioxide at 37 ° C. for 72 hours and 0.4 μl of 3H-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 countertop device. influence

  beta. The results are collated in the following table.

  No. Compound Conc. of Coups / minute Rating of (name) comp. active (cpm) effect (ig / g) I Control 8969 + 2984 (Con A) 2 L-tyrosine 0.1 12915 2045 insignificant growth 3 L-lysine 0.1 11063 + 1528 not significant 1.0 11833 +1823 insignificant growth 4 Na salt of a 0.1 12332 + 2235 not significant mixture of fatty acids w-3 (see 1.0 12039 1640 significant step in ex 1) - Control 25 8396 2233 (Con A)

  6 Prod. from the ex. 5 0.1 14605 1747 strong growth-

  Significantly 1.0 12010 +2285 not significant

  7 Prod. from the ex. 1 0.1 16085 + 2063 strong growth

  Significantly 1.0 14681 1769 cr. significant 8 Prod. from the ex. 3 0.1 12661 + 1548 cr. significant 1.0 12420 + 1452 cr. significant 9 Prod. from the ex. 4 0.1 11920 + 2005 cr. significant 1, 0 12240 1950 believe. 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, i.e. a strong immunostimulatory effect, while the separate components of the test materials according to the invention in themselves had only one action

low or no biological

  It is appropriate to use as a starting material unsaturated C 18-24 fatty acids representing one component of the composition, mainly oils obtainable from North Sea fish such as salmon, cod, sardines. or their liver, but oils from freshwater fish may also be used. Fatty acids

  w-3 polyunsaturates can be obtained from the oils

  above by a known method [J. Am. Chem. Soc. 59, 117 (1982)].

  These fatty acids are susceptible to oxidation. To inhibit oxidation, antioxidants such as vitamin E should be used. The above-mentioned active components can be formulated into capsules, tablets, dragees or other pharmaceutical compositions formulated according to a known method.

in herself.

  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 synergistically increased.

  (b) It can be used against acute viral infections, especially infections. infections with a herpes virus, for

  remove the infection during an initial stage.

  c) Thanks to the immunostimulatory effect, it can be preferably used against retroviruses, in particular against the immunodeficiency syndromes (for example AIDS) induced by the

viruses of types HTLV III and IV.

  (d) It contains only natural active compounds that are biologically essential; thus, it is useful for prophylactic, long-lasting, cure-type treatment in case of risk of viral infection as well as in diseases

of the immune system.

  e) It also acts internally; In this way it is possible to avoid the inconvenient external treatment which is commonly used in antiviral treatment. The compositions according to the present invention are

  illustrated in detail in the following non-limiting examples.

EXAMPLE 1

  164 g (1 mole) of L-lysine monohydrate and 320 g (approximately 1 mole) of a mixture of polyunsaturated fatty acids (containing 27.6% EPA and 44.6% DOHA are mixed with The homogeneous mixture thus obtained is introduced by a known encapsulation process into capsules of

  hard gelatin capable of receiving 500 mg of active component.

EXAMPLE 2

  165 g (1 mole) of L-histidine are mixed with 320 g (approximately 1 mole) of a mixture of polyunsaturated fatty acids (containing 27.6% of EPA and 44.6% of DHA). . The procedure described in

Example 1 is then followed.

EXAMPLE 3

  41.0 g (0.25 fnole) of L-lysine monohydrate are mixed with 320 g (approximately 1 mole) of a mixture of polyunsaturated fatty acids (containing 90.0% EPA and 0.1% by weight). % of vitamin E), then the

  The procedure described in Example 1 is followed.

EXAMPLE 4

  The procedure of Example 3 is followed, except that 164 g (1 mol) of L-lysine monohydrate and in the form of polyunsaturated fatty acid, 328 g (1 mol) of docosahexaenoic acid (1HA).

  [manufactured by Sigma, St Louis, USA, under catalog number D-

6508 (1987)] are used.

EXAMPLE

  The procedure of Example 1 is followed except that the

  L-lysine is replaced by 181 g (1.0 mol) 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) L-threonine.

EXAMPLE 7

  The procedure of Example 3 is followed except that 164 g (1 mole) of Lysine monohydrate and 80 g (about 0.25 mole) of a mixture of polyunsaturated fatty acids w-3 (containing about 90.0%

  DFHA and 0.1% vitamin E) are used.

EXAMPLE 8

  The procedure of Example 3 is followed, except that 164 g (1 mole) of Lysine monohydrate and in place 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 (1 987)] 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-ornithine.

EXAMPLE 10

  The procedure of Example 3 is followed, except that L-lysine is replaced by 33 g (0.25 mole) of L-ornithine,

EXAMPLE I 1

  The procedure of Example 4 is followed except that the

  L-lysine is replaced with 132 g (1.0 mole) of L-omrnithine.

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 L-lysine is replaced with 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

  Tablet preparation Tablets are prepared from the composition described in Example 1; they each contain the following components: mg Composition of Example 1 600 Lactose 120 Starch 63 Polyvinylpyrrolidone 3.5 Magnesium Stearate 3.6 Tablets may optionally be coated

  a coating of sugar with a suitable machine.

Claims (10)

  An antiviral and / or immunostimulatory pharmaceutical composition, characterized in that it comprises as active components; amino acids occurring in living organisms and / or their derivatives in which the carboxy group is substituted with a C14 alkyl group or an amino group or with an alkali metal, alkaline earth metal or ammonium cation; or the hydrates and / or salts of these compounds, with one or more C 18-24 fatty acids containing an alsyl group bearing at least two double bonds, optionally mixed with carriers and / or additives and antioxidants commonly used in the pharmaceutical industry, in which the molar ratio of   the 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 oils   of fish as unsaturated fatty acids.   3. Composition according to claim 1, characterized in that it comprises eicosapentaenoic acid (EPA) and the L-lysine as active components.   4. Composition according to claim 1, characterized in that it comprises docosahexaenoic acid (DHA) and the L-lysine as active components.   5. Composition according to claim 1, characterized in that it comprises the docosahexaenoic acid, the acid   eicosapentaenoic and L-lysine as active components.   Process for the preparation of an antiviral and / or immunostimulatory pharmaceutical composition, characterized in that as active components: amino acids occurring in living organisms and / or their derivatives in which the carboxy group is substituted by a C1-C4 alkyl group or an amino group or an alkali metal, alkaline earth metal or ammonium cation; or the hydrates and / or salts of these compounds, with one or more C 18-24 fatty acids containing an alkyl group bearing at least two double bonds, optionally with carriers and / or additives and antioxidants commonly used in the pharmaceutical industry, and put them under the form of a pharmaceutical composition, in which   The molar ratio of amino acid and fatty acid (s) is from 1: 4 to 4: 1.   7. Process 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-lysine are used as the active components.   9. Process according to claim 6, characterized in that docosahexaenoic acid (DHA) and L-lysine are used as active components.   10. Process according to claim 6, characterized in that the docosahexaenoic acid, the acid   eicosapentaenoic acid and L-lysine as active components.