THERAPEUTIC USE OF PHOSPHORYL-L-SERINE-N-ACYL-SPHINGOSINE

Abstract: The present invention relates to the therapeutical use of phosphoryl-L-serine-N-acyl-sphingosine composed of a mixture of compounds of formula (I) or (II), in which n = 6-16 and R means an acyl residue of a higher aliphatic acid having between 16 and 24 carbon atoms, the distribution of the values for n and R being those of a natural sphingomyelin. The invention also includes a novel process for the preparation of these compounds, as well as pharmaceutical preparations containing them as active ingredients.

Description: THERAPEUTIC USE OF PHOSPHORYL-L-SERINE-N-ACYL-SPHINGOSINE BACKGROUND AND SUMMARY OF THE INVENTION The present invention concerns the therapeutical use of phosphoryl-L-serine-N-acyl-sphingosine composed of a mixture of comoounds of formula I or II, SUBSTITUTESHEET in which n is an integer of 6-16 and R means an acyl residue of a higher aliphatic acid having between 16 and 24 carbon atoms, the distribution of the values for n and R being hose of a natural sphingomyelin. The invention also includes a new process for the preparation of these compounds, as well as pharmaceutical preparations containing said compounds as active ingredients. The ratio of the mixture of compounds of formulas (I) and (II) is not critical in order to use the compound of the present invention. The compound, as previously defined, was prepared by treatment with phospholipase D, as described in Biochemistry vol. 28, No. 8, 1989, 34-57, starting from a derivative of sphingomyelin (i.e. phosphorylcholine-N- acyl-sphingosine, in which the acyl group was derived from higher aliphatic acids of different molecular size and was variable between C12 and C24) , and particularly to an analog of a sphingomyelin having an acyl group derived from 4-doxyl-pentanoic acid on the sphingosine amino group instead of the acyl residue R. In the cited paper there are no indications about pharmacological properties of the derivative. DETAILED DESCRIPTION OF THE INVENTION It has now been found that the compound has various interesting pharmacological properties and can be used therapeutically, especially in human medicine. More precisely the phosphoryl-L-serine-N-acyl-sphingosine of SUBSTITUTESHEET the present invention has an inhibiting action on the activation of protein kinase C, which activation can be undesired or have negative effect, ir- particular conditions of unbalance of the normal mechanisms of neurotransmittal functions. Activation is caused by an increased concentration of excititatory amino acids, like giutamic and/or aspartic acid; these acids have a direct toxic action on neuronal cells in the above mentioned abnormal conditions. Phosphoryl -L-serine-N-acyl -sphingosine can therefore be used in the therapy of many diseases of the central nervous system, as those arising after cerebral degenerations or lesions, like ischaemia, hypoxia, epilepsy, traumas and compressions, metabolic dysfunctions, aging, toxic-infective and chronic degenerative diseases, like Alzheimer's, Parkinson's and Huntington's diseases. * Phosphoryl-L-serine-N-acyl-sphingosine is moreover able to increase glycemia and histamine levels, resulting in cerebral glucose accumulation, which has important reflexes in the functionality of CNS. Also an activating influence on cerebral neurotransmitter systems was shown; particularly at cholinergic level, the compounds of the present invention can increase the release of acetylcholine (ACh) in the cerebral cortex area. The action on the cholinergic system is also reinforced by the efficacy of the aforesaid compounds in influencing cognitive functions, like e.g. learning and SUBSTITUTESHEET memorizing, in various behavioral tests (e.g. the capacity t invert scopolamine* caused amnesia) . The compounds I and II can also be used in therapies associated with slowdown of cerebral metabolism, in invoiutive cerebral syndromes* of different origins (due e.g. to senile decay and/or vascular pathologies), in Alzheimer's disease, memory loss and senile dementia, anoxic and oedematous conditions, cerebral tiredness, chronic vascular cerebropathies of aging, and pre-seniie dementia following trauma, postanoxic cerebropathies and extrapyramidal syndromes. Moreover the compounds of the present invention perform a modulatory action on the immune system and, particularly, an inhibitory action on the secretion of TNF (tumor necrosis factor) . They can therefore be favorably used in the therapy of pathclogies characterized by a modified immunological reactivity and/or by autoimmunological symptoms like multiple sclerosis, cachectic situations associated with infections and neoplastic diseases, pulmonary fibrosis, rheumatoid arthritis, bacterial and protozoic infections, HIV (human immunodeficiency virus) , cerebral complication of malaria, insulin dependent diabetes and several pathologies linked to organ transplantation or allergy caused pathologies. Another object of the invention is directed to the use of phosphoryl-L-serine-N-acyl-sphingosine of the formula I or II as defined above, in the preparation of pharmaceutical compositions to be employed in the SUBSTITUTESHEET treatment o seases or con t ons as exp a ne a ove. Another object cf the present invention relates to pharmaceutical preparations containing phosphoryl-L- serine-N-acyi-sphingosine of formula I as active ingredient, optionally ■ together with usual pharmaceutical excipients to be administered to a patient in need thereof. These pharmaceutical preparations can be for oral, rectal, parenteral, local or transdermic use. The pharmaceutical preparations can be presented in solid or semisolid form, e.g. as pills, tablets, gelatine covered capsules, or soft gelatine capsules. For the parenteral use it is possible to use formulations for intramuscular, subcutaneous or transdermic use, or formulations suitable for infusions or intravenous injections, and they can therefore be prepared as solutions of active components or as a lyophilized powder of the active components, eventually to be added to one or more excipients or pharmaceutically acceptable solvents, which are usable for the aforesaid purpose and osmolar with physiological fluids. For the local application spray preparations, e.g. nasal sprays, can be considered, or ointments for topical use, or plasters for transdermal administration. The preparations of the invention can be used both in humans and animals. They contain preferably between 0.01 % and 10 % of the active components for solutions, sprays, ointments and creams, and between 1 % and 100 %, preferably between 5 % and 50 % of the active component, SUBSTITUTESHEET for the solid form preparation, the remaining being made up cf excipients . The dosage will be determined according to the indication, the desired effect and the route of administration. The daily dosage for the i jectable (subcutaneous * or intramuscular) or transdermic or oral administration in humans of phosphoryl-L-serine-N-acyl-sphingosine of formula I varies between 0.5 and 12 mg/kg body weight of active substance. Phosphoryl-L-serine-N-acyl-sphingosine , as described by formulae I or II, is a derivative of the natural compound sphingomyelin and, concerning the N-acyl-sphingosine component, it is identical with the composition of the natural product, i.e. it is a mixture of different acylamino-alkenyl residues corresponding to the above mentioned formula, in which both n and R vary within the defined ranges, which are those of sphingomyelin. According to the starting material from which sphingomyelin is extracted, R can be a mixture of acyl residues belonging to palmitic, stearic, nervonic, lignoceric and, in minor amounts, to other acids of different saturated and unsaturated molecular species of the same order. The present invention also concerns the corresponding compounds of formulae I and II in unitary composition, i.e. in which both n and R have a defined value within in the defined range. These compounds are novel. The compounds I and II can be prepared according SUBSTITUTESHEET to already known methods starting from ai.cor.oi formula III or of formula IV where n = 6 - 16, which is reacted with phosphoric acid or a reactive derivative thereof, L-serine and a fatty acid R-COOH, where R has between 16 and 24 carbon atoms, in random sequence. The process involves acylating the a ino-alcohol on the nitrogen atom with the mentioned acid and then reacting the obtained derivative with phosphoric acid and L-serine, or the steps can be carried out in different sequences. These new derivatives have the same pharmacological activities as reported for the product of formula I or II and can be used in therapy for the same indications. The following scheme shows a preferred sequence of the aforesaid preparation, which can also be used for the preparation of comoounds of formula I or II SUBSTITUTE SHEET where Ph designates a phenyl group and TPS designates trϋsopropyl-benzenesulfonyl chloride. SUBSTITUTESHEET Phosphoryl-L-serine-N-acyl-sphingcεine of formula I or II is generally present in the betaine form, i.e. according to formula of the serine component, and that is also true for the aforesaid chemically unitary compound. If desired, also these betaines can be transformed into acid addition salts or basic salts, i.e. into alkaline, alkaline-earth or magnesium or heavy metal salts, or into salts of hydrohalogenic acids, as e.g. chlorides, bromides or iodides, in sulfates, sulfonates, or in organic acid salts. Also these salts, if therapeutically compatible, can be used for therapeutic applications. Therapeutically non-acceptable salts are also part of the invention, and can optionally be used for the purification of the compounds. Compounds of formula I or II can be prepared by an already known procedure, e.g. according the aforesaid method and described in Biochemistry, vol. 28, No. 8, 1989, 3457. It has now been found that the exchange between choline and serine can be done also directly utilizing phospholipase D, reacting sphingomyelin and serine, without the use of the above mentioned intermediate, 10 SUBSTITUTESHEET i.e. the N-acyl derivative of 4-doxyi-pentanoic acid of iyso-spnmgomyelin. This process is, tnerefore, still anotner 00 ect of tne present invention, ana the resulting great tecnnicai advantage is clear. This procedure ■ • s illustrated in Ex.l. The starting materials for the preparation of phosphoryl-L-serine-N-acyi-sphmgosine and the other analogous compounds are known or can fie obtained according methods described in literature. The invention is illustrated in the following non-limiting Examples: Example 1 : 5 gr of chromatcgrapnically pure sphingomyelin were dissolved in a mixture of 80 ml of ethyl acetate and 80 1 t- ml of acetate buffer pH 5.6, 0.2 M, CaCl 2 0.04 M saturated with serine. 320 U of phospholipase D from Streptomyces sp. were added and the reaction was conducted for 24 hours at 45 C. At the end 80 ml of chloroform/methanol 2:1 were added in order to extract 20 the compound. After partitioning with 50 ml of HC1 0.5N, the organic layer was washed with 50 ml of water and then precipitated in 500 ml of ethanol containing 10 ml of sodium acetate 4.5M. The final purification was performed through chromatography on silica gel using a f 25 mixture of chloroform/methanol/water 60:30:3 as eluent. The pure fractions were gathered, concentrated and finally precipitated in 250 ml of acetone. Yield: 2.7 11 SUBSTITUTESHEET gr. After thin layer chromatography on silica gel, the compound is ninhydrir.ε and phosphorus positive having Rf = 0.27 (sphingomyelin 0.20) and 0.11 (sphingomyelin 0.13) respectively, utilizing as eluent chioroform/methanoi/water 6*0:30:4 and chloroform/ethyl acetate/n-propanol/methanol/ NH3 0. IN 25 :25 :25 :22 : 13 > Example : Pharmaceutical preparations in solution for injection Preparation No. 1 A vial of 2 ml contains : phosphoryl-L-serine-N-acyl-sphingosine mg 5 sodium chloride mg 16 citrate buffer pH 6 in distilled water, ad 2 ml Preparation No. 2 A vial of 2 ml contains : phosphoryl-L-serine-N-acyl-sphingoεine mg 50 sodium chloride mg 16 citrate buffer pH 6 in distilled water, ad 2 ml Preparation No. 3 A flacon of 4 ml contains: phosphoryl.-L-serine-N-acyl-sphingosine mg 100 sodium chloride mg 32 citrate buffer pH 6 in distilled water, ad 4 ml 12 SUBSTITUTESHEET The invent on e ng escribed in t s way, t s clear that these methods can be modified in several ways. These modifications cannot be considered different from the concept of the invention and all modifications, which can be ' considered as evident for an expert in the field, are included in the following claims. 13 SUBSTITUTESHEET

Claims: CLAIMS 1. Use of phosphoryl-L-serine-N-acyl-sphingosine formed' by a mixture of compounds of formula I or II R in which n is 6-16 and R means an acyl residue of a higher aliphatic acid having between 16 and 24 carbon 14 SUBSTITUTESHEET atoms, the distribution of the values for n and R being those of a natural sphingomyelin, or salts thereof, in the preparation of pharmaceutical compositions. 2. use of phosphoryl-L-serine-N-acyl-sphingosine according to claim 1 wherein the pharmaceutical compositions are for the therapy of diseases or disorders of the nervous system. 3. Use of phosphoryl-L-serine-N-acyl-sphingosine according to claim 1 wherein the pharmaceutical compositions are for the therapy of pathologies characterized by a modified immunological reactivity and/or symptoms of autoimmunity. . Use according to claim 2 wherein the pharmaceutical compositions are for the treatment of the nervous system as outcomes of neuronal lesions or degenerations. 5. Use according to claim 2 wherein the pharmaceutical compositions are for the treatment of one of the following affections or diseases: ischemia, hypoxia, epilepsy, traumas or compressions, metabolic dysfunctions, aging, toxic-infective and chronic neurodegenerative diseases. 6. Use according to claim 5 wherein the 15 SUBSTITUTESHEET pharmaceutical compositions are for the treatment of Alzheimer's, Parkinson's and Huntmgton's diseases. 7. Use according to claim 2 wherein the pharmaceutical compositions ' are for the treatment of psychomotric involutive senile syndromes, chronic vascular cerebropathies of aging, senile dementia, and pre-senile dementia following trauma, postanoxic cerebropathies and extrapyramidal syndromes . 8. Use according to claim 3 wherein the pharmaceutical compositions are for the treatment cf one of the following disorders or diseases: multiple sclerosis, cachectic situations associated with infections and neoplastic diseases, pulmonary fibrosis, rheumatoid arthritis, bacterial, protozoic and HIV (human immunodeficiency virus) infections, cerebral complications of malaria, insulin dependent diabetes. 9. Use according to any of claims 1-8 in which acid addition salts or salts of metal bases or organic bases of phosphoryl-L-serine-N-acyl-sphingosine are used. 10. A process for the preparation of phosphoryl-L- serine-N-acyl-sphingosine formed by a mixture of compounds of formula I or II 16 SUBSTITU EE in which n is 6-16 and R means an acyl residue of a higher aliphatic acid having between 16 and 24 carbon atoms, the distribution of the values for n and R being those of a natural sphingomyelin, or salts thereof, n which sphingomyelin is reacted with phospholipase D. 11. A process for the preparation of phosphoryl-L- serine-N-acyl-sphingosine formed by a mixture of compounds of formula I or II 17 SUBSTIT in which n is 6-16 and R means an acyl residue of a higher aliphatic acid having between 16 and 24 carbon atoms, the distribution of the values for n and R being those of a natural sphingomyelin, or salts thereof, in which N-4-doxyl-pentanoyl-lyso sphingomyelin is reacted with phospholipase D. 12. Compounds of formula I or II R I 18 SUBSTITUTE SHEET wherein n = 6 - 16 and R means an acyl residue of a higher aliphatic acid having between 16 and 24 carbon atoms, in chemically unitary form. 13. A process for the preparation of compounds defined in claim 12 or a mixture of these compounds, in which an aminoalcohol of formula III or IV in which n = 6 - 16, is reacted with phosphoric acid or a reactive derivative thereof, L-serine and a fatty acid R-COOH, in random sequence. 14. A pharmaceutical preparation containing a compound of formula I or II in which n is 6-16 and R means an acyl residue of a higher aliphatic acid having 19 SUBSTITUTESHEET between 16 and 24 carbon atoms, the distribution of the values for n and R being those of a natural sphingomyelin,or a salt thereof as active ingredient together with a pharmacologically acceptable excip ent. 15. A pharmaceutical composition containing a compound according to claim 12 as active ingredient together with a pharmacologically acceptable excipient. 16. A method for treating disorders of the nervous system or pathologies characterized by a modified immunological reactivity and/or symptoms of autoimmunity whereinphosphoryl-L-serine-N-acyl-sphingosine formed by a mixture of compounds of formula I or II 20 SUBSTITUTESHEET in which n is 6-16 and R means an Ξicyl residue of a higher aliphatic acid having between 16 and 24 carbon atoms, the distribution of the values for n and R being those of a natural sphingomyelin, or salts thereof is administered to a patient in need thereof . 17. A method for treating disorders of the nervous system or pathologies characterized by a modified immunological reactivity and/or symptoms of autoimmunity wherein compounds of formula I or II in which n is 6-16 and R is an acyl residue of a higher aliphatic acid having 16-24 carbon atoms, in chemically unitary form, is administered to a patient in need thereof. 21 SUBSTITUTESHEET

Inventors: KIRSCHNER GUENTER, MENON GIANPAOLO, ROMEO AURELIO, KIRSCHNER, GUENTER, MENON, GIANPAOLO, ROMEO, AURELIO
Applicants: FIDIA SPA, KIRSCHNER GUENTER, MENON GIANPAOLO, ROMEO AURELIO, FIDIA S.P.A.
Application year: 1993
Main classification: A61K31
IPC codes: A61K31, A61P3, A61P9, A61P11, A61P25, A61P29, A61P31, A61P33, A61P35, A61P37, A61P43, C07F9