GB2216418A

GB2216418A - Antiviral and immunostimulating compositions comprising amino acids and polyunsaturated higher fatty acids.

Info

Publication number: GB2216418A
Application number: GB8905385A
Authority: GB
Inventors: Nagy Peter Literati; Maria Boros; Jeno Szilbereky; Istvan Racz; Gyoengyver Soos; Miklos Koller; Alan Pinter; Gabor Nemeth
Original assignee: BIOREX KFT
Current assignee: BIOREX KFT
Priority date: 1988-03-09
Filing date: 1989-03-09
Publication date: 1989-10-11
Anticipated expiration: 2009-03-09
Also published as: HUT60432A; JPH01316316A; FI891145A0; CA1334576C; FI94591B; LU87471A1; IT8919704A0; SE8900828L; CH678918A5; IT1229562B; DE3907649A1; SE8900828D0; KR890014103A; GB2216418B; FR2628324B1; BE1002890A3; HU209973B; GB8905385D0; FI94591C; NL8900574A

Description

ANTIVIRAL AND IMMUNOSTIMULATING PHARtvlADEUTICAL COMPOSITIONS AND PROCESS FOR PREPARING SAt2,E This invention relates to antiviral and immunostimulant pharmaceutical compositions.

According to an other aspect of the invention, there is provided a process for the preparation of these compositions.

The advantageous anti viral action of GJ-3 polyunsaturated fatty acids / 5,B,11,14,17-eicosapentsenoic acid (hereinafter: EPA) and 4,7,10,13,16,19-docosahexaenoic acid (hereinafter: DHA)7 called a considerable attention. Szads / Antimicrobial Agents and Chemoterapy 12, 523 (1977)7 showed by in vitro experiments that polyunsaturated fatty acids, e.g. both EPA and DHA, are able to inhibit virus replication. This fact was supported by Reinhardt et al.

[J. of Virology 25, 479 (1977)7 investigating the inhibition of the replication on PR 4 bacteriophage.

The antiviral effect of polyunsaturated fatty acids, among them that of EPA and OHA, is described in detail in the United States patent specification No. 4,513,008. The effect of EPA and DHA was compared in animal experiments on mice and guinea pigs to Acyclovir / 9-(2-hydroxyethoxy- methyl)uanine7, being the most frequently used antiviral agent at present. It has been stated that the compositions particularly containing OHA showed a more preferable action against herpes virus than that of acyclovir.

A number of articles have been devoted in the literature to the antiviral effect of OHA and EPA, such as: Whitaker et ai. L Proc. Natl. Acad. Sci. USA 76, 5919 (1979)7 as well as Goodnight et al. / Arteriosclerosis, 2, 87 (1982)7 and Yoshiaki /-Biochimica et Biophysica Acta 793, 80 (1984)7.

Prickett et al. /~Immunology 46, 819 (1982)7 showed that the humoral immune response was stimulated by eicosapentaenoic acid as an arachidonic acid analogue. Under an EPA-rich diet, the specific IgG and IgE production as a response given to egg albumin was increased in inbred Sprague-Dawley rats to a 4 to 8-fold value in comparison to the control. According to this paper, an enhanced antibody response was induced by the EPA-rich diet.Further on, it was shown by the investigations that EPA acts through an inhibition of the suppressive prostaglandin system whereby it is able to inhibit or correct, respectively, the immune deficiency accompanying aging and other pathological processes (autoimmune processes, tumourogenesis These statements were supported by the articles of Kelley et al. /-J. of Immunol. 134, 1914 (1985)7 as well as Homey et al. L Clin. Exp. Immunol. 65, 473 (1986)7.

It is long known on the basis of in vivo investigations of Pearson et al. /-Proc. Soc. Exp. Biol. Med. 79, 409 (1952)7 that L-lysine has an Effect inhibiting the encephalomyelitis virus. Later on, Tankersley /. Bact. 87, 609 (1964)7 called attention to the fact that the replication of herpes simplex virus (hereinafter: HSV) is inhibited by lysine in human cells under in vitro conditions.

Based on human examinations, Kagan published / The Lancet, 1, 137 (1974)7 that both the oral and genital HSV-induced laesions very rapidly disappeared on effect of a treatment with L-lysine.

Griffith et al. /-Dermatologica, 156, 257 (1978)7 studied the therapeutic action of L-lysine on 45 patients infected by HSV I and HSV II in various doses, with varied treatment periods. The age of the patients (predominantly women) varied between 8 and 60 years. It was stated that L-lysine exerted only a suppressive but not healing effect on HSV.

The aim of the present invention is to provide a novel pharmaceutical composition combining the advantageous therapeutical effects of b/-3 unsaturated fatty acids with those of amino acids and certain essential amino acids, particularly lysine, ornithine and histidine, and showing a stronger action than that of any anti viral agent known till now.

The invention is based on the recognition that the advantageous antiviral effects separately known in se of -3 -3 unsaturated fatty acids, particularly EPA and DHA, as well as of amino acids, respectively, particularly of lysine, are synergistically increased in a mixture formed therefrom and, in addition, the mixture shows an immunostimulating action, too.

Thus, the present invention relates to an anti viral and/or immunostimulating pharmaceutical compositon, which comprises as active ingredients amino acids occurring in living organisms (such as taurine, cycloserine, homcserine, homocysteine, canavanine, sarcosine, hydroxyproline, hydroxylysine, 4-hydroxyphenylalanine, essential amino acids) and/or their derivatives the carboxyl group of which is substituted by a C1 4alkyl group or amino group or an alkali metal, alkali earth metal or ammonium cation, preferably by sodium or potassium cation; or the hydrates and/or salts of these compounds together with one or more C18 24 fatty acid(s) containing an alkyl group bearing at least two double bonds, optionally in admixture with carriers and/or additives and antioxidants commonly used in the pharmaceutical industry, wherein the molar ratio of the amino acid and the fatty acid(s) is from 1:4 to 4:1.

C1B 24 iL-3 fatty acids containing at least two double bonds and amino acids, basic in character, are preferably used as components of the compositions according to the invention. It is suitable to use lysine, histidine or ornithine, or their derivatives as essential amino acids having a basic character.

Substances usually employed in the pharmaceutical industry such as lactose, starch or magnesium stearate may be used as carriers and additives in the compositions according to the invention.

For inhibiting the oxidation of the composition, the use of ol-tocopherol (vitamin E), glutathione or traditional antioxidants such as butylhydroxytoluene is suitable.

Among the compositions according to the invention, the activity and eventual toxicity of mixtures containing an X -3 polyunsaturated fatty acid mixture (containing 27.6 % of EPA and 44.6 % of DHA) and L-lysine in molar ratios of 1:1, 1:4 and 4:1, respectively, (test compositions) were studied on the basis of their effect exerted on the propagation and morphology of Hep 2 cells (human epithelial tumour cell line). These experiments were carried out on a plastic tissue-cultivating sheet containing 6x4 hollow spaces (holes with a ground surface of 1.9 cm2 each).

A stock solution containing the test composition in a concentration of 10 mg/ml was prepared in Eagle MEM medium (manufactured by Serva GmbH Co., Heidelberg, German Federal Republic). An aliquot part of the stock solution was diluted 10-fold, the solution obtained was diluted 2-fold and then, by repeating twice the 2-fold dilution of the latter solution, solutions with successively decreas- ing concentrations of the test composition (solutions Nos.

1 to 54 were prepared. The cells were treated with 1 ml of a solution each containing the active ingredient in the following concentrations: Solution Concentration No. /ug/ml 1 10000 2 1000 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: PBS), whereafter a nutritive medium was added to the cultures.

After 24 hours and fixation by methanol, the cultures were dyed by ethanolic Giemsa solution (manufactured by Reanal, Budapest, Hungary) and the morphology of the cells was evaluated under light microscope. It could be stated that the test compound proved to be toxic only above a concentra- tion of 1000/ug/ml.

The virus replication-inhibiting effect was examined on Hep 2 cells as described above. Type I of HSV was used for infection. The concentration of the virus amounted to about 1000 PFU (plaque forming unit). Solutions containing the test composition in a concentration of 1000, 500 and 250 1ug/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 OC for 1 hour. Then, the cells were treated by the virus pre-incubated with the test composition and the development of cytopathologic (CP) alterations was observed for 7 days. It could be stated that the virus replication was directly inhibited by a 50û /ug/ml or 250 /ug!ml dose of the test composition: the infective titre value of the cytopathologic dose (neg.

log. Cud50) was 1.75 to 2.04 calculated for 0.1 ml, in comparison to the neg. log. GEO50 value of 4.5 of the untreated control virus culture. The value of the virus inhibition exceeds by two orders of magnitude that of the control.

Under the same conditions, EPA and OHA did not show any valuable virus inhibition; and lysine in itself inhibited the virus replication to an extent being only by about one order of magnitude higher than that of the control.

Vaccine virus was also treated in an in vitro experiment carried out in the above manner. The neg. log.

CPO50 value of the test composition measured on vaccine virus proved to be 1.75 to 2.15 in comparison to the 5.5 value of the untreated control virus, i.e. the test composition showed a virus-inhibiting effect which was by three orders of magnitude stronger 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 the lymphocytes was investigated in such a way that a lymphocyte cell population obtained on a Fico Uromiro gradient /Scand. 0. Clin. Lab. Invest.

21, 97 (1966)7 was pipetted into the hollow hcles of flat-bottom sheets, 25 ,ug/ml of Concanavaline A (hereinafter: Con A) (manufactured by Pharmacia, Uppsala, Sweden) and then a solution containing the compositions according to the invention in a concentration of 0.1, 1.0 or 10 /ug/ml, respectively, were added to each of the parallel cultures.

A culture containing 25 /ug/ml of Con A without test composition was used as control. The sheets were maintained under an air atmosphere containing 5% of carbon dioxide at 37 C for 72 hours and 0.4 /uci of 3H-thymidine was added to each sample after 64 hours, before ending the cultivation.

After 72 hours, the cultures were filtered through a glass filter, the filters were put into a scintillation cuvet and the radioactivity was measured in 5 ml of toluene solution each by using a beta-counter device. The results are summarized in the following Table.

No. Compound Concentration Counts/minute Evaluation of name of the active cpm the effect ingredient /u9/9 1 Control 25 8969 + 2984 (Con A) 2 L-tyrosine 0.1 12915 + 2045 weakly significant growth 3 L-lysine 0.1 11063 + 1528 not significant 1.0 11833 + 1823 weakly significant growth 4 Na salt of an G;;-3 0.1 12332 + 2235 not significant fatty acid mixture (see the composition 1.0 12039 + 1640 not significant in Example 1) 5 Control 25 8396 + 2233 (Con A) 6 Product of Example 5 0.1 14605 + 1747 highly significant growth 1.0 12010 + 2285 not significant 7 Product of Example 1 0.1 16085 + 2063 highly significant growth 1.0 14681 + 1769 significant growth 8 Product of Example 3 0.1 12661 + 1548 significant growth 1.0 12420 + 1452 significant growth 9 Product of Example 4 0.1 11920 + 2005 significant growth 1.0 12240 + 1950 significant growth It is obvious from these examinations that the compositions according to the invention, particularly the polyunsaturated fatty acid mixture used together with lysine and tyrosine, gave a significant test result, i.e.

a strong immunostimulating effect, while the separate components of the test materials according to the invention in themselves proved to have no or only a weak biological action.

It is suitable to use as starting material of C18 24 '=y-3 - 3 unsaturated fatty acids, representing one component of the composition, first of all oils which can be obtained from fishes of the northern seas such as salmon, codfish, sardine or from their liver but oils arising from fresh -water fishes may also be utilized. The Cly-3 polyinsaturated fatty acids may be obtained from the above oils by using a known method /-J. Am. Chem. Soc. 59 117 (1982)7. These fatty acids are liable to oxidation. For the inhibition of oxidation, it is suitable to use antioxidants such as e.g.

vitamin E.

The active ingredients mentioned above can be transformed to capsules, tablets, drags or other pharmaceutical compositions formulated in a manner known per se.

The main advantages of the pharmaceutical composition according to to the invention can be summarized as follows: a) The antiviral therapeutic effect of the active ingredients of the composition is synergistically increased.

b) It can be used against acute virus infections, particularly with herpes virus infections, for suppressing the infection in an initial stage.

c) Owing to the immunostimulating effect, it can preferably be used against retroviruses, especially against the immunodeficiency syndromes (e.g. AIDS) induced by HTLV III and IV type viruses.

d) It contains exclusively native active compounds which are essential from a biological viewpoint; thus, it is useful for a prophylactic, long-lasting, cure-like treatment in case of a danger of virus infection as well as in diseases of the immune system.

e) It acts also internally; thus, the inconvient external treatment commonly used in the antiviral therapy can be avoided.

The compositions according to the invention are illustrated in detail by the following non limiting Examples.

Example 1 164 g (1 mole) of L-lysine monohydrate and 320 g (about 1 mole) of an Ot-3 polyunsaturated fatty acid mixture (containing 27.6 % of EPA and 44.6 % of DHA) are admixed at room temperature. The homogeneous mixture thus obtained is filled by using a known encapsulation process into hard gelatine capsules able to receive S00 mg of active ingredient.

Example 2 155 g (1 mole) of L-histidine are mixed with 320 g (about 1 mole) of an OS-3 polyunsaturated fatty acid mixture (containing 27.6 % of EPA and 44.6 % of DHA).

Further on, the process described in Example 1 is followed.

-Example 3 41.0 g (0.25 mole) of L-lysine monohydrate are mixed with 320 g (about 1 mole) of an o3-3- polyunsaturated fatty acid mixture (containing about 90.0 % of EPA and 0.1 % of vitamin E), then the process described in Example 1 is followed.

Example 4 The process described in Example 3 is followed, except that 164 g (1 mole) of L-lysine monohydrate and, as a polyunsaturated fatty acid, 328 g (1 mole) of docosahexaenoic acid (DHA) / manufactured by Sigma, St. Louis, USA under the catalogue number D-6508 (1987)7 are used.

Example 5 The process described in Example 1 is followed, except that 181 g (1.0 mole) of L-tyrosine are used, instead of L-lysine.

Example 6 The process described in Example 1 is followed, except that 120 g (1.G mole) of L-threonine are used, instead of L-lysine.

Example 7 The process described in Example 3 is followed, except that 164 g (1 mole) of L-lysine monohydrate and 80 g (about 0.25 mole) of an & -3 polyunsaturated fatty acid mixture (containing about 90.0 % of OHA and 0.1 % of vitamin E) are used.

Example 8 The process described in Example 3 is followed, except that 164 g (1 mole) of L-lysine monohydrate and, instead of a polyunsaturated fatty acid mixture, 302 g (1 mole) of eicosapentaenoic acid (EPA) / manufactured by Sigma, St. Louis, USA under the catalogue number E-706 (1987)7 are used.

Example 9 The process described in Example 1 is followed, except that 132 g (1.0 mole) of L-ornithine are used, instead of L-lysine.

Example 10 The process described in Example 3 is followed, except that 33 g (0.25 mole) of L-ornithine are used, instead of L-lysine.

Example 11 The process described in Example 4 is followed, except that 132 g (1.0 mole) of L-ornithine are used, instead of L-lysine.

Example 12 The process described in Example 1 is followed, except that 133 g (1.0 mole) of L-aspartic acid are used, instead of L-lysine.

Example 13 The process described in Example 1 is followed, except that 211 g (1.0 mole) of L-arginine hydrochloride are used, instead of L-lysine.

Example 14 The process described in Example 1 is followed, except that 105 g (1.0 mole) of L-serine are used, instead of L-lysine.

Example 15 The process described in Example 1 is followed, except that 145 g (1.0 mole) of L-glutamine are used, instead of L-lysine.

Example 16 Preparation of tablets From the composition as described in Example 1 tablets are prepared each of which contains the following components: mg Composition according to Example 1 500 Lactose 120 Starch 63 Polyvinylpyrrolidone 3.5 Magnesium stearate 3.5 If desired, the tablets are covered with a sugar coat by using a panning machine.

Claims

1. An antiviral and immunostimulating pharmaceutical composition, which c o m p r i S e s as active ingredients: amino acids occurring in living organisms and/or their derivatives containing the carboxyl group as substituted by a C1 4alkyl group or amino group or by an alkali metal, alkali earth metal or ammonium cation; or the hydrates and/or salts of these compounds together with one or more C1824 fatty acid(s) containing an alkyl group bearing at least two double bonds, optionally in admixture with carriers and/or additives and antioxidants commonly used in the pharmaceutical industry, wherein the molar ratio of the amino acid and the fatty acid(s) is from 1:4 to 4:1.

2. A composition as claimed in claim 1, which c o m p r i s e s fatty acids obtained from the oils of fishes as unsaturated fatty acids.

3. A composition as claimed in claim 1, which c o m p r i s e s eicosapentaenoic acid (EPA) and L-lysine as active ingredients.

4. A composition as claimed in claim 1, which c o m p r i s e s docosahexaenoic acid (DHA) and L-lysine as active ingredients.

5. A composition as claimed in claim 1, which c o mp r i s e s docosahexaenoic acid, eicosapentaenoic acid and L-lysine as active ingredients.

6. A process for the preparation of an antiviral and immunostimulating pharmaceutical composition, which c o m p r i s e s mixing as active ingredients: amino acids occurring in living organisms and/or their derivatives containing the carboxyl group as substituted by a C1 4alkyl group or amino group or by an alkali metal, alkali earth metal or ammonium cation; or the hydrates and/or salts of these compounds together with one or more C18-24 fatty acid(s) containing an alkyl group bearing at least two double bonds, optionally with carriers and/or additives and antioxidants commonly used in the Fharcaceutical industry, and transforming them to a pharmaceutical composition, wherein the molar ratio of the amino acid and the fatty acid(s) is from 1:4 to 4:1.

7. A process as claimed in claim 6, which c o m p r i s e s using fatty acids obtained from the oils of fishes as unsaturated fatty acids.

8. A process as claimed in claim 6, which c o m p r i s e s using eicosapentaenoic acid (EPA) and Llysine as active ingredients.

9. A process as claimed in claim 6, which c o m p ri e s using docosahexaenoic acid (DHA) and L-lysine as active ingredients.

10. A process as claimed in claim 6, which c o m p r I s e s using docosahexaenoic acid, eicosapentaenoic acid and L-lysine as active ingredients.

11. A co,.osition substantially as hereinbefore described in any one of Examples 1 to 16.

12. A process as claimed in claim 6 substantially as hereinbefore described in any one of Examples 1 to 16.

13. A composition as claimed in any one of claims 1 to 5 or 11 for use in therapy.

14. Use of a mixture of amino acids occurring in living organisms and/or their derivatives containing the carboxyl group as substituted by a C1 4aikyl group or amino group or by an alkali metal, alkali earth metal or ammonium cation (or the hydrates and/or salts of these compounds) together with one or more C18 24 fatty acid(s) containing an alkyl group bearing at least two double bonds in the preparation of a medicament for curing viral disease and/or for stimulating the immune system of a patient.