MXPA97009788A - Analogues of nucleotides for topical treatment of proliferative diseases of the p - Google Patents

Abstract

Pharmaceutical compositions are provided which contain mono, di and triphosphate esters of antiproliferative mononucleoside analogs, dideoxynucleoside analogs terminating in DNA strand and other nucleoside analogues for the topical treatment of hyperproliferative skin diseases, including psoriasis. The phosphate-useful esters of the nuclideside analogs include phosphoroamidates and phosphoracytoses, as well as polyphosphates having atoms that form bridges C and

Description

NUCLEOTIDE ANALOGS FOR TOPICAL TREATMENT OF PROLIFERATIVE SKIN DISEASES DESCRIPTION OF THE INVENTION The present invention provides formulations for the topical treatment of psoriasis and other diseases caused by hyperproliferation of skin cells. Specifically, the invention relates to the treatment of psoriasis or other skin diseases, with formulations containing phosphate esters of nucleoside analogs, antiproliferative nucleosides and dideoxynucleosides which terminate in DNA strand. Psoriasis is a benign disease of human skin characterized generally by plaques covered by thickened scales. This disease is caused by an increased proliferation of epidermal cells of unknown cause. In normal skin, the time required for a cell to move from the basal layer to the upper granular layer is approximately 5 weeks. In psoriasis, this time is only 6 to 9 days, partly due to an increase in the number of proliferating cells and an increase in the proportion of cells which are in division (G. Grove, Int. J. Dermatol. : 111, 1979). Approximately 2% of the population of the REF: 26288 United States has psoriasis, which occurs in approximately 3% of Caucasian Americans, approximately 1% in African Americans and rarely in Native Americans. Epidermal hyperplasia associated with inflammation is characteristic of psoriatic skin. Skin biopsies of affected areas show hyperkeratosis with nuclear fragment restraint, increased proliferation with defective keratinization and chronic inflammatory infiltrates E.A. Bauer, M. Tabas and J.B. Goslen, in Textbook of Internal Medicine, W.N. Kelly (ed.), 1989, pp 1042-1045). With increased cell proliferation, there is an increased synthesis of DNA in the affected tissue which has been the basis for trials evaluating the efficacy of antipsoriasis agents. Although rare primates with clinical and histopathological features of psoriasis have been reported (N.J. Lo e, Drug Dev. Res. 13: 147-155, 1988), there is no recognized animal model for psoriasis. Accordingly, the investigation of anti-psoriasis drugs has been based on experimentally induced hyperplasia in animals or in a mouse strain having the spontaneous mutation (FSN) for squamous skin (JP Sundberg et al., J. Invest, Dermatol 92: 414 , 1989). Another mouse model for the disease associated with epidermal proliferation is the hairless mouse deficient in essential fatty acids (EFAD). The experimentally induced animal models also include nude nude mice, which are immunologically defective and are grafted with diseased human skin. Current therapies consist of efforts to reduce rapid cell proliferation and reduce inflammation. These therapies include the use of active agents in a topical and systematic manner, or both, optionally combined with irradiation. Topical treatments include the use of steroid creams and the use of coal tar ointments or mineral tar followed by ultraviolet irradiation (UV B, 290-320 nm). Topical 5-fluorouracil has been used with some success but the treatment causes severe erythema, edema, bullae formation and ulceration of the skin in treated areas and therefore has not been well accepted by patients (CJ McDonald, Pharmac. Ther. 14: 1-24, 1981). TriazureKR (6-azauridine triacetate) has been tested topically on the skin of patients with psoriasis but has been rendered ineffective (William Drell, personal communication, May 1993). Antiproliferative agents including methotrexate, 6-azauridine and triazure have also been used systemically. Extensive psoriasis has been treated with oral administration of 8-methoxypsoralen, a photosynthesis, followed by ultraviolet A irradiation (320 nm) or administration of retinoids, such as etretinate, followed by ultraviolet A irradiation (CJ McDonald, Pharmac. Ther. 14: 1-24, 1981; E.A. Bauer, M. Tabas, and J.B. Goslen, in Textbook of Internal Medicine, W.N. Kelly (ed.), 1989, pp 1042-1045). Other diseases caused by hyperproliferation of skin cells include atopic dermatitis, lichen planus, actinic keratosis, basal cell carcinoma and squamous cell carcinoma. The topical use of phosphate esters of antiproliferative nucleosides and of dideoxynucleosides and their analogs for the treatment of psoriasis or other diseases caused by hyperproliferation of skin cells has been reported. Related PCT publication No. WO 94/26273 discloses that phosphate esters of antiherpes nucleosides such as azycloi are effective in animals infected with strains of mutant virus in which acyclovir can be converted to acyclovir phosphates due to the mutation that affects the viral enzyme, thymidine kinase. According to the invention, there is provided a method of treatment for psoriasis and other diseases caused by hyperproliferation of skin cells, comprising topically applying a composition containing as an active ingredient a nucleoside analogue phosphate ester having the formula: wherein N is a nucleoside analog having antiproliferative activity; Z is 0 or S or NH; and n is 1, 2 or 3; or a pharmaceutically acceptable salt thereof. The method can also be carried out using a composition containing as an active ingredient a nucleoside analogue phosphate ester having the formula: wherein N is a nucleoside or nucleoside analogue having antiproliferative activity; X is 0 or CH2 or S; Z is O or S or NH; and n is 2; or a pharmaceutically acceptable salt thereof. In preferred embodiments, the nucleoside or nucleoside analogue is selected from the group consisting of cytosine arabinoside, guanosine arabinoside, 5-fluorodeoxyuridine, 5-fluorouridine, 6-azouridine, 2-chlorodeoxyadenosine, 2-chloro-2'-fluoroaradeoxyadenosine. , 2 -fluoroaradeoxydenosine, 6-methylmercaptopurine, riboside, dideoxycytidine, dideoxythymidine, dideoxyguanosine, dideoxyinosine, dideoxydenosine, 2'-deoxitubercidine, 2'-deoxyformicin, 2'-deoxy- (3,4-d) pyrimidine, acyclovir and ganciclovir. According to the method of the invention, the active ingredient in the composition is in a concentration from 0.001 g% to 100 g%. In a preferred embodiment, the active ingredient in the composition is in a composition from 0.01 g% to 10 g%. In a particularly preferred embodiment, the active ingredient in the composition is in a concentration of 0.1 g% up to 5 g%. According to one aspect of the invention, the composition is applied to an area of the skin from 1 to 10 times daily in a dosage from 0.001 g% to 100 g% per application. Alternatively, the composition is applied to an area of the skin from 1 to 10 times daily in a dosage from 0.01 g% to 10 g% per application. In a preferred embodiment, the composition is applied to an area of the skin from one to ten times daily in a dosage from 0.1 g% to 5 g% per application. According to a further aspect of the invention, pharmaceutical compositions comprising the nucleoside analog phosphate ester of the method are provided as an active ingredient. In preferred embodiments, the pharmaceutical compositions additionally comprise propylene glycol, polyethylene glycol 400 and polyethylene glycol 3350. In particularly preferred embodiments, the pharmaceutical compositions additionally comprise an ingredient for improving skin penetration during topical application. The invention also provides antiproliferative nucleoside analogue phosphate esters used in the methods of the invention. In a preferred embodiment, the nucleoside analog is a phosphoroamidate or phosphorothiorate ester and / or is a methylene phosphonate or thiophosphonate ester. The invention provides phosphate esters of antiproliferative nucleosides such as 2-chlorodeoxyadenosine, 2-chloro-2'-fluoroaradeoxyadenosine, cytosine or guanosine arabinoeido, 6-azauridine, 6-mercaptopurine riboside, 5-f luorour idine, 5-fluorodeoxyuridine, acyclovir, ganciclovir, dideoxycytidine and other dideoxynucleosides and nucleoside analogs that end in chain which are then applied topically and are surprisingly and unusually effective in reducing the rate of cell proliferation and knocking down lymphocytes in psoriatic skin. The monophosphate, diphosphate and triphosphate esters have the general formula wherein N is an antiproliferative nucleoside analogue; Z is 0, S or NH; and n is 1 or 2; or alternatively, they have the following formula wherein N is an antiproliferative nucleoside analogue; Z is 0, S or NH; X is 0, CH2 or S; and n is 1, 2 or 3. Accordingly, the phosphoesters may be phosphate, phosphothiorate or phosphoroamidate and the diesters and triesters may have atoms that form bridges other than oxygen, for example, esters of 2,3-thiotriphosphate, or 2, 3 -μ-methylenediphosphonate. Contrary to expectations, these nucleoside analogue phosphates surprisingly pass through the cell membrane of hyperproliferative skin cells and reduce the rate of cell division by inhibiting various enzymatic steps in the biosynthetic purines and pyrimidines., nucleotides, RNA and DNA. In addition, the phosphate compounds of 2-chlorodeoxyadenosine and 2-chloro-2'-fluoroaradesoxiadenosine phosphate are particularly useful for treating the inflammatory component of psoriasis due to their ability to inhibit the growth of lymphocytes and mononuclear white blood cells present in inflammatory infiltrates in the skin of psoriatic patients. The invention also provides pharmaceutical formulations of nucleoside analogs mono-, di- and triphosphates in concentrations that can be applied topically to effectively reduce the proliferation of psoriatic skin cells. Dideoxynucleoside phosphates that terminate in DNA strand, when applied to the skin in a suitable topical formulation, will similarly reduce the proliferation of psoriatic cells. These include acyclovir, ganciclovir, dideoxycytidine, dideoxythymidine, dideoxyguanosine, sodium diisoxaniline, sodium oxazole, 3'-azidodidesoxythymidine (AZT) and other dideoxynucleoside analogues such as those described in the application for U.S. Patent No. Copending No. Serial No. 07 / 373,088, which is incorporated herein by reference. Additional phosphate esters of nucleosides useful as anti-psoriasis agents, when administered topically, include those mentioned in Nucleotide Metabolism (J.F. Henderson &; A.R.P. Paterson, Academic Press, 1973, to pp. 266-271). Salts of these compounds can be easily prepared and such salts can show increased solubility in aqueous media, for example, creams, gels or other aqueous dispersions. Typically, useful salts of these compounds include sodium, potassium, lithium, ammonium or hydrogen salts. Any physiologically acceptable cation known to those familiar with the art can also be used. In addition, such salts are usable and effective in polyethylene glycol creams and lotions which provide a favorable mucous or cutaneous dispersion.

Topical compositions According to another aspect of the invention, there is provided a method for the topical treatment of psoriasis, which comprises applying a composition containing phosphate esters of nucleoside analogue of the invention to psoriatic lesions on the skin of an affected patient. The nucleoside analog phosphate, dideoxynucleoside and nucleoside phosphate esters terminating in the chain of the invention, as previously described, can be prepared for topical use by incorporation into a variety of compositions known to those familiar in the art. which are useful and convenient for dermatological use. The nucleoside phosphate esters are often more soluble in water than the corresponding bases, so formulations of aqueous solution, water-in-oil emulsion or an aqueous cream are largely preferred. The water solubility of the nucleoside analogue phosphate esters of the invention can be increased by the preparation of salts, such as sodium, potassium, lithium, ammonium or hydrogen. In a particularly preferred composition, the active ingredient is prepared in a polyethylene glycol (PEG) carrier. Alternatively, the active ingredients can be applied topically in a dry powder formulation, using an insoluble powder, such as starch or talcum as a diluent or carrier. The vehicle is an important component of some topical compositions, because it can be selected to improve penetration, to prolong the duration of activity or to satisfy requirements of the application site. For example, a formulation for application to the corpus calloses of the body, such as the palms of the hand or the sole of the foot, may include a penetration enhancing agent such as dimethyl sulfoxide, propylene glycol or Azone "R. Alternatively, a powder composition can be selected for application in intertriguous areas such as crotch, inner elbow or between fingers or toes.The composition can also be made to contain various organic polymers or other compositions known to those familiar with the art to provide sustained release of the active anti-psoriasis derivatives Many of the topical compositions suitable in the formulation known to those pharmaceutical chemists can be found: Blaug, S., Ch. 87 in Remington's Pharmaceutical Sciences (15th Ed., 1975, Mack Publishing Company, Easton, Pennsylvania 18042) Compositions include, for example, powders, pastes, ointments, j alloys, waxes, oils, lipids, anhydrous absorption bases, oil-in-water or water-in-oil emulsions, carbocera emulsions (polyethylene glycols of various molecular weights), semisolid gels and semisolid mixtures containing carbocera. The concentration of active ingredient in the topical compositions of the invention may be between about 0.001 g% up to 100 g%; preferably from about 0.01 g% to 10 g%; more preferably from about 0.1 g% to about 5 g%. The compositions may further comprise effective concentrations of other agents which help to promote skin penetration and healing, as described in the aforementioned form and are well known to those usually familiar with the art. The efficacy of topical compositions containing the active nucleoside analogue phosphates of the invention can be evaluated using conventional test procedures, known to those familiar with the art. These include the use of stimulated animal models (e.g., epidermal hyperplasia) and other predictive in vivo assays (N.J. Lowe, Drug Dev. Res. 13: 147-155, 1988). The compositions can be applied to the psoriatic lesions of the affected skin repeatedly; for example 1, 2 or several times a day, the treatment can be prolonged for several days until the healing takes place. The risk of toxicity and irritation incidence is minimal. In the particularly preferred embodiments of the invention, the nucleoside consists of 2-chlorodeoxyadenosine, 2-chloro, 2'-fluoro-ara-adenosine, 6-azauridine, 5'-fluoroureidine or 5-fluorodeoxyuridine, guanosine arabinoside or a dideoxynucleoside in a topical cream consisting of a mixture containing polyethylene glycol 3350, polyethylene glycol 400 and propylene or ethylene glycol. The nucleoside analogues consist of cytosine arabinoside or guanosine, 5-fluorodeoxyuridine, 5-fluorouridine, 6-azauridine and dideoxynucleosides terminating in DNA strands such as dideoxycytidine, dideoxythymidine, dideoxyguanosine, dideoxyinosine and dideoxynucleoside analogues as described in the publication PCT WO 90/00555, and related compounds 2-chlorodeoxyadenosine and 2-chloro-2'-arafluorodeoxydadenosine and 2-fluoroaradesoxiadenosine. Additional nucleoside analogs that inhibit epidermal cell DNA polymerase when supplied as mono-, di-, or triphosphates include 2'-deoxytubercidin, 2'-deoxyformicin, and 2'-deoxy- (3,4-d) pyrimidine. Additional compounds useful as anti-psoriasis agents, when administered topically, include the mono, di and triphosphate esters of the deoxyribonucleoside ribonucleoside derivatives of 8-azaguanine, 6-mercaptupurine, 6-thioguanine, 6-methylmercaptopurine, 2,6-diaminopurine. , 8-azaxanthin, formicin, psychofuranin, decoinin, xylosiladenine, 6-chlorpurine, 6-azauracil, 5-fluorocitodine or 5-fluorocytosine, 5-fluorouracil, 5-iodouracil and 5-bromouracil; and the esters of 3'-mono-, di-, and triphosphate-deoxyadenosine, deoxyxopyranosylimine, deoxyglucopyranosylimine, and cytosine arabinoside (for chemical structures see Nucleotide Metabolism, JF Henderson &ARP Paterson, Academic Press, 1973, pp. 266-271 ).

Tests for topical medication evaluation l. Assay of suppression of epidermal DNA synthesis. Because the increased DNA synthesis characteristic of epidermal hyperplasia in psoriatic skin, the incorporation of radioactive DNA precursors into the skin tissue treated with a pharmacological agent has been used to evaluate the degree of DNA synthesis suppression caused by the pharmacological agent. Briefly, an antipsoriasis agent is applied in a topical carrier to the area of an athymic mouse in which psoriatic human dermis and epidermis has been grafted using techniques well known in the art. Similarly, the EFAD hairless mouse model can be used in this assay to measure the effects of antiproliferative drugs. Approximately 6 hours after treatment, the treated tissue is cut and placed in tissue culture medium containing 3H-thymidine deoxyribose or 3H-bromodeoxyuridine for pulse labeling for 0.1-5 hours. The DNA is then isolated from the tissue using conventional methods and the deoxyribose incorporation of 3H-thymidine or 3H-bromodeoxyuridine in the DNA is measured by determining the radioactivity (cpm / μg of DNA) in the isolated DNA. The efficacy of the anti-psoriasis agent for proliferative suppression is determined by comparing the incorporation of radioactivity into DNA from untreated tissue with the DNA of tissue treated with different compositions (% increase in active ingredient) of the anti-psoriasis agent. 2. Inhibition assay of polyamine biosynthesis. This assay is based on the amount of ornithine decarboxylase (ODC) present in epidermal cells. ODC is a rate-limiting enzyme in the formation of polyamines which generally increases in hyperplasia and includes epidermal hyperplasia associated with psoriasis. Increased ODC activity can also be induced by strips of stratum corneal tape (Lesiewicz, et al., In Models in Dermatology, Vol. 2, pp. 112-116 (HI Maibach &NJ Lo e, eds.), 1985) or by application of phorbol ester (12-0-tetradecanoylphorbol-13-acetate or TPA). 3. Skin biopsy. EFAD hairless mice, mice with scaly skin fsn / fsn or a nude mouse grafted with psoriatic human skin are treated with the antipsoriatic drug substance and in the following 1-15 days the treated skin is examined for general indications of psoriasis (scaly of skin, redness of inflammation, etc.), and then analyzed by biopsy so that the number of layers of epidermal cells can be cut under a microscope using conventional histological procedures. The number of epidermal cell layers is compared to a normal untreated psoriatic skin, examined in a similar manner, to determine the effect of the treatment with the antipsoriatic drug substance. In addition, the relative degree of inflammation in the tissue can be quantified by determining the amount of white blood cells (ie, neutrophils and / or monocytes) in the capillaries of the dermis and dilatation of the capillaries in relation to the psoriatic skin. treated and normal skin control. Similarly, human clinical trials can be conducted using skin biopsies and microscopic examination using bilateral paired comparisons with small amounts of antipsoriatic pharmaceutical agents applied to areas of the skin. (for example in the forearms or legs) of approximately 3 cm in diameter. Paired bilateral comparisons are particularly preferred to avoid the risk of a cross effect from a treated area to an untreated control area. 4. Mice with scaly skin. Inbred mice for spontaneous squamous cutaneous mutation (fsn / fsn) develop a scaly skin after weaning that becomes progressively thicker with age, so remember the proliferative skin diseases, in particular psoriasis. At 42 days of age, the mice show epidermal hyperplasia with a multilaminated basomental membrane and increased inflammation measured in soft blood cells and in tortuous dermal blood vessels, all characteristic of human psoriasis. These mice can serve as a model for human psoriasis in order to determine the efficacy of the treatment using topically applied nucleoside phosphate. A composition that includes the nucleoside phosphate is applied to a portion of 1-5 mm in the dural or ventral skin of a fsn / fsn mouse and the skin is observed in a general and microscopic manner, from 12 hours to 15 days later to determine the degree of epidermal hyperplasia and other psoriatic skin symptoms presented earlier using standard dermatological and histological methods. The relevant comparisons are for a similar portion of the mouse that has not been treated, by another fsn / fsn mouse that has not been treated with an fsn / fsn treated only with the carrier ingredients without the nucleoside phosphate or with non-mutant mice for the locus fsn treated similarly. 5. Markers of keratinocytes. Changes in protein expression are consistent with changes in hyperplasia associated with psoriatic skin. Two useful molecules to detect abnormal conditions in the skin are keratin and filaggrin. The accumulation of mouse-specific keratin, K6, in the suprabasilar epidermis is a molecular change typically associated with hyperplasia. In the EFAD mouse without hair or in the mouse fsn / fsn and the antibody-based methods to detect K6 are useful to determine the presence and quantity of the molecule in the suprabasilar epidermis and therefore can serve as efficacy indicators of antipsoriasis compositions . Filaggrin is a protein in the epidermis which is normally associated with kerato-aline granules in the stratum granulosum and in the transition cells, but which disappears in the cornified cells. However, in the fsn / fsn mouse, filaggrin remains present in the layers of surface cells in a characteristic pattern of transition cells. Therefore, the presence of filaggrin in the upper cornified cells, detected by antibodies against filaggrin and / or by microscopic inspection, is characteristic of psoriatic cells and can be used to evaluate the efficacy of antipsoriasis pharmaceutical preparations applied to the skin of the mouse fsn / fsn. Synthesis of selected purines and purine nucleoside analogues. Specific methods for synthesizing some of the anti-proliferative purines and purine nucleoside analogs useful as phosphate esters for the treatment of psoriasis are summarized in the following. Additional compounds and synthesis methods are known to those familiar with the art.

Representative procedures are described in R.K. Robins and G.D. Kini, in The Chemistry of Antitumour Agents (D.E.V. Wilman, ed.), 1990, pp.299-321. (1) Purine analogues Synthesis of 6-mercaptopurine is carried out by treatment of hypoxanthine with phosphorus pentasulfide in tetralin at elevated temperature. 6-Thioguanine is synthesized by treatment of guanine with phosphorus pentasulfide in pyridine under reflux. 3-adenosine, 3-hypoxanthine and 3-desase-6-mercaptopurine are synthesized by ring closure of the 3,4-diaminopyrimidines appropriately substituted in the seven-ring imidazo [4,5-c] pyridine ring (Robbins, RK et al., J. Org. Chem. 28: 3041, 1963). 3-desaguanine is synthesized when dehydrated (4) -carbamoylmethylimidazole-4 (5) -carboxylic acid methyl ester with phosphorus oxychloride to produce methyl 5- (4) -cyanomethylimidazole-4 (5) -carboxylate which cyclizes by treatment with liquid ammonia. (2) Purine nucleosides 6-methylmercaptopurine riboside is synthesized by condensing 6-methylmercaptopurine with 2,3,5-tri-O-acetyl-D-ribofuranosyl chloride using the mercuric chloride catalyst followed by deacetylation. Alternatively, the 6-methylmercaptopurine riboside can be produced directly by methylation of 6-thioinosine (Fox, J.J. et al., J. Am Chem. Soc. 80: 1669, 1958). 9- / SD-arabinofuranosyladenine (ara-A) is synthesized when the 3 ', 5'-di-O-isopropylidene derivative of 9- (/-. -D-xylofuranoyl) adenine is treated with methanesulfonyl chloride to convert it to the corresponding 2'-O-methyl derivative, which is separated by acetic acid to produce 9- (2'-O-mesyl-jS-D-xylofuranosyl) adenine, which is treated with sodium methoxide in methanol to produce the 2 ', 3' -epoxide, which is treated with eodium in aqueous dimethylformamide to open the ring and produce ara-A, which is purified by crystallization. The related compound, 9-3-D-arabinofuranosyl-2-fluoroadenine (2-chloro-ara-A) is synthesized by acetylating 2,6-diaminopurine with acetic anhydride in pyridine under reflux to produce 2,6-diacetamidopurine, which is condensed with 2, 3, 5-tri-O-benzyl-aD-arabinofuranosyl chloride to produce the blocked β anomer of the nucleoside, which is deacetylated by treatment with methylamine in ethanol to produce the diaminonucleoide, which is treated with a mixture of fluoboric acid and tetrahydrofuran with aqueous eodium nitrate to produce 2 ', 3', 5 '-tri-O-benzyl-jS-D-arabinofuranosyl-2-fluoroadenine, which is treated with boron trichloride to produce 2- fluoro-ara-A. 2-Chloro-2'-deoxyadenosine is synthesized by fusing 2,6-dichloropurine with 1, 3,5-tri-0-acetyl-2-deoxy-D-erythropentafuran and, on the other hand (Robine and Kini, in The Chemietry of Antitumor Agents, 1990, ap 308). (3) Purine cyclic nucleotides and cyclic nucleotide analogues 8-chloro-cAMP is synthesized by cAMP bromination in the presence of aqueous sodium hydroxide to produce 8-bromo-cAMP, which is treated at elevated temperatures with thiourea to produce -thio-cAMP, which is chlorinated with chlorine gas in a methanolic HCl solution. (4) Other antiproliferative nucleosides related to purines Thiazofurin (2 - / -.- D-ribofuranoylthiazole-4-carboxamide) is synthesized by treatment of 2,5-anhydro-3,4,6-tri-O-benzoyl-D- alonothioamide with ethyl bromopyruvate to promote ring closure that produces ethyl 2- (2, 3, 5-t ri-O-benzoyl-β-D-ribofuranosyl) thiazole-4-carboxylate as the main product, which is separate and treated with methanolic ammonia.

A related compound, seleno thiazofurin, is synthesized by treatment of 2,5-anhydro-3,4,6-tri-0-benzoyl-D-alonitrile with liquid H2Se to produce the corresponding selenamide, which is reacted with ethyl bromopyruvate to promote ring closure that produces ethyl 2- (2, 3, 5-tri-O-benzoyl-β-D-ribofuranoyl) eelenazole-4-carboxylate ethyl as the main product, which is treated with methanolic ammonia. Pyrazofurin (3- (l-8-D-ribofuranosyl) -4-hydroxypyrazole-5-carboxamide) is a naturally occurring nucleoside that can be isolated from Streptomyces candidus (Gutowski, GE et al., Biochem. Biophye, Ree. Commun. 51: 312, 1973). Alternatively, pyrazofurin is synthesized by treatment of the a-ketoeter with the hydrazine derivative to produce the diazo intermediate, which is heated with acetic anhydride and sodium acetate to produce cyclized hydroxypyrazole nucleoside, which is aminated with methanolic ammonia. and then benzylated to produce pyrazofurin. It synthesizes 2-deoxycophormycin, (8R) -3- (2-deoxy-jS-D-erythropentofuranosyl) -3,6,7,8-tetrahydroimidazo [4, 5-d] [1,3] -diazepin-8- ol by glycosylation of aglycone, 6,7-dihydroimidazo [4, 5-d] [1,3] diazepin-8 (3H) -one with 2-deoxy-3,5-di-Op-toluoyl-D- chloride erythropentafuranoeil (Chan, E. et al., J. Org. Chem. 47: 3457, 1982). Synthesis of nucleoside monophosphate, diphosphates and triphosphates and nucleoside phosphate analogues: Methods for synthesizing nucleoside monophosphates by reacting the nucleoside with phosphorus oxychloride are described in WO 90/00555 and WO 94/26273 and as described previously (Yoshika a et al., Bull. Chem. Soc. Japan 42: 3505-3508, 1969; Toorchen, D. and Topal, M., Carcinogenesis 4: 1591-1597, 1983). The nucleoside diphosphates are prepared by the methods of Ott, D.G. et al. (Anal Biochem 21: 469-472, 1967). The nucleoside triphosphates are prepared by the method of Seela and Rdling (Nuc Acide Ree., 20: 55-61, 1992) or from nucleophilic monophosphate by the method of Moffat and Khorana (J. Am. Chem. Soc. : 663, 1991), or by the method of Hoard and Ott (J. Am. Chem. Soc. 87: 1785-1788, 1963). Examples 1-3 below present the details of some useful syntheses for preparing the phosphate esters of nucleosides and nucleoside analogues. Other nucleoside phosphate analogs include nucleoside phosphorothioatoe, nucleoside phosphoroamidate, nucleophilic phosphonate and nucleophilic foefofoforuride which can be synthesized using methods well known to those well acquainted with the art and summarized, for example, by D.W. Hutchinson (The synthesis, reactions and properties of nucleoeide mono-, di-, tri-, and tetraphoephatee and nucleotides with changes in the phosphoryl reeidue, In Chemistry of Nucleotides and Nucleosides, L. Townsend, ed., 1991 at pp. 81 -146 and references therein). The common symptoms are summarized as follows. (1) The foeforotioatoe of nucleoeides are nucleotide analogues in which one or more of the phosphoryl oxygen atoms have been replaced by sulfur. The first synthesis methods reacted a protected nucleoside and tris (l-imidazolyl) phosphane sulfur, while the most recent syntheses constitute the latter reagent with thiophosphoryl chloride (PSC13). A nucleoside phosphoroanilidate can be converted to a phosphorothioate by treatment with eodium hydroxide and carbon dieulfide. Nucleic acid 5 '-foeforothioatoe can be re-propelled from direct eulfurization of 5'-nucleoside phosphites. The 2 '(3') -foeforothioatoe of purine nucleoide can be synthesized by reacting 2 ', 3' -O-di-n-butylenetetanylene derivative with thiophenophoryl chloride followed by alkaline hydrolysis. (2) The nucleoside phosphoramidates are analogs in which one or more oxygen atoms or foephoryl has been replaced by nitrogen, which generates a PN bond which is considerably more labile than the PS bond in the phosphorothioates of nucleoeidum under the same conditions moderately acidic Synthesis of these compounds includes the phosphorylation of aminonucleosides and the treatment of nucleoside azides with phosphorous acid triesters. The lipophilic nucleoside phosphoroamidates may be particularly useful antipsoriatic compounds because of their ability to be more readily captured by cells when they are hydrolyzed into biologically active compounds. (3) The nucleoside phosphonates are composed in which the phosphoryl oxygen is replaced by carbon which generates a stable PC bond and which have decreased acidity of the P-OH groups when the phosphorus atom is substituted with an alkyl group which is a donor. electrons instead of oxygen. Nucleoside phosphonates are easily prepared from nucleoside halides by those familiar with the art either by the Arv or Michaelis-Becker reactions. The nucleoside 5'-phosphonates can be synthesized from 2 ', 3'-protected 5'-iodo-5'-deoxynucleotide using method well known to those familiar with the art. The 5'-nucleoside and isoetherphosphonates in which the 5'-oxygen is substituted with a methylene group are synthesized by coupling a 5'-suitably protected nucleoside aldehyde with diphenyl triphosphoxyphenidimethylphthalate to provide a di-ester of foefonate a, β - unsaturated which is then reduced and deprotected in the phosphoryl residue to provide the phosphonate. The isosteric nucleoside 3'-phosphonates are synthesized from the riboea-1-phenylethylated chloride which is coupled to the heavy metal salt of a purine or pyrimidine. Phosphonates are generally less polar than their phosphate counterparts and therefore are useful as antipsoriatic agents because they are easily captured by the cells when they are applied topically. (4) The nucleoede foeforofluoridate are analogs of mononucleotides. The treatment of 5'-nucleoside phosphates with 2, 4-dinitrofluorobenzene produces the 5'-phosphorofluoridates of nucleoside by means of 2,4-dinitrofeniléeter of the nucleotide. (5) Other analogues of nucleophilic polyphosphate include those in which atoms other than oxygen have been substituted between the α, β-phosphorus atoms in di and triphosphate of nucleoside or between β atom, and phosphorus in nucleoside triphosphates ( which include those listed in Table III on page 119 of DW Hutchinson, In Chemietry of Nucleotides and Nucleosides, L.

To neend, ed. , 1991). Usually, analogous to, ß ee are prepared by condensing a 2 ', 3' -O-protected nucleoside with the pyrophosphate analogue with the help of DCC or by nucleophilic displacement actions involving the displacement of a toluene-sulfonyl residue (tosyl) from the 5 'position of the sugar residue of the tosyl nucleoside by the methylene biphosphonate ion. The preparation of a β analog, and are presented in Example 4. Preparation of a topical polyethylene cream: 5 grams of nucleoside phosphate are dissolved in 5 ml of propylene glycol and 60 grams of polyethylene glycol 400 (both from Spectrum Chemicals Inc. Angeles, CA) at 70 °, with agitation. 40 grams of polyethylene glycol 3350 (Spectrum Chemicals Inc.) are added with stirring, until the mixture becomes completely clear. After the composition is placed in tubes or other containers and allowed to cool to room temperature and sealed. The effective concentration of the active ingredient in the composition can vary from 0.001 g% to 50 gl, and preferably from 0.01 g% to 5 g%. The composition may also comprise other ingredients for improving skin penetration (eg, dimethyl sulfoxide or DMSO) and / or storage stability of the composition as is known to those usually familiar with the art.

Treatment of psoriasis: The topical compositions of the invention comprising an antiproliferative or anti-inflammatory nucleoside phosphate in an effective concentration against psoriasis ranging from 0.01 g% to 10 g% are applied to the affected areas from one to six days per day. The dosing protocols and optimal concentration of the active nucleoside phosphate compounds can be readily determined by clinicians familiar with the technique for the treatment of psoriasis.

EXAMPLE 1 PREPARATION OF NUCLEOSIDE TRIFOSPHATES FROM MONONUCLEOSID The preparation of 5'-trifoefatoe of deeoxirribonucleotide, dideoxyribonucleotides and analogues involves a series of reactions, as indicated immediately below.

II III O O O IV The synthesis of mononucleosides ee is described in the application for US patent Serial No. 08 / 060,258, filed on May 12, 1993. A nucleotide (I) and an excess of 1,1 '-carbonylimidazole (II) are reacted during about 1 hour at room temperature to form an imidazolidate (III). The unreacted 1,1'-carbonyldiimidazole is decomposed with methanol before an access of inorganic pyrophosphate (IV) is added. This eliminates the formation of inorganic polyphosphates which would have to be removed out of the reaction materials. The phosphorylation is allowed to proceed until approximately 24 hours after the addition of the inorganic pyrophosphate (IV) and after the product of the nucleoide diphosphate (V) is purified by anion exchange chromatography on DEAE-cellulose followed by conversion of the product to a salt such as a sodium salt. Because the nucleotide (I) and the imidazolidate (III) can react together to form a by-product of symmetric pyrophosphate, the anion exchange chromatography on DEAE-cellulose at a lower pH, when the desired product (V) has a lower charge that the unwanted byproduct, which allows the separation of the two compounds. A reagent used in the tributylammonium pyrophosphate solution which is produced by the following procedure. To an aqueous solution of pyridinium phosphate, obtained by passing a solution of tetrasodium pyrophosphate decahydrate (446 mg, 1 mmol) through a column of Do ex 50W-X4MR (pyridinium) resin (17 ml) is added tributylamine ( 0.24 ml, 1 mmol). The solution is concentrated under vacuum and the residue is then dried by consecutive addition and evaporation of anhydrous pyridine followed by addition and evaporation of two 10 ml portions of N., N-dimethylformamide (DMF). The synthesis of nucleoside triphosphates is completed by the following method. To a solution or suspension of mono-oligonucleotide (0.1 mmol) such as the anhydrous tributylammonium salt in 1 ml of DMF, 1,1'-carbonyldiimidazole (80 mg, 0.5 mmol) was added with 1 ml of DMF. The mixture is mixed for 30 minutes and then kept in a desiccator at room temperature for 4-12 h before it is treated with 33 μl (0.8 mmol) of methanol and allowed to react for 30 minutes at room temperature. Tributylammonium pyrophosphate (0.5 mmoles) is added in 5 ml of DMF and mixed vigorously and then the mixture is kept in a locker at room temperature for 24 hours to allow imidazolium pyrophosphate to precipitate. The precipitate is separated and washed with four portions of 1 ml of DMF by centrifugation and the re-melting in DMF is about 80-100% pure. The copolymer is treated with an equal volume of methanol and evaporated to dryness under vacuum. The residue is chromatographed on a 2 x 20 cm column of DEAE in cellulose with a linear gradient of triethylammonium bicarbonate (a gradient of 3 1 of about 0 to 0.4 M) at pH 5-7.5 and the fractions are collected and harvested. Spectrophotometrically to identify the fractions containing the nucleoside triphosphates. The appropriate fractions are evaporated under vacuum and the triethylammonium nucleoside triphosphate is dissolved in methanol to a concentration of approximately 0.05 M and five volumes of an acetone solution of sodium perchlorate (15 equivalents) are added to form a salt precipitate. of sodium nucleoside triphosphate. It will be understood by those familiar with the art that other salts of nucleoside triphosphate can be made by the appropriate precipitation reactions. The precipitated salt is collected by centrifugation, washed with four portions of 1 ml of acetone and dried under vacuum over phosphorus pentoxide. Additional procedures are available for synthesis of nucleoside triphosphates including those set forth in the following example.

EXAMPLE 2: SYNTHESIS OF MONO-, DI- AND NUCLEOSIDE TRIFOSPHATES USING FOSFOROMORFOLINO- 2, 2, 2-TRIBROMOETILO CHLORHYDRATE By using essentially the method of van Boom, et al. (Tetrahedron Lett 32: 2779-2782, 1975), ribonucleoside mono-, di-, and triphosphates and their derivatives are prepared by a single intermediate. In general, reactions include reacting a monofunctional reagent (2,2,2-tribromoethyl phosphoromorpholinohydrochloride) with a ribonucleotide (or its derivative) to produce phosphotherer derivatives with a 2,2,2-tribromoethyl protecting group attached to the ribonucleoside ( that is, to produce 5'-ribonucleoside phosphorofolidates or 5'-mononucleoside phosphoromorpholide derivatives). The protecting group is removed by a coupling reaction of Cu / Zn with an acidic deblocker and neutralization to produce the mono-, di- and triphosphate based on the acid used and the deblocking step, and the ammonium salt used in the step neutralizing That is, to obtain the monophosphate ribonucleoside, HCl and ammonia are used; to obtain the diphosphate ribonucleoside ee use the mono (tri-n-butylammonium) salt of the foephoric acid; Bis (tri-n-butylammonium) pyrophosphate is used to obtain the triphosphate ribonucleotide. The general reactions are indicated as follows: vi The monofunctional reagent (I), 2,2,2-tribromoethyl phosphoromorpholinohydrochloride ee prepares by reacting 2,2,2-tribromoethyl and morpholine foeforodichlorhydrate in an anhydrous ether, from which the reaction product is separated and recrystallized. using cyclohexane / n-pentane using methods well known in the art. The crystalline 2,2,2-tribromoethyl phosphoromorpholinohydrochloride has a m.p. of 79 ° C.

The monofunctional reagent (2 mmoles) is mixed with 1 mmole of nucleoside or its derivatives in anhydrous pyridine at 20 ° C for 48 h; after the reaction mixture is fractionated chromatographically (B.J. Hunt &W. Rigby, Chem. &Ind. 1868, 1967) to give colorless solid nucleotide (III). The treatment of nucleotides with Cu / Zn coupling in anhydrous DMF for 10 minutes at 20 ° C followed by filtration to remove excess Cu / Zn provides the nucleoside phosphoromorpholidats. Subsequently, the nucleoside phosphoromorpholidats are treated with different acids and ammonia sources to provide the mono-, di- or triphosphates. For the monophosphate, the phosphoromorpholidate is treated with 0.01 N HCl, pH 2 for 2 h at 20 ° C, and subsequently neutralized with aqueous ammonia (pH 9) and purified on a Sephadex G-25"R column. , the 5'-nucleoside triphosphate is obtained by reacting the phosphoromorpholidate (0.1 mmol) in 2 ml of anhydrous DMF with 0.5 mmoles of bis (tri-α-butylammonium) pyrophosphate in 2 ml of anhydrous DFM at 20PC for 3 hours under conditions that include moisture The reaction product is concentrated under vacuum, treated with Do ex 50HR (ammonium form) and purified on a 2 x 25 cm column of DEAE celluloea using 3 1 linear gradient from 0.0 to 0.3. M of Et3N? 2C03 solution The 5'-nucleoside diphosphate is obtained by reacting the phosphoromorpholidate (0.1 mmolee) with 0.6 mmoles of mono (tri-n-butylammonium) phosphate in 4 ml of anhydrous pyridine at 20 ° C during 3 hours under conditions that exclude moisture and the reaction product is conceived ntra and purify in a similar way. Alternatively, the derivatives of foefotriester (III) can be directly converted to the corresponding nucleoside diphosphates by treatment with Zn powder in pyridine solution containing mono (tri-n-butylammonium) -phosphate. That is, 1 mmol of reagent III is added to a stirred solution of 15 ml of anhydrous pyridine containing 0.1 g of finely divided Zn and 2 mmoles of mono (tri-n-butylammonium) phosphate under conditions that exclude moisture for approximately 48 hours at 20 ° C. Subsequently the reaction mixture is centrifuged to sediment the Zn and the supernatant is co-evaporated three times with 15 ml of water in each stage before purification in DEAE cellulose.

EXAMPLE 3: SYNTHESIS OF 2'- DESOXYRIBONUCLEOSIDE 7-DESAZAPURINE THYPHOSPHATES (1) Synthesis of the triethylammonium salt of 7'-desaza-2'-deoxyinosine 5'-triphosphate. 7-Desea-2'-deoxyinosine (25 mg, 0.1 mmol) is dissolved in trimethyl phosphate (250 μL, 1.07 mmol) and P0C13 (18.5 μL, 0.2 mmol) is added. The mixture is stirred for 1.5 h at 0 ° C and then a mixture of 0.5 M bis (tri-n-butylammonium) pyrophosphate in 1 ml of anhydrous DMF and 1 ml of tri-O-butylamine is added with vigorous stirring, for 1 min.; The solution is neutralized with an aqueous solution of 1 M Et3NH2C03, pH 7, and evaporated to dryness under vacuum. The residue is purified on a 2.6 x 30 cm column of DEAE-sephadex using a linear gradient of a solution of Et3NH2C03, pH 7 (11 H20 / 11 0.7 M TBK) to give a colorless solid with a UV (H20)? ^ 258 nm. (2) Synthesis of the triethylammonium salt of 7'-desase-2'-deoxydedene 5'-triphosphate. This compound is prepared in a similar manner from 7-dease-2'-deoxyadenosine to provide a colorless solid with a UV (H20) λ, 270 nm. (3) Synthesis of the triethylammonium eal of 5'-trifoefato of 7-deesasa-2 '-deoxyguanosine. This compound is prepared in a similar manner from 7-deea-2'-deeoxiguanodine to give a colorless solid with a UV (H20) λ, of 259 nm.

EXAMPLE 4: SYNTHESIS OF 2-CHLORODESOXY-3-ADENOSIN PHOSPHETHYLENE DIFOSPHONATE (2CdAPMDP) Nucleoside polyphosphate analogs have been synthesized in which the atom between the β-phosphorus atoms, and in a nucleotide triphosphate, such as deeoxiadenosine trifoephate, has been replaced by another oxygen atom (TC Myers et al., Phosphonic analogue of nucleoeide phosphates I. The synthesis of 5'-adenylyl methylene diphosphonate, to phosphonic analogue of ATP J. Am. Chem. Soc. 85: 3292-3295, 1963; RG Yount, ATP analogues, Adv. Enzymol. : 1-56, 1975). Substitution produces a stronger bond than the P-0 bond that occurs in a nucleoside phosphate molecule. The synthesis is described essentially in method 1 or method 2 of Myers et al. (J. Am. Chem Soc. 85: 3292-3295, 1963). In Method 1, 2-chlorodeoxyadenoein 5'-phosphoroamidate is reacted with methylene diphenyleonic acid to produce phosphonic acid analogues of the nucleoide pyrophosphate. Alternatively, by using method 2, 2-chlorodeoxyadenosine monophosphate is reacted with methylene diphosphonic acid using dicyclohexylcarbodiimide (DCC) as the condensing agent. According to method 1, methylene diphosphonic acid is obtained by hydrolysis in concentrated HCl or its tetraethyl ester is prepared by reaction of methylene iodide with excess of triethyl phosphite. L-3-dicyclohexylguanidinium 5'-phosphoroamidate is treated (3.6 mmoles) and methyldiphosphonic acid (10.8 mmoles) with 54 ml distilled o-chlorophenol; The mixture is cooled in ice and 36 ml of dry pyridine are added. The resulting solution is allowed to stand at room temperature with occasional stirring for 48 h, when 300 ml of water is added while cooling on ice. The solution is extracted six times with ether (850 ml total). The aqueous solution is adjusted to pH 2 with 1 N HCl and then treated with 30 g of acid-washed activated carbon (Norit A) and stirred for 30 minutes; Subsequently, the coal is collected by filtration and thoroughly washed with water (5 1, total). The nucleotide derivatives are eluted with 50% aqueous ethanol-5% concentrated ammonium hydroxide (3 1 total) and the eluate is concentrated to a volume of 400 ml by evaporation at 35 ° C. The concentrated eluate is applied to a 2.7 cm x 31 cm column of Dowex-2MR (chloride, 8% crosslinking) and eluted with a linear gradient made by mixing 2 N 0.003 N HCl (in the mixing vessel and 2). 1 HCl 0.003 N plus LiCl (0.45 N) in the tank); ee collect fractions of 10 ml and identify fractions containing phosphomethylene diphosphonate 2-chlorodeoxyadenosine (2CdAPMDP) using paper chromatography or using ultraviolet absorbance methods well known in the art. The fraction containing 2CdATMDP-ee neutralizes with 1 N LiOH and is concentrated by evaporation at 30 ° C.; the concentrated solution is treated with 250 ml of acetone-10% methanol to precipitate a solid which is separated by centrifugation and washed with the mixture of acetone-10% methanol until no chloride is detected in the washings. The Li salt of 2CdATMDP can be further purified by dissolving the salt in 100 ml of water adjusted to pH 8 with LiOH and by eroding the solution through a Dowex-2MR column as described above, using an elaborate gradient. 1.5 1 HCl of 0.003 N in the mixing chamber and 1.5 N of 0.003 N HCl plus 0.45 N LiCl in the tank and treat the eluate as described above, followed by dissolution by the precipitate in 6 ml of water and precipitation with 40 ml of methanol. The final precipitate is dissolved in 15 ml of water and lyophilized to produce a tetralithium powder of 2CdATMDP. Using method 2, methylene diphosphonic acid (11.4 mmol) and 2-chlorodeoxyadenosine monophosphate (2.6 mmol) are dissolved in pyridine (30 ml) and water (4 ml) to produce a mixture of two faees to which DCC is added. room temperature with vigorous stirring in 3 aliquots (29 mmol) at the beginning of the reaction; 48 mmoles after 4 hour and 19 mmoles after 12 hours). After 24 hours, the reaction is terminated and the precipitated dicyclourea is filtered off and washed with water. The filtrate and the washing are adjusted to a total volume of 150 ml with water and extracted five times with ether (300 ml, total). The solution is adjusted to pH 8 and chromatographed on a 2.5 cm x 17.5 cm column of Do ex-l "R (formate, 2% crosslinking), the column ee washed with 1.5 1 of water to remove pyridine. Elution of the column is carried out using a gradient generated by successively adding to a mixing chamber containing 500 ml of water, the following solutions: formic acid 4 N (500 ml), formic acid 4 Nmae ammonium formate 0.1 M (500 ml) and formic acid 4 N marate 0.2 M ammonium formate (1500 ml) and collecting 15 ml fractions The fractions containing 2CdATMDP (approximately in tubes 115-134) were identified using ultraviolet absorption, by using of methods well known in the art Combined fractions containing 2CdATMDP are freeze-dried to a volume of up to about 200 ml and treated with 7 g of acid-washed activated carbon (Norit A) and stirred for 15 minutes; Collect activated carbon by filtration and wash with water (800 ml, total). The product elutes with 50% aqueous ethanol-5% concentrated ammonium hydroxide (600 ml total) and the eluate is concentrated to a volume of 200 ml by evaporation at 20 ° C, filtered to remove traces of carbon. activated and lyophilized to a powder. The powder is dissolved in 4 ml of water and the solution is treated with excess 1 M barium acetate; The resulting precipitate is collected by centrifugation, washed with water and dried in 0.1 N HBr at 0 ° C. The pH of the solution is adjusted to 6.5 with 1 N NaOH and the resulting precipitate is collected by centrifugation, washed successively with 2 x 2 ml each of water, ethanol and ether. The sample is dried at room temperature over P204 for 12 hours to provide dibary 2CdATMDP hydrate. Other nucleophilic analog phosphomethylene diphosphonates of the invention are prepared in a similar manner.

EXAMPLE 5: APPLICATION OF PHOSPHATE ESTERS THAT HAVE AN ANTIPROLIFERATIVE EFFECT USING THE EFAD MOUSE MODEL Ester of mono-, di and trifoefato of antiproliferative nucleoside analogues as described in Example 2 are prepared in an essential manner. These compounds are formulated invidually in topical polyethylene cream as described above, containing from 0.001 g% to 10 g% of active ingredient in a propylene glycol cream, polyethylene glycol 400 and polyethylene glycol 3350 to produce a cream mixture. Similar formulations are also produced which contain an ingredient for improving skin penetration (for example dimethyl sulfoxide or Azone ™). The compositions are applied in aliquots of μl at 6-hour intervals on the dorsal side of the mouse without hair EFAD for a period of 6 h to 5 days. At the end of each time interval, a mouse is sacrificed and the treated tissue is cut and placed in tissue culture medium containing 3H-thymidine deoxyribose for pulse marking for 2 hours to measure the effects of the mono-, di- and trifoefato eobre the proliferation of cutaneous cells. The DNA is then isolated from tissue using standard procedures and the incorporation of 3H-thymidine deoxyribose into DNA is measured by determination of radioactivity (cpm / μg of DNA) on the isolated DNA. The efficacy of the mono-, di-, and triphosphate esters of anti-pustulosis composition for proliferative region was determined by comparing the incorporation of radioactivity in the DNA of untreated tissue with the DNA of tissue treated with different formulations of antipsoriae agents. The total amount of active ingredient was determined for each skinned skin biopsy from g% of the active ingredient in the applied cream formulation and the amount of applications administered before the mouse skin enema was performed. For each experimental time point, a corresponding untreated time point assay was performed using skin from the ventral side of the mouse. We used the difference in the incorporation of 3H-thymidine deoxyribibe into the DNA of the doreal skin and the ventral control skin to calculate the total suppression of cell proliferation (in percent) as a function of the total amount of each of the esters of phosphate administered. The 3H incorporation results were also used to determine the percent suppression (comparison of the dorsal skin with incorporation into the ventral skin) as a function of the time of each of the individual compositions tested. As a function of time and as a function of the total amount of active ingredient administered, the incorporation of 3H into DNA corresponding to the dorsal skin treated with phosphate esters is significantly less than the incorporation of 3H into DNA corresponding to the ventral skin. not treated.

EXAMPLE 6: THE APPLICATION OF DIDESOXYTIDINE TRIFOSPHATES INHIBITS THE POLYAMINE BIOSYNTHESIS IN A MOUSE MODEL The polyamine biosynthesis inhibition assay was performed by measuring the amount of ornigine decarboxylase (ODC) present in epidermal cells after the topical administration of dideoxytidine triphosphate to the mouse skin previously treated with the formalin ether of 12-0- tetradecanoylphorbol-13-acetate (TPA, applied to increase ODC activity). Briefly, an athymic knot mouse or an EFAD mouse has been topically treated with a TPA solution to increase the concentration of ODC in the epidermal tissue and mimic the elevated concentrations of this enzyme as they are generally found in hyperplasia associated with psoriasis. In the next 30 minutes to 1 hour after treatment with TPA, a cream containing dideoxycytidine triphosphate was applied as the active ingredient together with 0.01% DMSO to nourish the skin penetration, in the area treated with TPA of the skin . The cream contains dideoxycytidine triphosphate prepared essentially as described in Example 2 and which is prepared in a pharmaceutically acceptable cream at a concentration of 5 g%, which is applied to the skin in an aliquot in 20 μl.; Additional aliquots are applied every hour for a period of 6 hours for a total of 6 applications. At the end of 6 hours, ODS concentrations in the skin were determined using techniques well known in the art (J. Lesie icz et al., In Modele in Dermatology, Vol.2 (HI Maibach &NJ Lo e, ede .), 1985, pp 112-116). As controls, untreated skins of nude nude mice were tested for normal concentrations of ODS and skin from athymic study mice treated with TPA and tested to determine the concentration of ODS elevation following administration of TPA. As an additional control, skin treated with TPA and normal skin is treated in parallel with a cream formulation containing DMSO but without dideoxycytidine triphosphate. The concentration of ODS in the skin treated with TPA and the cream containing dideoxycytidine triphosphate, with normal skin and with the skin treated with TPA is compared. The efficacy of treatment with dideeoxicitidine trifoefato is determined by a decrease in ODC in relation to that observed in the skin treated with TPA and by means of the concentration of ODS in relation to normal skin. After treatment with the cream containing dideoxycytidine trifoephate, the concentrations of ODS decrease significantly in relation to ADC concentrations in skin treated with TPA that did not receive cream or that received cream without dideoxycytidine triphosphate as an active ingredient. It will be understood by those familiar with the art that treatments and analysis of treatment efficacy can be determined with other esterase of dideoxynucleoside phosphate in a similar manner. These didexynucleotide phosphate esters include esters of dideoxythymidine mono-, di-, and triphosphates, dideoxyguanosine, dideoxydenosine, dideoxyinoine, and dideoxynucleoside analogue including azidodideoxythymidine (AZT) and dideoxydidehydrotimidine.

EXAMPLE 7: APPLICATION OF 5'- FLUORODESOXYURIDINE PHOSPHATE ESTERS AND 5-FLUOROURIDINE PHOSPHATE ESTERS WHICH HAVE AN ANTIPROLIFERATIVE EFFECT USING GRAFTED MICE WITH HUMAN PSORIATELY SKIN A percutaneous biopsy is used to detect the antiproliferative effect of 5'-fluorodeoxyuridine triphosphate and 5-fluoruridine triphosphate when applied topically to human psoriatic skin inserted in a nude mouse. Human pessimistic skin approximately 1-10 mm in diameter is inserted into the doreal side of an athymic mouse using techniques well known in the art. After the graft has healed sufficiently, psoriatic human skin is treated with psoriatic pharmaceutical substances containing as an active ingredient 5-fluorodeoxyuridine trifoefate, 5-fluorouridine trifoephate or eue mono- or diphosphates. These compounds are synthesized essentially as described in Example 1. The pharmaceutical compositions are made to include the active ingredients in a topical polyethylene cream essentially as described above in the description with the active ingredients at a concentration of 0.1 g% to 10 g%. The cream containing the active ingredient is applied to the skin sections grafted in 20 μl aliquots at 6 hour intervals for 1-15 days. After each 24-hour period of treatment, the treated skin is examined for general indications of psoriasis (condition of the skin or redness of inflammation). After day 15, the mouse skin is excised and biopsied to determine the number of epidermal cell layers and the relative degree of capillary dilation within the skin by microscopic inspection using standard histological techniques. The number of epidermal cell layers with normal human skin and untreated human psoriatic skin, similarly examined, that has been grafted to nude nude mice is compared to determine the effect of treatment with 5-fluorodeoxyuridine triphosphate and trifoephate 5- fluorouridine containing pharmaceutical compoeicionee. The relative degree of inflammation in the tissue is quantified by determining the amount of neutrophils and monocytes in the capillaries of the dermis under appropriate staining conditions. The relative degree of dilatation of the capilaree with untreated psoriatic skin and with normal control skin is compared and noted. The number of cell layers in the epidermis, the degree of inflammation and the degree of capillary dilation are determined and correlated with the days of treatment and the amount of active ingredient, determined from the concentration in the composition and the amount administered . Compositions having 5'-fluorodeoxyuridine triphosphate, 5-fluorouridine triphosphate or dream mono- or diphenates all show a significant reduction in psoriasis symptoms in relation to untreated malolarypal skin.

EXAMPLE 8: APPLICATION OF 2'-DESOXIRRIBGNUCLEOSIDE PURINE 7-DESASA TRIFOSPHATES THAT HAVE AN ANTIPROLIFERATIVE EFFECT ON HUMAN PSORIATELINE SKIN A human volunteer having patches of psoriatic skin in the forearm and treated with a cream composition including as an active ingredient a triethylammonium salt of 7'-triphosphate 7-dease-2'-deoxyinosine, synthesized essentially as described in Example 3. The active ingredient is a concentration of 50 g% in a pharmaceutically acceptable cream such as the polyethylene glycol cream described above in the description. The skin of both arms is examined macroscopically to determine the squamous condition and inflammation, and the results are recorded (read and write) before starting the treatment with the cream containing 5'-trifoefato de 7-desasa-2'-desoxiinoeina . One or two drops of cream are applied to the areas of the psoriatic skin approximately 3 cm in diameter on one of the forearms, at 6-hour intervals during the course of 14 days. The other forearm remains untreated during the 14-day period.

At daily intervals during the 14-day periods, the skin of both arms is examined macroscopically to determine the squamous condition and inflammation, and the results are recorded as in the above for bilateralee paired tests. If a sufficient number of psoriatic patches are available in the human volunteer, similar preparations containing triethylammonium salts of 7-desesia-2'-deoxydenosine 5'-triphosphate and 5'-desassane-2 'triphosphate are simultaneously tested. -deoxyguanosine at a concentration of 50 g%, to determine antipsoriatic activity. At the end of the 14-day period, a section of 1-2 mm in diameter is removed from the dermis and epidermis for biopsy and microscopic examination (to determine the number of cell layers in the epidermis, the degree of inflammation and the degree of of capillary dilatation in the dermie) as described in Example 7, using conventional and conventional procedures. The skin of the worsened skin of the untreated arm is taken as a negative control, of the normal skin of the untreated arm as a poetic control of the lae area treated with 7-desasapurin-2'-deoxyribonucleoside triphosphate containing creams for determination of efficacy of the active ingredients in relation to the control samples.

The macroscopic obervations of the bilaterally paired areas of treated psoriatic skin and psoriatic skin untreated in the same volunteer show that treatment with 2'-desoxirribonucleoeidoe triphosphate of 7-desasapurin significantly decreases psoriatic systems over a period of 14 days . The macroscopic observations are supported by the results of the microscopic examination of the skin taken from treated and untreated areas. Similarly, mono- and diphosphate also have activity.

EXAMPLE 9: APPLICATION OF PHOSPHATE ESTERS OF 2-CHLORODESOXIADENOSINE THAT HAVE ANTIPROLIFERATIVE EFFECT ON THE SKIN OF A MOUSE WITH ESCAMOSA SKIN Fsn / fsn mice are treated in dogs of 35-42 days of age, which show epidermal hyperplasia with inflammation, with a pharmaceutical composition containing as the active ingredient the mono-, di- or triphosphate ester of 2-chlorodeoxyadenoein, prepared essentially as described in Example 2. The compositions contain the active ingredient of mono-, di- or triphosphate at a concentration of 5 g%. The composition is applied in 5 μl aliquots at 12 hour intervals to a 1-5 mm portion of the ventral skin of the fsn / fsn mice, and it is treated in a manner similar to control fsn / fsn mice with the composition, but which lacks the active ingredient. The skin is generally observed microecopically at daily intervals that begin 12 hours after the first application and extend up to 15 days afterwards. Observations are used to determine the degree of epidermal hyperplasia and inflammation characteristic of psoriatic skin that occur during standard dermatological and histological methods. For each subsequent observation point in the treatment, the treated skin is compared to a similar portion of the same mouse which has not been treated, and with skin treated only with the carrier ingredients, if the mono-, di- or triphosphate ester of 2-chlorodeoxyadenosine. After a few days, a significant improvement in the inflammation and scaling condition of the skin is observed for the skin portions treated with compositions containing the mono-, di- or triphosphate ester of 2-chlorodeoxyadenosine as compared to the untreated skin. or the skin treated with the composition but lacking the active ingredients. They are performed in similar analyzes using fsn / fsn mice treated with compositions containing as an active ingredient the mono-, di- or triphosphate ester of 2-chloro-2'-fluoroadeoxydaenoein. A similar reduction in psoriatic symptoms is observed using compositions over a period of 14 days.

EXAMPLE 10: APPLICATION OF 3 '-AZAURIDINE THYPHOSPHATE WHICH HAS AN ANTIPROLIFERATIVE EFFECT ON HUMAN PSORIATELINE SKIN A human volunteer having psoriatic skin parts on the arms and legs is treated with a cream composition which includes as an active ingredient 6-azauridine triphosphate, synthesized essentially as described in Example 1. The active ingredient is at a concentration of 25 g% in a pharmaceutically acceptable cream such as a polyethylene glycol-based cream. The skin of the arms and legs was examined macroscopically to detect the flaky condition of inflammation, and the observations were recorded (in writing and with photographs) before starting treatment with the cream containing 6-azauridine triphosphate. Dosed aliquots of the cream, each containing 50 μl, were applied to areas of the psoriatic skin of approximately 5-10 cm in diameter in one of the spleens and one of the legs at 3-hour intervals during the course of 14 days . The other arm and leg remained untreated during the 14-day period to serve as negative controls. At daily intervals during the period of 14 days, the skin was examined macroscopically in the arms and legs to determine the squamous condition and inflammation, and the results were recorded as in the above for bilateral paired comparisons. At the end of the 14-day period, a section of 1-2 mm in diameter is removed from the skin (dermie and epidermie for biopsy and macroscopic examination to determine the number of cell layers in the epidermis, the degree of inflammation and the degree of capillary dilatation in the dermis, as described in Example 7 using standard histological procedures.The skin is taken from the psoriatic skin of the untreated arm and / or the leg as a negative control, from normal skin of an untreated area. as a poetic control, and from the areas that were treated with 6-azauridine triphosphate containing cream for determination of effectiveness of the active ingredient in relation to the control samples.The macroscopic observations of the bilaterally paired area of psoriatic skin treated and psoriatic skin not treated in the same volunteer show that treatment with 6-azauridine triphosphate significantly decreases the symptoms of psoriasis during the period of 14 days. The macroscopic obeervations are supported by the results of the microscopic examination of the skin taken from treated and untreated areas. Similar results are obtained with the 6-azauridine mono and diphosphate.

EXAMPLE 11: HUMAN PSORIATIC SKIN TREATMENT USING 2-CHLORODESOXIADENOSIN PHOSPHETHYLENE DIFOSPHONATE (2CdATMDP) Male human skin graft is used in a nude mouse to detect the antiproliferative effect of 2CdATMDP when applied topically. The macroscopic obervation and microecological cutaneous biopeia analysis were used to detect the efficacy of the antipsoriatic composition. Human psoriatic skin approximately 1-10 mm in diameter is grafted to the dorsal side of an athymic mouse using techniques well known in the art. After the graft has proven efficient, the human skin is treated with an anti-psoriatic drug substance containing the nucleoside analogue 2CdATMDP as an active ingredient, essentially as described in Example 4. The pharmaceutical composition includes the active ingredient in a topical polyethylene cream essentially as described in the foregoing in the description, with the active ingredient at a concentration of about 0.5 to 5 g%. The cream containing the active ingredient is applied to sections of skin grafted in 10 μl aliquot at a 12-hour interval for 1-30 days. The treated skin is examined for general indications of psoriasis (e.g., eucalyose condition and inflammation) once during each 24-hour treatment period. On the 15th and the 30th day, the skin is removed from the mouse grafted area and standard histological analysis is used to determine the amount of layers of epidermal cells in relative degree of dilatation of the capillaries with dermis of the skin. The number of epidermal cell layers is compared with normal human skin and untreated human psoriatic skin which has been grafted onto the mouse and atomic lens, examined in a similar manner, to determine the effect of the treatment with the pharmaceutical composition containing 2CdATMDP. In addition, the relative degree of capillary dilation quantifies tissue inflammation by determining the amount of neutrophilia and monocytoe in the capillaries of the dermis compared with untreated psoriatic skin and with normal control skin. The number of cell layers in the epidermis, the degree of inflammation and the degree of capillary dilation are correlated with the day of treatment in the amount of active ingredient (determined from the concentration in the composition and the amount administered). After the entire treatment period, a significant reduction in inflammation and squamous condition was observed in treated psoriatic grafted skin, compared with the untreated control psoriatic skin grafted to a nude nude mouse. A human volunteer with skin psoriatic patches on both forearms was similarly treated on a forearm twice daily (using 50 μl per treatment) of the pharmaceutical composition containing the nucleoside analog 2CdATMDP. The condition of the treated psoriatic skin compared to the control areas of the other forearm (untreated skin or skin treated with the cream formulation lacking the active ingredient), is observed daily and a skin biopsy of approximately 1-3 mm is taken on day 15 and on day 30 for standard hietological analysis as described in the above for the mouse model. After 15 days, less inflammation is observed in the skin treated with the composition containing 2CdATMDPcompared with the untreated skin or the skin treated only with the inert mixture of formulation without the active ingredient. After day 30, a significant change is observed both in the inflammation and in the skin condition treated with the composition having the active ingredient, compared with the skin control patches of the other forearm.

EXAMPLE 12: TREATMENT OF HUMAN ATOPIC DERMATITIS WITH 2-CHLORO-2'-FLUOROARADESOXIADENO PHOSPHATE ESTER The ethers of mono-, di- or triphosphate of 2-chloro-2'-fluoroaradeoxyadenosine are essentially synthesized as described in the US Pat. Example 2. An adolescent patient with atopic dermatitis of unknown origin and characterized by extreme itching and lesion of eczema on the wrists and neck is treated with a composition of notion containing a mixture of esters of mono-, di-, and triphosphate 2. -chloro-2'-fluoroaradesoxiadenosine, each at a concentration of 5 g%. The lotion is applied to affected areas of the skin at will up to 12 times per day in portions approximately 1-3 ce. After 1-4 weeks of treatment, the itching and inflammation of the skin with dermatitis decreases significantly in relation to the one presented by the patient at the beginning. In addition, after treatment, lesions of eczema on the treated skin have healed or healed. Subsequently, the lotion is applied at will only when the itch recurs.

EXAMPLE 13: TREATMENT OF FLAT LIQUEN DISEASE WITH DIDESOXINUCLEOSIDE PHOSPHATES The dideoxycytidine mono-, di- or triphosphate esters are synthesized essentially as described in Example 2. Each dideoxycytidine phosphate ester is formulated in a cream composition based on polyethylene glycol at a concentration of 0.5 g%. Human volunteers who have chronic lichen planus characterized by inflammation and pruritic skin rashes of papules in sue brazoe and piernae are divided into three groups and each group is treated with one of the compositions: the esters of mono-, di- or dideoxycytidine triphosphate. Each group is treated using the cream only on one leg or one arm, leaving the other leg or arm untreated as a bilateral control compared to the treated skin. The cream is applied in volumes of about 1 cc at intervals of about 6 hours for a period of 7 days to 3 months. The treated and untreated areas are examined visually every day during the week and at least once a week or as required (determined by the treating physician). As the inflammation and / or formation of papules seems to diminish, the doctor who performs the treatment also decreases the number of applications per day as required, to keep the disease under control. At the time when, the treated area, the inflammation seems to decrease in the amount of pruritic skin rashes seems to have decreased, a skin biopsy (approximately 1-3 mm) of the volunteer is taken in both the treated extremity and the extremity untreated in the affected area, for histological examination of both inflammation (for example dilation of capillaries of the dermis) and carcteríeticae of the papules (for example, number of layers of the epidermis and evidence of whitish lines or puncta aeociadae with the papules ). For each of the three groups some members of the group show significant improvement in the inflammation and pruritic skin rashes of the lichen planus papules on the skin of their treated arm or leg compared to the skin of the corresponding untreated limb. It will be understood by one familiar with the art that similar phosphate esters of other nucleoside phosphate analogous of the invention and compotetoe dideoxy including dideoxytimdine, dimethoxyguanosine, dimesoxiadenosine, dideoxyinosine and dideoxynucleoside analogs including AZT and d4T can be used similarly in compositions Pharmaceutical EXAMPLE 14: TREATMENT OF ATYPICAL KERATOSIS WITH ACICLOVIR PHOSPHATES Acyclovir triphosphate is synthesized essentially as described in Example 1 and a cream composition containing 0.1-0.5 g% acyclovir triphosphate and about 0.1 g% Azone ™ is formulated to improve skin penetration, using methods well known in the art. A group of middle-aged human volunteers with patches of actinic keratosis on the skin that has been exposed to the sun (for example in the nose, hands, and arms or leg) is divided into tree groups: a group in which actinic keratosis is frozen with nitrogen and liquid to deconstruct the cell, a group which was treated with the composition of acyclovir triphosphate and a control group which is treated with the composition containing inert ingredients and Azone "R but not containing acyclovir triphosphate. The groups treated with the cream compositions with or without acyclovir triphosphate are applied the cream twice a day (in the morning and at night) in sufficient volumes to cover the affected area (approximately 0.01-0.5 ce) during a period of The treated skin of each month is examined every third day by the doctor and the observations are recorded (in writing and with a photograph). volunteers every 6 months to determine the recurrence of actinic keratosis in the treated areas. For the group treated with the cream containing acyclovir triphosphate, a significant number of patients show decreased keratotic growth and decreased redness of the affected area at the end of the one-month treatment. Treatment may continue as determined by the doctor based on the verifications every 6 months. It will be understood by those familiar with the art that acyclovir mono, and diphosphate can also be used in similar pharmaceutical formulations and that ganciclovir mono-, di- and triphosphates or other antiproliferative nucleoside analogs of the invention can also be used in compositions that they have antiproliferative activity for skin diseases associated with hyperproliferation. For the group treated with the cream composition without acyclovir triphosphate, no change in actinic keratosis was observed after one month and the affected area was subsequently removed using clinically acceptable methods. For the group treated by freezing the keratotic area with liquid nitrogen, all show death of the cells in the treated area in the following 24 hours followed by detachment of the dead cells and associated pain during healing. In some cases there is itching in the treated area. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property:

Claims (22)

1. A method for treating diseases defined by cutaneous cell hyperproliferation, the method is characterized in that it comprises topically administering to the cutaneous or mucosal lesions of the disease a composition containing as an active ingredient a phosphate ester of nucleoside analogue having the formula : wherein: N is a nucleoside analog having antiproliferative activity; Z is O, S or NH; and n is 1, 2 or 3; or a pharmacologically acceptable salt thereof.

2. A method for treating diseases defined by cutaneous cell hyperproliferation, the method is characterized in that it comprises topically administering to cutaneous or mucosal recesses of the disease a composition containing as an active ingredient a phosphate ester of nucleoside analogue having the formula : wherein N is a nucleoside or nucleoide analog having antiproliferative activity; X is O, CH2 or S; Z ee 0, S or NH; and n ee 1 or 2; or a pharmacologically acceptable salt of the mole.

3. The method according to claim 1 or 2, characterized in that the nucleoside or nucleoside analogue is selected from the group consisting of cytosine arabinoside, guanosine arabinoside, 5-fluorodeoxyuridine, 5-fluorouridine, 6-azauridine, 2-chlorodeoxyadenosine, 2-Chloro-2'-fluoroaradeoxyadenosine, 2-fluoroaradeoxydenoein, 6-methylmercaptopurine, riboide, dideoxycytidine, dideoxythymidine, dideoxyguanosine, dideoxyinosine, dideoxydenoeine, 2'-deoxytubercidine, 2'-deoxyformicin, 2'-deoxy- (3,4-d) ) pyrimidine, acyclovir and ganciclovir.

4. The method according to claim 1 or 2, characterized in that the disease is selected from the group consisting of psoriasis, atopic dermatitis, lichen planus, actinic keratosis, basal cell carcinoma and eecamoeae cell carcinoma.

5. The method according to claim 4, characterized in that the disease is peoriaeis.

6. The method according to claim 1 or 2, characterized in that the active ingredient in the composition is in a concentration from 0.001 g% to 100 g%.

7. The method according to claim 1 or 2, characterized in that the active ingredient in the composition is in a concentration from 0.01 g% to 10 g%.

8. The method according to claim 1 or 2, characterized in that the active ingredient in the composition is in a concentration from 0.1 g% to 5 g%.

9. The method according to claim 1 or 2, characterized in that the composition is applied to an area of the skin from one to ten times daily in a dosage from 0.001 g% to 100 g% per application.

10. The method according to claim 1 or 2, characterized in that the composition is applied to an area of the skin from one to ten times daily in a dosage from 0.01 g% to 10 g% per application.

11. The method according to claim 1 6 2, characterized in that the composition is applied to an area of the skin from one to ten times daily in a dosage from 0.1 g% to 5 g% per application.

12. A pharmaceutical composition characterized in that it comprises as an active ingredient a phosphate ester of a nucleoside analogue having the formula wherein: N is a nucleoside analog having antiproliferative activity; Z is 0, S or NH; and n is 1, 2 or 3; or a pharmacologically acceptable salt thereof.

13. A pharmaceutical composition, characterized in that it comprises as an active ingredient a phosphate ester of a nucleoside analogue having the formula: wherein N is a nucleoside or nucleoside analogue having antiproliferative activity; X is 0, CH2 or S; z is 0, S or NH; and n is 1 or 2; or a pharmacologically acceptable salt thereof.

14. The pharmaceutical composition according to claim 10 or 11, characterized in that it additionally comprises propylene glycol, polyethylene glycol 400 and polyethylene glycol 3350.

15. The pharmaceutical composition according to claim 10 or 11, characterized in that it additionally comprises an ingredient for improving the penetration of the skin during topical application.

16. The pharmaceutical composition according to claim 10 or 11, characterized in that the nucleoside analogue is selected from the group consisting of cytosine arabinoside, guanosine arabinoside, 5'-fluorodeoxyuridine, 5-fluorouridine, 6-azauridine, 2-chlorodeoxyadenosine, 2-chloro-2'-fluoroaradeoxydenoein, 2-fluoroaradeoxyadenosine, 6-methylmercaptopurine riboside, dideoxycytidine, dideoxythymidine, dideoxyinosine, dideeoxiadenoein, 2'-deoxytubercidine, 2'-deoxyformicin, 2'-deoxy- (3, 4-d) pyrimidine , aciclovir and ganciclovir.

17. The pharmaceutical composition according to any of claims 10 to 14, characterized in that the active ingredient is in a concentration from 0.001 g% to 50 g%.

18. The pharmaceutical composition according to any of claims 10 to 14, characterized in that the active ingredient is in a concentration from 0.01 g% to 10 g%.

19. The pharmaceutical composition according to any of claims 10 to 14, characterized in that the active ingredient is in a concentration from 0.1 g% to 5 g%.

20. An antiproliferative nucleoside analogue phosphate ester, characterized in that it has the formula wherein N is an antiproliferative nucleoside analogue; Z is S or NH; X is C or S; and n is 1 or 2.

21. The compound according to claim 18, characterized in that N is 2-chloro-deoxyadenoein.

22. The composition according to claim 19, characterized in that Z ee NH, X ee CH2 and n is 2.