USE OF ADENOSINE DERIVATIVES IN PREPARING PHARMACEUTICAL COMPOSITIONS POSSESSING IMMUNOSTIMULANT ACTIVITY

Abstract: The use of adenosine derivatives in preparing pharmaceutical compositions possessing immunostimulant activity, comprising a pharmacologically acceptable carrier and an effective quantity of at least one of said adenosine derivatives of general formula (I): in which: R = C₁-C₁₈ linear or branched alkyl radical, or phenylalkylene radical in which the alkylene chain has 1-6 C atoms R₁ = H, C₁-C₆ aliphatic acyl radical, or C₇ aromatic acyl radical; R₂ = H, C₁-C₆ aliphatic acyl radical, or C₇ aromatic acyl radical, or the two R₂ radicals together form an isopropylidene chain.

Description: This invention relates to the use of adenosine derivatives for the preparation of pharmaceutical composition for the immunotheraphy of congenital and acquired deficiency and of tumours. The therapeutic uses of the product class containing the products of the present invention as antivirals, cytostatics and oncogenic tissue transformation inhibitors are already known and widely described in the literature, for example in GB patent No. 1,555,991 in which the antitumoral activity of said compound is disclosed, but it is only directed to the prevention and treatment of a limited number of tumours that is viro-induced tumours. The therapeutic uses of this product class as antiinflammatories, antipyretics, platelet antiaggregation agents and sleep inducers, claimed by the present Applicant in US patents 4,454,122 and 4,373,097, are also known. J. Supramolecular Structure and cellular Biochemistry vol. 17 pages 11-17, 1981 discloses a generic study and hypothesis on the possible role of agents such as sulphydril compounds, disulfides, macrophages and methylthioadenosine in the proliferation of both normal and malignant cells. The information relative to methylthioadenosine which is the only compound named in this reference and related to the adenosine derivatives are only generic information about its hypothetic role as methylthiogroup donor in certain type cells. In this reference there is no indication of any therapeutical use. Reference should be made to the aforesaid patents for preparation details of the product of the present invention. The immunostimulants form a heterogeneous category of medicaments used for potentiating inadequate or depressed immune responses. Of various nature and origin, they all have the capacity to activate both specific and natural immunity cell functions, either directly or via activation of endogenous mediators. In the great majority of cases they recognise more than one point of connection to the immune system, and are therefore able to stimulate various functions. For many of them the real action mechanism is substantially unknowns, and this poses a limit to better understanding of their pharmacological potential and the interpretation of frequent side-effects which are often of considerable importance. Immunostimulants are classified according to their origin, namely endogenous such as thymic hormones, monokines, lymphokines, interferons, or natural exogenous (generally obtained from microorganisms) such as BCG, Corynebacterium Parvum, Muramyl dipeptide, Bestatin and Lentinan, or also synthetis exogenous such as Levamisole, Isoprenosine and Tuftsin. As the constantly increasing consumption figures prove, the interest in these medicaments is considerable but up to the present time more on a hypothetical use level rather than on the level of consistent results and real use. The main fields of application of immunostimulants are the immunotherapy of congenital and acquired immunodeficiencies and tumour immunotherapy. In the immunodeficiency field there is a current widespread use of thymic hormones, which restore normal resistance to infecting agents but raise considerable problems of selectivity, bioavailability and toxic effects which have not yet been overcome. With regard to tumour therapy, since the immunogenicity of certain experimental tumours and the ability to induce their remission by acting on the immune functions of the host were demonstrated several years ago, numerous attempts have been directed towards establishing an immunotherapy for human tumours. These results have been discouraging, but in reality these were premature attempts the failure of which was caused by empiricism, poor knowledge of tumour biology and the interactions between the tumour and host, and the indiscriminate and improper use of immunostimulants. Very intensive basic research is currently in progress directed towards an understanding of how tumours are generated and develop, the mechanisms by which they evade immunitary supervision, the reasons for their poor immunogenicity, and the importance of specific and natural immunity in protection against tumours. This is a subject dense with unknowns, but undoubted successes obtained in some patients by immunotherapy formulas which included the use of LAK (lymphokine-activated killer cells), interleukin-2 and interferon have shown that immunotherapy can be effective (Rosemberg S.A. et al., A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukine-2 or high-dose interleukine-2 alone, N. Engl. J. Med. 316, 889, 1987; Goldstein D., Laszlo J., Interferon therapy in cancer: from imaginon to interferon, Cancer Res. 46, 4315, 1986). In the present state of things it can be said that if immunotherapy is used rationally and is subjected to critical methodological selection, it opens extremely important perspectives, with considerable expectancy of the introduction of immunostimulants of known action mechanism, tested efficiency and low toxicity. This invention relates to the use of adenosine derivatives as active principle in preparing pharmaceutical compositions possessing immunostimulant activity, for the immunotherapy of congenital and acquired immuno deficiencies and of tumours comprising a pharmacologically acceptable carrier and an effective quantity of at least one of said adenosine derivatives of general formula: in which: R = C₁-C₁₈ linear or branched alkyl radical, or phenylalkylene radical in which the alkylene chain has 1-6 C atoms R₁ = H, C₁-C₆ aliphatic acyl radical, or C₇ aromatic acyl radical; R₂ = H, C₁-C₆ aliphatic acyl radical, or C₇ aromatic acyl radical, or the two R₂ radicals together form an isopropylidene chain. Moreover, when R₁ = H the invention also relates to pharmacologically acceptable acid addition salts of the compounds of formula (I). Preferred meanings of R are: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, pentyl, hexyl, heptyl, octyl, decyl, hexadecyl, octadecyl, benzyl. Preferred meanings of R₁ are: hydrogen, acetyl, propionyl, butyryl, succinyl, glutaryl, benzoyl, tosyl. Preferred meanings of R₂ are: hydrogen, acetyl, propionyl, butyryl, succinyl, glutaryl, benzoyl, tosyl. The preferred acid addition salts of the compounds of formula (I) are: chloride, sulphate, hydrogensulphate, phosphate, formate, acetate, citrate, tartrate, lactate, methanesulphonate, p-toluenesulphonate. According to the present invention, we have now discovered that the adenosine derivatives of formula (I) possess considerable immunostimulant activity in addition to their already known pharmacological activities. The immunostimulant activity was initially demonstrated for certain of the terms of general formula (I) by using four experimental tests, the results of which are given in Tables A, B, C and D. The following experimental tests were used: 1. Primary antibody response against thymus-dependent antigen, evaluated as the number of plaque-forming splenic cells (PFC) Mice B6 D2 F1, antigen SRBC (red sheep corpuscles) Jerne N.K and Nordin A.A., Plaque formation in ager by single antibody-producing cells Science, 140, 405, 1963. Table A 2. Delayed hypersensitivity against cell antigen (DTH) Mice B6 D2 F1, antigen SRBC (red sheep corpuscles) Lagrange P.H., Mackaness G.B and Miller T.E., potentiation of T-cell-mediated immunity by selective suppression of antibody formation with cyclophosphamide. J. Exp. Med., 139, 1529, 1974. Table B 3. Macrophage cytotoxic activity against target tumour induced by Corynebacterium Parvum Mice C57 BL/6; tumour L1210 pretagged with ⁵¹Cr; attack:target ratio 80:1; tumouricide activity evaluated as release of tagging compound and expressed as % of specific cytotoxicity. Herscowitr H.B. et al., Eds., Manual of macrophage methodology, M. Dekker 1981. Table C 4. Splenic NK (natural cytotoxic cell) activity potentiated with polynucleotide (Poly I:C) Mice C57 BL/6; tumour YAC-1; evaluation as in test 3. Herbeman R.B. Ed., "Natural cell-mediated immunity against tumors". Academic Press, 1980. Table D All the examined products are able to potentiate the primary antibody response to thymus-dependent antigen, as shown by the percentage increases in the splenic PFC number given in Table A. The most effective compound of the series appears to be MTA which at doses of 20 and 40 mg/kg doubles the number of plaque forming cells. The treatment was carried out during the inductive stage of the response. The effect of a known immunostimulant, Tuftsin, is shown at the foot of Table A. Similar potentiation is observed in a cell immunity model for which daily treatment during the sensitization period results in amplified DTH response, as shown by Table B. In this case MTA is again seen to be the most active compound of the series under examination. with maximum efficiency for a dose of 10 mg/kg. The effect of another known immunostimulant, Muramyl dipeptide (MDP), is shown at the foot of the table. For certain of the products under examination immunostimulant capacity was also evaluated on a model of macrophage cytotoxic activity against target tumour induced by Corynebacterium Parvum. Table C shows that all the tested compounds are able to potentiate the inducing effect of Corynebacterium Parvum. By associating adenosine derivatives of general formula (I) with a sub-optimum dose of Corynebacterium, the obtained microphage activation is equal to or very often greater than that obtained by an optimum dose of Corynebacterium. Compounds administered in the absence of activator are unable to stimulate the microphage cytotoxic function. Of all the prepared products, that which proved of particular interest from the point of view of the present invention was 5′-deoxy-5′-methylthioadenosine (MTA) of formula (II): which is a physiological compound already present in living organisms and proved to be the most active in every test carried out. A further evaluation of the stimulant capacity of MTA was made on an NK activity model. Table D shows that when administered alone to the animal, the product does not modify the spontaneous NK activity. However, when associated with Poly I:C it potentiates its effect by reducing the polynucleotide dose required to obtain maximum activation. In general, the tested products influence specific immunity by amplifying both humoral and cell-mediated immune responses. They do not appear to directly influence natural immunity, but when associated with conventional macrophage and NK function inducers they considerably potentiate their effect, so greatly reducing the activator dose required for optimum response. The compounds of the present invention are practically free of acute toxicity when administered orally. They are also practically free of toxicity at therapeutic doses for any method of administration. The following values apply to MTA: The adenosine derivatives of formula (I) can be administered orally, parenterally, intravenously or rectally in any therapeutically useful form, either alone or diluted with suitable pharmacologically acceptable excipients. They can also be used in products for external topical application, and in other forms normally used in the pharmaceutical field. The active principle content of the pharmaceutical compositions of the present invention corresponds to a therapeutic administration of between 5 and 20 mg/kg of body weight. Some examples of typical pharmaceutical compositions comprising MTA are given hereinafter by way of illustration:

Claims: Claims for the following Contracting States : AT, BE, CH, DE, FR, GB, IT, LI, LU, NL, SE The use of adenosine derivatives of general formula (I): in which: R = C₁-C₁₈ linear or branched alkyl radical, or phenylalkylene radical in which the alkylene chain has 1-6 C atoms; R₁ = H, C₁-C₆ aliphatic acyl radical, or C₇ aromatic acyl radical; R₂ = H, C₁-C₆ aliphatic acyl radical, or C₇ aromatic acyl radical, or the two R₂ radicals together form an isopropylidene chain; as active principle for the preparation of pharmaceutical compositions for the immunotherapy of congenital and acquired immunodeficiencies and of tumours. The use of adenosine derivatives as claimed in claim 1, characterised in that R is a group chosen from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, pentyl, hexyl, heptyl, octyl, decyl, hexadecyl, octadecyl, benzyl; R₁ is a group chosen from hydrogen, acetyl, propionyl, butyryl, succinyl, glutaryl, benzoyl, tosyl; and R₂ is a group chosen from hydrogen, acetyl, propionyl, butyryl, succinyl, glutaryl, benzoyl, tosyl. The use of adenosine derivatives as claimed in claim 1, characterised in that R₁=R₂=H, R=CH₃. The use of adenosine derivatives as claimed in claim 1, characterised by including the use of a pharmacologically acceptable acid addition salt when R₁ = H. The use of adenosine derivatives as claimed in claim 4, characterised in that the acid addition salt is chosen from chloride, sulphate, hydrogensulphate, phosphate, formate, acetate, citrate, tartrate, lactate, methanesulphonate, p-toluensulphonate. The use of adenosine derivatives as claimed in claim 1, characterised by the fact that said compositions are suitable for administration by oral, parenteral, intravenous, rectal or external topical route. The use of adenosine derivatives as claimed in claim 1, characterised by the fact that said compositions are prepared in the form of capsules containing from 50 to 100 mg of derivative of formula (I) where R₁=R₂=H, R=CH₃. The use of adenosine derivatives as claimed in claim 1, characterised by the fact that said compositions are prepared in the form of tablets containing from 50 to 100 mg of derivative of formula (I) where R₁=R₂=H, R=CH₃. The use of adenosine derivatives as claimed in claim 1, characterised by the fact that said compositions are prepared in the form of suppositories containing from 50 to 100 mg of derivative of formula (I) where R₁=R₂=H, R=CH₃. The use of adenosine derivatives as claimed in claim 1, characterised by the fact that said compositions are prepared in the form of injectable vials containing from 25 to 50 mg of derivative of formula (I) where R₁=R₂=H, R=CH₃. The use of adenosine derivatives as claimed in claim 1, characterised by the fact that said compositions are prepared in the form of vials for oral use containing from 50 to 100 mg of derivative of formula (I) where R₁=R₂=H, R=CH₃. Claims for the following Contracting States : GR, ES A method for preparing pharmaceutical compositions for the immunotherapy of congenital and acquired immunodeficiencies and of tumors containing as active principle an adenosine derivative of general formula (I): in which: R=C₁-C₁₈ linear or branched alkyl radical, or phenylalkylene radical in which the alkylene chain has 1-6 C atoms; R₁ = H, C₁-C₆ aliphatic acyl radical, or C₇ aromatic acyl radical; R₂ = H, C₁-C₆ aliphatic acyl radical, r C₇ aromatic acyl radical, or the two R₂ radicals together form an isopropylidene chain, in combination with suitable pharmaceutically acceptable excipients, comprising the admixture of said adenosine derivative with said excipients. A method as claimed in claim 1, characterised in that R is a group chosen from metyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, pentyl, hexyl, heptyl, octyl, decyl, hexadecyl, octadecyl, benzyl; R₁ is a group chosen from hydrogen, acetyl, propionyl, butyryl, succinyl, glutaryl, benzoyl, tosyl; and R₂ is a group chosen from hydrogen, acetyl, propionyl, butyryl, succinyl, glutaryl, benzoyl, tosyl. A method as claimed in claim 1, characterised in that R₁=R₂=H, R=CH₃. A method as claimed in claim 1, characterised by including the use of a pharmacologically acceptable acid addition salt when R₁= H. A method as claimed in claim 4, characterised in that the acid addition salt is chosen from chloride, sulphate, hydrogensulphate, phosphate, formate, acetate, citrate, tartrate, lactate, methanesulphonate, p-toluensulphonate. A method as claimed in claim 1, characterised by the fact that said compositions are suitable for administration by oral, parenteral, intravenous, rectal or external topical route. A method as claimed in claim 1, charactetrised by the fact that said compositions are prepared in the form of capsules containing from 50 to 100 mg of derivative of formula (I) where R₁=R₂=H, R=CH₃. A method as claimed in claim 1, characterised by the fact that said compositions are prepared in the form of tablets containing from 50 to 10 mg of derivative of formula (I) where R₁R₂=H, R=CH₃. A method as claimed in claim 1, characterised by the fact that said compositions are prepared in the form of suppositories containing from 50 to 100 mg of derivative of formula (I) where R₁=R₂=H, R=CH₃. A method as claimed in claim 1, characterised by the fact that said compositions are prepared in the form of injectable vials containing from 25 to 50 mg of derivatives of formula (I) where R₁=R₂=H, R=CH₃. A method as claimed in claim 1, characterised by the fact that said compositions are prepared in the form of vials for oral use containing from 50 to 100 mg of derivative of formula (I) where R₁=R₂=H, R=CH₃.

Inventors: BASSI, LUIGI, GENNARI, FEDERICO
Applicants: BIORESEARCH S.P.A.
Application year: 1989
Main classification: A61K31
IPC codes: A61P37, C07H19, A61K31