MXPA97009782A - Aciclovir derivatives for application top - Google Patents

Abstract

The present invention relates to compositions for topical application in infections caused by the herpes virus, which comprise phosphate esters of nucleoside analogues against herpes, such as acyclovir monophosphate, acyclovir diphosphate and acyclovir triphosphate, which show a greater activity against the natural strains of the herpes virus as well as against resistant strains, particularly negative strains for the enzyme thymidine kinase of the virus. Phosphate esters of nucleoside analogs include phosphorates and phosphothiarates, as well as polyphosphates having C-like bridges and

Description

ACICLOVTR DERIVATIVES FOR TOPICAL APPLICATION DESCRIPTION OF THE INVENTION Acyclovir (ACV) is an antiviral nucleoside analogous to guanosine, which contains a portion of unusual incomplete sugar (acyclic). Nucleoside analogs interrupt the process of DNA replication in cells and for that reason they are useful as antiviral and antineoplastic agents. ACV is particularly effective in the treatment of infections caused by herpes simplex viruses types I and II. Activity against ACV herpesvirus in cells occurs with low toxicity, since ACV is selectively phosphorylated by the HSV thymidine kinase enzyme, but not by the thymidine kinase of host cells. As a consequence, only cells infected with HSV can form ACV monophosphate (ACV-MP). Then, ACV-MP is anabolically transformed by cellular enzymes into ACV triphosphate, which is the active agent that interferes with viral replication (Fyfe, et al., J. Biol. Chem. 253: 8721-8727 (1978 ); Furman, P., et al., J. Virol. 32: 72-77 (1979)). Acyclovir herpes virus activity has been demonstrated by inhibiting the replication of the herpes simplex virus in cell cultures (O'Brien, REF: 26305 W., et al., Antimicrob Agents and Chemother, 34: 1178-1182 (1990 )); It has also been demonstrated in clinical studies, where patients infected with HSV were treated by oral administration of ACV (Whitley, R., Imbioliby and Prophylaxis of Human Herpesvirus Infections, C. Lopez et al., (eds) Plenum Press , NY 1990; and Straus, S., Sexually Transmitted Diseases 16 (2): 107-113 (1989)). Acyclovir is the treatment to be selected for mucosal and cutaneous HSV infections; although patients receiving topical therapy with acyclovir experience some reduction in their symptoms, healing is slow and incomplete (Spruance, S., et al., J. Infect. Dis. 146: 85-90 (1982); and Spruance, S., et al., Antimicrob, Agents Chemother, 25: 553-555 (1984)). The combination treatment using ACV with interferon for cultured cells infected with the herpes virus (O'Brien, W., et al., Antimicrob.Ampets and Chemother, 34 (6): 1178-1182 (1990)) or using ACV together with A1110U, which is an HSV inactivator, as topical therapy for herpetic keratitis in athymic mice (Lobe, D., et al., Antiviral Research 15: 87-100 (1991)), demonstrated synergistic activity against herpes virus I regarding the use of ACV alone. Acyclovir has been used with qualified success in clinical trials for the treatment of another viral disease, varicella (Whitley, R., et al., Immunobiology and Prophylaxis of Human Herpesvirus Infections, C. Lopez (ed.), Plenum Press, New York (1990) pp. 243-253). It has also been used experimentally, but without success, in the treatment of other disorders in which there is a hypothesis of a viral etiology, such as aplastic anemia (Gómez-Almaguer, D., et al., Amer. J. of Hematology 29 : 172-173 (1988)) and duodenal ulcer (Ruñe, SJ, et al., Gut 31: 151-152 (1990)). Acyclovir phosphates have been shown to be effective against wild-type or laboratory isolates of cultured cells infected with HSV-1, but have little or no efficacy against HSV thymidine kinase mutants under the same conditions (see data of Figures 1 and 2). In immunosuppressed patients, such as those suffering from infections caused by the HIV virus (AIDS) or in transplant recipients who are being treated with immunosuppressive drugs to prevent rejection of the transplant, the CVA has been chronically administered to prevent problematic herpes outbreaks . Such treatment provides a selective pressure that causes mutations in HSV thymidine kinase (90% frequency) as well as in DNA polymerase (10% frequency) which in turn produces viral strains resistant to ACV. There is no effective topical treatment for these herpes virus strains resistant to acyclovir. In accordance with the present inventionPhosphate esters of acyclovir and other phosphate esters of antiviral nucleoside analogues against herpes are provided, which are effective in the treatment of herpetic lesions in mucous membranes and cutaneous lesions caused by herpes virus infections. These agents, surprisingly, demonstrate antiviral activity against lesions caused by herpes virus strains defective in thymidine kinase, even when relatively inactive against these mutant viruses in cultured cells. The present invention also provides pharmaceutical formulations comprising an effective antiviral concentration of an acyclovir derivative, which may be acyclovir monophosphate, acyclovir diphosphate, acyclovir triphosphate, acyclovir glycerol monophosphate, acyclovir glycerol diphosphate, acyclovir morpholidate monophosphate, morpholidate acyclovir diphosphate, acyclovir isopropylidene glycerol monophosphate, acyclovir isopropylidene glycerol diphosphate, acyclovir phosphomethylene diphosphonate, or a mixture thereof, in a pharmaceutical carrier suitable for topical application. Other nucleosides against herpes simplex based on thymidine kinase phosphorylation will also exhibit increased activity when applied to the skin of infected patients in the form of their phosphate ester derivatives, in a suitable topical formulation. In accordance with another aspect of the present invention, there is provided a method for the topical treatment of a viral infection, comprising applying a formulation containing any of the acyclovir phosphate derivatives of the present invention, or a mixture thereof, to mucosal or cutaneous lesions of an animal infected with the virus, including humans or other mammals. In a preferred embodiment of the method, the animal is infected with a herpes virus. In a particularly preferred embodiment, the animal is infected with a herpes virus strain that is resistant to acyclovir. The herpes virus strain resistant to acyclovir may be a viral strain whose resistance to the antiviral agent is due to an alteration or defect in the thymidine kinase gene. In accordance with the present invention, at least one nucleoside analogue phosphate is used against herpes in the preparation of a medicament for the treatment of a mucosal or cutaneous viral infection. In a preferred embodiment, the nucleoside phosphate is a water-soluble salt. In another preferred embodiment, the viral infection is caused by the herpes simplex virus type 1 or type 2. In another aspect of the present invention, the phosphate esters of nucleoside analogues against herpes according to the present invention, are used together with a pharmaceutically acceptable vehicle, in the preparation of a medicament for the treatment of a mucosal or cutaneous viral infection. In a preferred aspect, the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous cream and a polyethylene glycol. The present invention also provides phosphate esters of nucleosides against herpes such as phosphoramidates and phosphothiorates of acyclovir, and polyphosphate esters of nucleoside analogs against herpes comprising bridging and methylene bridging groups. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the comparative effect of acyclovir and acyclovir monophosphate on the replication of herpes simplex virus in Wi38 fibroblasts.

Figure 2 illustrates the comparative effect of acyclovir and acyclovir monophosphate on the replication of HSV-DM.21 TK-mutant in vitro (TK = thymidine kinase). Figure 3 illustrates the comparative effect of the topical application of acyclovir and acyclovir phosphate esters on infections caused by strains resistant to HSV-1 acyclovir, of the TK-deficient type in HRS / J mice. Figure 4 illustrates the comparative effect of the topical application of acyclovir and acyclovir phosphate esters on infections caused by strains of HSV-1 resistant to acyclovir, of the TK-altered type in HRS / J mice. Figure 5 illustrates the comparative effect of the topical application of acyclovir and acyclovir monophosphate on infections caused by strains of HSV-1 resistant to wild-type acyclovir in HRS / J mice. Figure 6 illustrates the comparative effect of topical application of acyclovir and acyclovir monophosphate on infections caused by HSV-1 strains resistant to TK-altered acyclovir in HRS / J mice. The present invention provides acyclovir phosphate derivatives which demonstrate excellent topical activity against infections caused by the herpes simplex virus (HSV), particularly against the ACV-resistant mutants of HSV. Acyclovir is an analogue of the guanine base, which has a substituent group at position 9 and which has a cyclic sugar group from which the common name is derived. The chemical name of acyclovir is 9- (2-hydroxyethoxymethyl) -guanine, which has the following structure: The acyclovir phosphate derivatives of the present invention have a substituent R at the O-terminal location of the cyclic sugar group, as illustrated in the following Formula: wherein the substituents R are as follows: - imonof? "sfato -diphosphate -glicßrol • g glycerol monophosphate diphosphate -morfolidate -morfolidate monophosphate diphosphate tto ° -isopro? püidan -iaopropilidßn monofoafato da gboarol - * + difbsf! ato dß glicßr_ol_ The related triphosphate derivatives have the corresponding structures but including the additional phosphate. In accordance with the present invention, acyclovir monophosphate (ACV-MP), acyclovir diphosphate (ACV-DP), acyclovir triphosphate (ACV-TP), acyclovir glycerol monophosphate (ACV-MP-G), acyclovir glycerol diphosphate (ACV-DP-G), acyclovir morpholidate monophosphate (ACV-MP-morpholine), acyclovir morpholidate diphosphate (ACV-DP-morpholine), acyclovir phosphomethylene diphosphonate (ACV-PMDP), the acyclovir isopropylidene glycerol monophosphate (ACV-MP-isoP-G), the acyclovir isopropylidene glycerol diphosphate (ACV-DP-isoP-G), either alone or in combination, are prepared in a pharmaceutical formulation for suitable topical application and are applied to the skin lesions of an individual infected with HSV. The compounds ACV-MP, ACV-DP, ACV-TP, ACV-DP-G, ACV-PMDP, morpholine derivatives of acyclovir and the isopropylidene glycerol derivatives of acyclovir described above, are non-lipidium water soluble phosphate esters, and thus, they are preferably provided in topical water-based formulations. Surprisingly, it was discovered that the acyclovir phosphate esters, and it is expected that also the monophosphate and polyphosphate derivatives of other nucleosides, exhibit a higher topical activity against HSV. It was also demonstrated that the monophosphate, diphosphate and triphosphate salts, and the phosphomethylene diphosphonate derivatives of the nucleoside analogs, can be easily prepared and that such salts exhibit greater solubility in aqueous media, for example, creams, gels or other aqueous dispersions. . In addition, such salts are soluble in polyethylene glycol media, which provides a unique mucosal or cutaneous dispersion. Other polyphosphate ethers of nucleotide analogs that are useful in the methods of the present invention include the methylene polyphosphates of nucleoside analogs and polyphosphates with thio bonds of nucleoside analogs, as well as the mono- and poly-phosphoramidates and mono- and poly-phosphothiorates of nucleoside analogues. Similarly, monophosphates, diphosphates, and other phosphate esters of other nucleosides against herpes simplex will exhibit better topical activity than those described above. The following nucleosides against the herpes virus exhibit better activity in the form of phosphate esters: l-β-D-arabinofuranosyl-E-5- (2-bromovinyl) -uracil; 2'-fluorocarbocyclo-2'-deoxyguanosine; 6'-fluorocarbocyclo-2'-deoxyguanosine; 1- (β-D-arbinofuranosyl) -5 (E) - (2-iodovinyl) -uracil; (1-la, 2β, 3a) -2-amino-9- (2, 3-bis (hydroxymethyl) -cyclobutyl) -6H-purin-6-one; 9 - ((2- (1-Methylethoxy) -1- ((1-methylethoxy) -methyl) -ethoxy) -methyl) - (9C1) of 9H-purin-2-amine; trifluorothymidine; 9- [(1, 3-dihydroxy-2-propoxy) -methyl) -guanine; 5-ethyl-2'-deoxyuridine; E-5- (2-bromovinyl) -2'-deoxyuridine; 5- (2-chloroethyl) -2'-deoxyuridine; 1- (2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5-iodocytosine (FIAC); 1- (2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5-iododidine (FIAU); buciclovir; 6-deoxyacyclovir; 9- (4-hydroxy-3-hydroxymethylbut-1-yl) -guanine; E-5- (2-iodovinyl) -2'-deoxyuridine; 5-vinyl-l-β-D-arabinofuranosyl-uracil (V-araU); 1-β-D-arabinofuranosyl-thymine (ara-T); 2'-nor-2 'deoxyguanosine (2'NDG); 9- (4-hydroxy-3-hydroxymethylbut-1-yl) -guanine (penciclovir, BRL 3912); 1-ß-D-arabinofuranosyl-adenine (ara-A; vidarabine). The monophosphate, diphosphate triphosphate esters have the General Formula wherein N is a nucleoside analogue against the herpes simplex virus; Z is 0, S, or NH; and n is 1 or 2; or alternatively has the Formula wherein N is a nucleoside analogue against the herpes simplex virus; Z is 0, S or NH; X is 0, CH2 or S; and n is 1 or 2. According to the above, the phosphoesters can be phosphate, phosphothiorate or phosphoramidate and the diesters and triesters can have bridge atoms other than oxygen, for example, esters of 2,3-μ-thiotriphosphate, or 2, 3-μ-methylene diphosphonate. Contrary to expectations, these nucleoside analog phosphates can pass through the cell membrane of HSV-infected skin cells and reduce the viral replication rate by inhibiting HSV DNA polymerase. The mono- and di-phosphate nucleosides of the present invention are transformed into their corresponding triphosphates by cellular anabolic phosphorylation (s), but the triphosphate analogs inhibit the HSV DNA polymerase directly without the need to inhibit the cellular DNA polymerase. The present invention also provides pharmaceutical formulations of the nucleoside analogues mono-, di- and tri-phosphates in concentrations that can be applied topically to effectively reduce the proliferation of HSV in infected skin cells. Similarly, dideoxynucleoside DNA chain termination phosphates, when applied to the skin in a suitable topical formulation, will reduce HSV replication. These include acyclovir, ganciclovir, penciclovir, BVaraii, dideoxycytidine, dideoxythymidine, dideoxyguanosine, dideoxydenosine, dideoxyinosine, 3'-azidodidesoxythymidine, dideoxyhydrodideoxythymidine (d4T), and other dideoxynucleoside analogs such as those described in copending US Patent Application Number. Series 07 / 373,088. The salts of these compounds can be prepared easily and these must exhibit a greater solubility in aqueous media, for example in creams, gels or other aqueous dispersions. Typically, useful salts of these compounds include the sodium, potassium, lithium, ammonium or hydrogen salts. Any physiologically acceptable cation known to those skilled in the art can also be used. In addition, these salts are useful and effective in polyethylene glycol creams and lotions, which provide a favorable dispersion in mucous or in cutaneous applications. The various phosphate esters of these compounds can be prepared essentially as described below for acyclovir. Synthesis of Acyclovir Phosphate Esters: The present invention provides methods for the synthesis of mono- and di-phosphates of acyclovir, morpholidate monophosphates of acyclovir, glycerol monophosphates and glycerol diphosphates of acyclovir, and 1,2-isopropylidene glycerol monophosphates and diphosphates of acyclovir . Acyclovir monophosphate can be prepared from acyclovir according to the method of Yoshika a, M., et al. , Bull. Chem. Soc. Japan 42: 3505-3508 (1969) modified by the method of Toorchen, D. and Topal, M., Carcinogenesis 4: 1591-1597 (1983) and exemplified in Example 1. Acyclovir diphosphate is can prepare, in the same way as other nucleoside analogues, by the method of Ott, D. G., et al. , Anal. Biochem. 21: 469-472 (1967), • using either tributylammonium phosphate or tributylammonium pyrophosphate as a phosphate donor. The methods for the preparation of acyclovir glycerol diphosphate are presented in Examples 2 to 4. In general, glycerol phosphates of nucleosides are prepared in a manner similar to the preparation of the phosphatidyl nucleosides. In the approach described in Example 3, acyclovir phosphate is activated by the addition of a leaving group, for example, morpholine, according to Example 2, and is condensed with the glycerol-3-dicyclohexylammonium phosphate salt in the presence of of N, N'-dicyclohexylcarbodiimide (DCC). Alternatively, as described in Example 4, the glycerol phosphate, having the reactive hydroxyl groups protected by an isopropylidene moiety, is activated by the addition of morpholidate and then condensed with the acyclovir monophosphate under the conditions described in Example 2 A number of acyclovir diphosphate diglycerides (ACV-DP-DG) containing several acyl chains has been prepared by the condensation of the appropriate diacylphosphatidic acid morpholidate (PA-morfolidate) and acyclovir monophosphate (ACV-MP). A method by which the procedure can be carried out is described by Agranoff, B. and Suomi, W., Biochem. Prep. 10: 47-51 (1963). Alternatively, the nucleoside monophosphate morpholidate is prepared and condensed with phosphatidic acid in the manner described in U.S. Patent Application Serial No. 07 / 706,873, entitled "Liponucleotide Synthesis" and by British Patent Application No. 2,168,350 of Hong et al. The above chemical methods are generally described in terms of their general application for the preparation of the compounds of the present invention. Occasionally, the reaction may not be applicable in the manner described for each compound included within the scope of the present. The compounds for which this occurs will be easily recognized by those skilled in the art. In all these cases, the reactions can either be carried out successfully by the conventional modifications known to those skilled in the art, for example by means of adequate protection of the interfering groups changing to conventional alternative reagents, or, by the routine modification of the reaction conditions. Alternatively, other reactions described herein or conventional in some other manner will be applicable for the preparation of the corresponding compounds of the present invention. In all preparation methods, all raw materials are known or easily prepared from known raw materials. Unless otherwise indicated, all parts and percentages are given by weight. Synthesis of Monophosphates, Diphosphates and Triphosphates of Nucleosides and Nucleoside Phosphate Analogs. Methods for synthesizing nucleoside monophosphates by reaction of the nucleoside with phosphorus oxychloride are described in copending US Patent Applications Serial Nos. 07 / 879,088 and 08 / 060,258; and in the manner previously described (Yoshikawa et al., Bull. Chem. Soc. Japan 42: 3505-3508, 1969; Toorchen, D. and Topal, M., Carcinogenesis 4: 1591-1597, 1993). The nucleoside diphosphates are prepared by the method 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 Acids Res. 20: 55-61, 1992), or from nucleoside monophosphates by the method of Moffat and Khorana (J. Am. Chem. Soc. 83: 663, 1991) or by the method of Hoard and Ott (J. Am. Chem. Soc. 87: 1785-1788, 1963). The following Examples present the details of some useful syntheses for preparing phosphate esters of nucleosides and nucleoside analogues. Other nucleoside phosphate analogs including the nucleoside phosphorothioates, nucleoside phosphoroamidates, nucleoside phosphonates and nucleoside phosphorofluoridates can be synthesized using methods known to those skilled in the art and summarized, for example, in D. Hutchinson (The synthesis, reactions and properties of nucleoside mono-, di-, tri-, and tetraphosphates and nucleotides with changes in the phosphoryl residue.) In Chemistry of Nucleotides and Nucleosides, L. Townsend, ed., 1991, pp. 81-146 and references therein). The common syntheses are summarized as follows: (1) Nucleoside phosphorothioates are nucleoside analogues in which one or more of the phosphoryl oxygen atoms have been replaced by sulfur. The first synthesis methods reacted a protected nucleoside with sulfur tris (l-imidazolyl) phosphine, while the more recent syntheses replaced the latter agent with thiophosphoryl chloride (PSCI3). A nucleoside phosphoramylidate can be converted to phosphorothioate by treatment with sodium hydroxide and carbon disulfide. The 5'-nucleoside phosphorothioates can be obtained from the direct sulfurization of 5'-nucleoside phosphites. The 2 '(3') purine nucleoside rhosphorothioates can be synthesized by reacting their 2 ', 3'-0-di-n-butylstanylene derivatives with thiophosphoryl chloride, followed by alkaline hydrolysis. (2) The nucleoside phosphoramidates are analogs in which one or more of the phosphoryl oxygen atoms have been replaced by nitrogen, creating a PN bond which is considerably weaker than the PS bond of the nucleoside phosphorothioates, even in moderately acidic conditions. Syntheses of these compounds include the phosphorylation of aminonucleosides and the treatment of nucleoside azides with phosphorous acid triesters. The lipophilic nucleoside phosphoroamidates can be particularly useful HSV compounds due to their ability to be more easily incorporated by the cells, where they are hydrolyzed to obtain biologically active compounds. (3) Nucleoside phosphonates are compounds in which an oxygen of the phosphoryl is replaced by carbon, creating a stable bond PC and decreasing the acidity of the P-OH groups when the phosphorus atom is replaced by an electron-donating alkyl group , instead of oxygen. Nucleoside phosphonates are readily prepared from the nucleoside halides by those skilled in the art, using either the Arbusov reaction or the Michaels-Becker reaction. The 5'-nucleoside phosphonates can be synthesized from 5'-iodo-5'-deoxynucleosides protected at the 2 ', 3' positions using methods known to those skilled in the art. The 5'-nucleoside phosphonates, in which the 5'-oxygen is replaced by a methylene group, are synthesized by coupling a 5'-suitably protected nucleoside aldehyde with a triphenylphosphoroanilidene diphenyl phosphonate, to obtain a phosphonate diester a , ß-unsaturated, which is subsequently reduced and deprotected in the phosphoryl residue, to obtain the phosphonate. The 3'-isoesteric nucleoside phosphonates are synthesized from 1-phosphorylated ribose chloride, which is coupled with the heavy metal salt of a purine or a pyridine. Phosphonates are generally less polar than their phosphate counterparts and, therefore, are useful as agents with HSV because they are more easily incorporated by cells when applied topically. (4) The nucleoside phosphorofluoridates are analogs of the mononucleotides. The treatment of nucleoside 5'-phosphates with 2,4-dimitrofluorobenzene produces the nucleoside 5'-phosphorofluorohydrates via the 2, 4-dinitrophenylester of the nucleotide. (5) Other analogs of nucleoside polyphosphates include those in which other atoms other than oxygen are substituted between the α, β-phosphorus atoms in diphosphates and nucleoside triphosphates, or between the β, β-phosphorus atoms in the nucleoside triphosphates (including those listed in Table III on page 119 of DW Hutchinson, in Chemistry of Nucleotides and Nucleosides, L. Townsend, ed., 1991). Typically, a, β-analogs are prepared by condensing a 2 ', 3' -O-protected nucleoside with the pyrophosphate analog, with the aid of DCC or by nucleophilic displacement reactions involving the displacement of a toluene residue. sulfonyl (tosyl) from the 5 'position of the sugar residue of the tosyl nucleoside, by a methylene biphosphonate ion. The acyclovir derivatives of the present invention, comprising ACV-MP, ACV-DP, ACV-TP, ACV-MP-glycerol, ACV-DP-glycerol, ACV-MP-isopropylidene glycerol, ACV-DP-isopropylidene glycerol and ACV -P-methylene diphosphonate was found to have particular efficacy in topical treatment of herpetic lesions caused by HSV-1 infections resistant to acyclovir. Example 7 demonstrates that infection of cells grown with wild-type isolates and HSV laboratory strains can be treated with equal success using acyclovir, acyclovir monophosphate (Example 7).; Figure 1) . For these viral infections in Wi38 fibroblasts, both acyclovir and acyclovir monophosphate have the same IC50 of approximately 1 6 2 μM concentration. However, when the same cell culture system is infected with a virus strain resistant to acyclovir, the HSV-DM.21 strain, which lacks the thymidine kinase necessary to transform acyclovir into acyclovir monophosphate, is acyclovir and the monophosphate of acyclovir are not effective in reducing the number of viral plaques (Example 7, Figure 2). The efficacy of acyclovir phosphate esters with respect to cutaneous infections by aciclovir resistant HSV-1 is surprising in view of the above in vitro cell culture data. The acyclovir phosphate esters applied in an aqueous cream to the herpetic lesions of mice infected with acyclovir-resistant HSV-1 were substantially more effective than natural acyclovir in reducing the number of such lesions (Example 9; Figures 3 and 4) . Accordingly, in view of these results, it is believed that the in vitro incorporation of acyclovir and acyclovir phosphates proceeds through a different mode of operation than the lotion applied topically in vivo. Topical Formulations of Nucleoside Analog Phosphates. The nucleoside analog derivatives of the present invention, as previously described, can be prepared for topical use by incorporating them into a variety of formulations known to those skilled in the art, which are useful and convenient for dermatological use. The nucleoside analogues are water-soluble and, accordingly, can be in aqueous solution, in oil-in-water emulsion or in an aqueous cream, and are the most preferred formulations. The water solubility of acyclovir and other nucleoside monophosphates can be increased through the preparation of salts, such as sodium, potassium, ammonium or hydrogen salts. In a particularly preferred formulation, 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 formulations, since it can be selected to improve penetration, to prolong the duration of the activity or to meet requirements of the application site. For example, a formulation for application to the corpus calloses of the body, such as the palms of the hands or soles of the feet, may include a penetration enhancing agent such as propylene glycol dimethyl sulfoxide or Azone®; A powder formulation can be selected to be applied to areas of the intertrigo such as the crotch, the elbows or between the fingers of the hands or between the toes. The formulation can also be prepared to contain various organic polymers or other compositions known to those skilled in the art, to obtain a sustained release of the active anticyclic acyclovir derivatives. A multitude of appropriate topical formulations can be found in the form known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, 15th edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042 (Chapter 87: Blaug, Seymour). These formulations include, for example, powders, pastes, ointments, gelatins, waxes, oils, lipids, anhydrous absorption bases, oil-in-water or water-in-oil emulsions, carbowax emulsions (polyethylene glycols of a variety of molecular weights), gels. semi-solids and mixtures are solid contain carbowax. The concentration of the active ingredient in the topical application formulations of the present invention can be found in a concentration of about 0.01 g% to 100 g%; preferably from about 0.1 to 50 g%; more preferably from about 1 g% to about 15 g%. In addition, the formulations may comprise effective concentrations of other agents that help promote skin penetration and healing, as described in the aforementioned form and which are well known to those skilled in the art. The efficacy of the topical formulations containing the active phosphate esters of the present invention can be evaluated using conventional test procedures known to those skilled in the art. For example, a particularly expedient method is the murine "orofacial model," such as that described by Ellis, M., et al. , Antimicrobial Agents and Chemotherapy 33: 304-310 (1989). In this test system, the pathogenesis of HSV in mice inoculated by scarification in the snout has been shown to be a reasonable model of the disease cycle of cutaneous HSV infection in immunocompromised hosts. The formulations can be applied to the herpetic lesions of the affected skin repeatedly; for example once, twice or several times a day and the treatment can be extended for several days until the healing is achieved. The risk of toxicity and irritation incidence is minimal. EXAMPLE 1 Phosphorylation of Acyclovir Nucleoside Derivatives Acyclovir nucleoside derivatives were prepared by the following methods. Unprotected acyclovir was reacted with POCI3 in trimethyl phosphate ((CH3?) 3PO) essentially in the manner described by Yoshikawa et al. , Tetrahedron Letters 50: 5065-5068 (1967); and Yoshikawa, M., Kato et al. , Bull Chem. Soc, Japan, 42: 3205-3208 (1967). To a cold (0 ° C) solution of 2 M POCI3 in 300-400 ml of trimethyl phosphate, acyclovir (1 M) was added dropwise with stirring, maintaining the reaction temperature between 0 ° and 5 ° C. The reaction process was monitored by CLAR using an Mono Q HR 5/5 anion exchange column (Pharmacia, üppsala, Sweden). Typically, 5 μl of the reaction mixture was neutralized with aqueous sodium hydroxide (final pH 7) and injected into the column. The elution was carried out as follows: washing with water, elution with 0.1 M ammonium carbonate, NH4HCO3, which elutes the acyclovir monophosphate, followed by a linear gradient of NH4HCO3 from 0.1 to 0.6 M, which elutes some phosphorylated products superiors The reaction was almost concluded in a period of 45 to 75 minutes, determined by this method, and the product of the reaction was hydrolyzed and neutralized with two volumes of aqueous sodium hydroxide to a final pH of 7. The purification of the product was carried out in the manner described above for the analysis of the reaction mixture. By this method, 0.8 moles of acyclovir monophosphate was prepared and purified with a fast flowing Q Sepharose column using the same elution conditions. Yields varied between 80 and 96% after repeated lyophilisations in water.

Thin-layer chromatography (TLC) analysis (silica plates 60 / F254, Merck) showed a single Pi stain positive in Ü.V., using the l-propanol / NH3 25% / H0 development system (20:20 : 3 in volume). EXAMPLE 2 Preparation of Acyclovir Monofosfo orfolidate Acyclovir monophosphate (5 mmol) and morpholine (20 mmol) were suspended in t-butanol (50 ml) and heated under gentle reflux while adding N, N'-dicyclohexylcarbodiimide (DCC, 20 mmol) dissolved in t-butanol (50 mmol), dropwise over a period of 1 hour. The mixture was stirred at reflux for 12 to 36 hours and evaporated to dryness. The residue was triturated with ether and washed by decantation with the same solvent. The product was purified by recrystallization from a methanol-ether mixture. EXAMPLE 3 Preparation of an-glycol-3-difoafoaciclovir a Starting from acyclovir monophosphomorpholidate Acyclovir monophosphomorpholidate (2 mmol), prepared in the manner described in Example 2, was dissolved in anhydrous pyridine (20 ml) and evaporated to dryness in vacuo. The process of dissolving the residue in pyridine and evaporation was repeated three additional times to remove traces of water from the compound. The dimethylcyclohexylammonium glycerol-3-phosphate salt (3 mmol) was added to the dehydrated residue and the mixture was dissolved in 20 ml of pyridine and stirred under an inert atmosphere at 60 ° C for 12 to 36 hours. The solvent was evaporated in vacuo and the residue was triturated with ether and the resulting solid was purified by ion exchange chromatography on a DEAE sephadex column (2.5 cm x 30 cm) using a linear gradient of ammonium bicarbonate (from 10 to 300 mmol , 500 ml each). The fractions containing the pure product (identified by TLC and analytical HPLC) were mixed and lyophilized to obtain the title compound. EXAMPLE 4 Preparation of sn-glycero-3-difosfoaciclovir from 1, 2-isopropylidene-sn-qlycero-3-monofoafomorfolidato A. Preparation of 1,2-isopropylidene-sn-qlycero-3-fos ate Oxychloride was added by drip of phosphorus (25 mmol), over a period of 30 minutes, to a mixture of 1,2-isopropylidene-glycerol (20 mmol) (Sigma, St. Louis, MO.) and triethylamine (100 mmol) cooled to 0 ° C. . After stirring the mixture at 0 ° C for 10 to 90 minutes, water (1 ml) was added to stop the reaction. Subsequently, the mixture was dissolved in chloroform (500 ml) and washed with water (3 x 100 ml). The water wash solutions were combined and the combined was extracted with chloroform (50 ml) and lyophilized. The product was used immediately for subsequent reactions without further purification. B. Preparation of 1,2-isoproiolidene-glycero-3-raonophosphomololidate The 1,2-isopropylidene-S2_-glycero-3-phosphate, prepared in the manner described in (A), was condensed with morpholine to prepare 1 , 2-isopropylidene-sn-glycero-3-monosphosphomorpholide, in accordance with the process described for the preparation of acyclovir phosphomorpholidate of Example 2. The reaction of intermediate compound 1,2-isopropylidensn-glycero-3-monophosphomorbide with acyclovir monophosphate under the conditions described in Example 2, yielded the compound 1,2-isopropylidene-sn-glycero-3-monophosphomorbide. 1, 2-isopropylidene-sn-glycero-3-diphospho-acyclovir (1 mmol) was dissolved in an aqueous 50-90% acetic acid solution and stirred at room temperature for a period of 4 to 12 hours and the glycerol The crude 3-diphosphocyclovir was purified in the manner described above.

EXAMPLE 5 Preparation of sn-glycero-3-phospho-acyclovir 1 mM L-2-isopropylidene-sn-glycero-3-phosphate (prepared as in Example 4B) and acyclovir (1 mM) were suspended in anhydrous pyridine (10 mM). ml) and DCC (4 mmol). Dissolved pyridine (4 ml) was added and the mixture was stirred at a temperature of 25 ° to 60 ° C for 12 to 72 hours. The solvent was evaporated and the residue was triturated with ether. The crude product was purified by ion exchange chromatography in the manner described in Example 3. Then, the isopropylidene protecting group was removed from the product by a treatment with aqueous acetic acid, to obtain the title compound. Alternatively, the title compound was also prepared using 2,4,6-triisopropylbenzenesulfonyl chloride (TPS-C1) as a condensing agent. EXAMPLE 6 Preparation of Phosphate Ester Mixture of Acyclovir by Alkaline Hydrolysis of Dipalmitoglycerol diphosphate of Acyclovir Dipalmitoglycerol diphosphate of acyclovir (1 mmol) was dissolved in chloroform, to which was added methanolic sodium hydroxide (2.1 mmol). The reaction was carried out for 20 to 90 minutes and the progress was monitored by CCF. Upon completion of the reaction, it was added Dowex-50 X-2 (H +) to the reaction mixture to adjust the pH to 7. The resin was separated by filtration and the filtrate was lyophilized and the crude product was purified in the manner described in Example 1. EXAMPLE 7 Preparation Nucleoside Triphosphates from Mbnonucleosides The preparation of 5 '-triphosphates from deoxyribonucleotides, dideoxyribonucleotides and analogs, involves a series of reactions that are indicated below. m 91 * vr The synthesis of nucleotide analogue monophosphate is described in the North American Patent Application Serial Number 08 / 060,258, filed on May 12, 1993, and in Example 1. Other methods are the following: A nucleotide (I) and an excess of 1,1'-carbonyldiimidazole (II) are reacted for about 1 hour at room temperature, to form an imidazlitol (III) . The 1, 1-carbonyldiimidazole that did not react is decomposed with methanol before adding an excess of inorganic pyrophosphate (IV). This eliminates the formation of inorganic polyphosphates, which would have to be subsequently removed from the reaction materials. The phosphorylation is allowed to proceed to completion in about 24 hours after the addition of the inorganic polyphosphate (IV) and then the product nucleoside triphosphate (V) is purified by anion exchange chromatography on a DEAE-cellulose column, followed by transformation of the product in a salt, such as the sodium salt. Because the nucleotide (I) and the imidazolidate (III) can be reacted together to form a by-product of symmetric pyrophosphate, the anion exchange chromatography on DEAE-cellulose is carried out at a lower pH, where the desired product (V ) has less charge than the undesirable byproduct, thus allowing separation of the two compounds. A reagent used in the synthesis is tributylammonium pyrophosphate, which is prepared by the following procedure. To an aqueous solution of pyridinium pyrophosphate, obtained by passing a solution of tetrasodium pyrophosphate decahydrate (446 mg 1 mmol) through a column of Dowex 50W-X4® (pyridinium) (17 ml), tributylamine (0.24 ml) is added. , 1 mmol). The solution is concentrated in vacuo and the residue is dehydrated by the consecutive addition and evaporation of anhydrous pyridine, followed by the addition and evaporation of two 10 ml portions of N, N-dimethylformamide (DMF). The synthesis of nucleoside triphosphates is achieved by the following method. To a solution or suspension of mononucleotide (0.1 mmol) in the form of the anhydrous tributylammonium salt, in 1 ml of DMF, is added 1, 1'-carbonyldiimidazole (80 mg, 0.5 mmol) in 1 ml of DMF. The mixture is mixed for 30 minutes and then kept in a desiccator at room temperature for 4 to 12 hours, before it is treated with 33 μl (0.8 mmol) of methanol and left to react for 30 minutes at room temperature. Tributylammonium pyrophosphate (0.5 mmol) is added in 5 ml of DMF and mixed vigorously, then the mixture is placed in a desiccator at room temperature for approximately 24 hours to allow the imidazolium pyrophosphate to precipitate. The precipitate is removed and washed with four 1 ml portions of DMF by centrifugation and resuspension in DMF, obtaining approximately 80 to 100% purity. The supernatant is treated with an equal volume of methanol and evaporated to dryness in vacuo. The residue is subjected to chromatography on a 2 x 20 cm column of DEAE-cellulose, with a linear gradient of triethylammonium bicarbonate (a gradient of 3 1 of about 0 to 0.4 M at a pH of 5 to 7.5) and the fractions were collected and evaluated spectrophotometrically to identify the fractions containing the nucleoside triphosphates. The appropriate fractions are evaporated in vacuo and the triethylammonium nucleoside triphosphate is dissolved in methanol at a concentration of approximately 0.05 M and five volumes of an acetone solution of sodium perchlorate (15 equivalents) are added., to form a precipitate of the sodium salt of the nucleoside triphosphate. Those skilled in the art will understand that other salts of the nucleoside triphosphates can be prepared by appropriate precipitation reactions. The precipitated salt is collected by centrifugation, washed with four 1 ml portions of acetone and dried under vacuum over phosphorus pentoxide. Additional procedures are available for the synthesis of nucleoside triphosphates, including that presented in the following Example. EXAMPLE 8 Synthesis of Nucleoside Monophosphates, Diphosphates and Triphosphates Using 2,2,2-tribromoethyl Phosphoromorpholino-Chlorhidate Using essentially the method of van Boom, et al. , (Tetrahedron Lett 32: 2779-2782, 1975), monophosphates, diphosphates and triphosphates of ribonucleosides and their derivatives were prepared from a single intermediate. In general, reactions include reacting a monofunctional reagent (2,2, 2-tribromoethyl phosphoromorpholinochloridate) with a ribonucleoside (or its derivative) to prepare phosphotriester derivatives with a 2,2,2-tribromoethyl protecting group attached to the ribonucleoside ( for example, to produce 5'-ribonucleoside phosphomorpolidates or 5'-phosphomorphollicide derivatives of ribonucleosides). The protecting group is removed by a coupling reaction with Cu / Zn with acid deblocking and neutralization, to produce the monophosphates, diphosphates and triphosphates, depending on the acid used in the deblocking step and the ammonium salt used in the neutralization step. That is, to obtain the ribonucleoside monophosphate, HCl and ammonia are used; to obtain ribonucleoside diphosphate, mono (tri-n-butylammonium) salt of phosphoric acid is used; to obtain the ribonucleoside triphosphate, bis (tri-n-butylammonium) pyrophosphate is used. The general reactions are outlined below: VI The monofunctional reagent (I) 2,2,2-tribromoethyl phosphomorpholinochloridate is prepared by reacting 2,2,2-tribromoethyl phosphodichloridate with morpholine in anhydrous ether, from this reaction the product is removed and recrystallized using cyclohexane / n -pentane, by the methods known in the art. The crystalline 2,2,2-tribromoethyl phosphomorpholinochloridate has a m.p. of 79 ° C. The monofunctional reagent (2 mmol) is mixed with 1 mmol of the nucleoside or its derivatives, in anhydrous pyridine at 20 ° C for 48 hours; then, the reaction mixture is fractionated by chromatography (B. J. Hunt &W. Rigby, Chem. &Ind. 1868, 1967), to obtain the nucleotides (III) as colorless solids. The treatment of nucleotides with Cu / Zn in DMF during minutes at 20 ° C, followed by filtration to remove excess Cu / Zn, yields the nucleoside phosphomorpolidates. Subsequently, the nucleoside phosphomorpolidates are treated with different acids and ammonia sources to obtain the monophosphates, diphosphates or triphosphates. For the monophosphate, the phosphomorpholide is treated with 0.01 N HCl, pH 2 for 2 hours at 20 ° C and then neutralized with aqueous ammonia (pH 9) and purified on a Sephadex G-25® column. Similarly, the 5'-triphosphate is obtained by reacting the phosphomorpholidate (0.1 mmol) in 2 ml of anhydrous DMF, with 0.5 mmoles of bis (tri-n-butylammonium) pyrophosphate in 2 ml of anhydrous DMF at 20 ° C. for 3 hours, under conditions that exclude moisture. The product of the reaction is concentrated in vacuo, treated with Dowex 50® (ammonium form) and purified on a 2 x 25 cm column of DEAE-cellulose, using a linear gradient of 3 1 of a solution of Et3NH2C03 from 0.0 to 0.3 M. The 5'-nucleoside diphosphate is obtained by reacting the phosphomorpholidate (0.1 mmole) with 0.6 mmole of (tri-n-butylammonium) monophosphate in 4 ml of anhydrous pyridine at 20 ° C for 3 hours , under conditions that exclude moisture, and. The product of the reaction is concentrated and purified in a similar manner. Alternatively, the phosphotriester derivatives (III) can be directly transformed into the corresponding nucleoside diphosphates, by treatment with Zn powder in pyridine solution containing (tri-n-butylammonium) monophosphate. That is, 1 mmol of the reagent (III) is added to a stirring solution of 15 ml of anhydrous pyridine containing 0.1 g of finely powdered Zn and 12 mmo of (tri-n-butylammonium) monophosphate, under conditions that exclude moisture , for 48 hours at 20 ° C. The reaction mixture is then centrifuged to package Zn on the bottom and the supernatant is coevaporated three times with 15 ml of water each time, before being purified on a DEAE-cellulose column. EXAMPLE 9 Synthesis of Acyclovir Triphosphates (1) Synthesis of the triethylammonium salt of 5'-acyclovir triphosphate. Acyclovir (25 mg, 0.1 mmol) was dissolved in trimethyl phosphate (250 μL, 1.07 mmol) and POCI3 (18.5 μL, 0.2 mmol) was added. The mixture was stirred for 1.5 hours 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-n-butylamine was added, with vigorous stirring in a period of 1 minute; the solution was neutralized with an aqueous solution of 3 M 3 Et 3 NH 2 C, pH 7, and evaporated to dryness in vacuo. The residue was purified on a 2.6 x 30 cm column of DEAE-sephadex using a linear gradient of Et 3 H 2 C 3, pH 7 (11 H 2 O / II TBK 0.7 M), to obtain a colorless solid which has a maximum absorbance of UV (H2O)? Max 258 nm. EXAMPLE 10 Synthesis of Acyclovir Phosphomethylene Diphosphonate The synthesis is essentially the same as that described in Method 1 of Myers et al. , (J. Am. Chem Soc. 85: 3292-3295, 1963). A 5'-nucleoside phosphoramidate was reacted with methylene diphosphonic acid to produce the phosphonic acid analogs of the nucleoside pyrophosphate.

Alternatively, using Method 2 of Myers et al. , (id.), a nucleoside monophosphate is reacted with methylene diphosphonic acid, using dicyclohexylcarbodiimide (DCC) as a condensing agent. According to method 1, methylene diphosphonic acid is obtained by hydrolysis in concentrated HCl of its corresponding tetraethyl ester, which is prepared by the reaction of methylene iodide with an excess of triethylphosphite. The 1,3'-phosphoramidate of 1,3-dicyclohexylguanidinium of acyclovir (3.6 mmol) and methylene diphosphonic acid (10.8 mmol) were treated with 54 ml of freshly distilled o-chlorophenol; The mixture was cooled on ice and 36 ml of anhydrous pyridine were added. This resulting solution was allowed to stand at room temperature with occasional stirring for 48 hours, after which 300 ml of water was added with the reaction mixture under ice. The solution was extracted six times with ether (total 850 ml). The aqueous solution was adjusted to pH 2 with 1 N HCl and then treated with 30 g of acid washed carbon (Norit A) and stirred for 30 minutes; Subsequently, the coal was collected by filtration and washed exhaustively with water (5 1 total volume). The acyclovir derivative was eluted with a 50% aqueous mixture of concentrated ethanol-5% ammonium hydroxide (3 total liters) and the eluate was concentrated to a volume of 400 ml by evaporation at 35 ° C. The concentrated eluate was applied to a 2.7 cm x 31 cm column of Dowex-2® (chloride, 8% cross-linking) and eluted with a linear gradient prepared by mixing 2 1 HCl 0.003 N (in the reaction vessel) and 2 1 HCl 0.003 N plus LiCl 0.45 N (in the receptacle); 10 ml fractions were collected and fractions containing acyclovir methylene diphosphate were identified using paper chromatography or UV absorbance, using methods known in the art. The fraction containing the acyclovir methylene diphosphonate was neutralized with 1 N LiOH and concentrated by evaporation at 30 ° C. The concentrated solution was treated with 250 ml of 10% acetone-methanol to precipitate a solid, which was separated by centrifugation and washed with 10% acetone-methanol, until no chloride was detected in the washings. The lithium salt of acyclovir methylene diphosphate can be further purified by dissolving it in 100 ml of water adjusted to a pH of 8 with LiOH, and chromatographing the solution through a Dowex-2® column in the manner described above, using a gradient of a mixture of 1.5 1 of 0.003 N HCl in the mixing chamber with 1.5 1 of 0.003 N HCl plus 0.45 N LiCl in the receptacle and treating the eluate in the manner described above, and then the precipitate was dissolved in 6 ml of water and was re-precipitated with 40 ml of methanol. The final precipitate was dissolved in 15 ml of water and lyophilized to obtain a methylene diphosphonate powder of acyclovir tetralithium. Using Method 2, methylenediphosphonic acid (11.4 mmol) and acyclovir monophosphate were dissolved. (2.6 mmol) in pyridine (30 ml) and water (4 ml), to produce a two phase mixture to which DCC was added at room temperature with vigorous stirring, in three aliquots (29 mmol at the start of the reaction; 38 mmol after 4 hours and 19 mmol after 12 hours). After 24 hours, the reaction was complete and the precipitated dicyclohexylurea was filtered and washed with water.

The filtrate and the washings were adjusted to a total volume of 150 ml with water and extracted five times with ether (300 ml of total volume). The solution was adjusted to pH 8 and subjected to chromatography on a 2.5 x 17.5 cm column of Dowex-1® (format, 2% cross-linking); the column was washed with 1.5 1 of water to remove the pyridine. Elution of the column was carried out using a gradient created by successively adding to a mixing chamber containing 500 ml of water, the following solutions: formic acid 4 N (500 ml), formic acid 4 N plus ammonium formate 0.1 M ( 500 ml) and formic acid 4 N plus 0.2 M ammonium formate (1500 ml) and 15 ml fractions were collected. Fractions containing 2CdATMDP (approximately in 115-134 tubes) were identified using ultraviolet absorption by methods known in the art. The combined fractions containing 2CdATMDP were lyophilized to a volume of approximately 200 ml and then treated with 7 g of acid washed carbon (Norit A) and stirred for 15 minutes.; then, the coal was collected by filtration and washed with water (800 ml of total volume). The product was eluted with 50% aqueous ethanol-5% concentrated ammonium hydroxide (600 ml total volume) and the eluate was concentrated to a volume of 200 ml by evaporation at 20 ° C, filtered to remove traces of charcoal and lyophilized to obtain a powder. The powder was dissolved in 4 ml of water and the solution treated with an excess of 1 M barium acetate; the resulting precipitate was collected by centrifugation, washed with water and dissolved in 0.1 N HBr at 0 ° C. The solution was adjusted to pH 6.5 with IN NaOH and the resulting precipitate was collected by centrifugation, washed successively with 2 x 2 ml of water, ethanol and ether. The sample was dried at room temperature over P2O for 12, to obtain the dibary hydrate of 2CdATMDP. Other nucleoside analog phosphomethylene diphosphonates of the present invention were prepared in a similar manner. EXAMPLE 11 Absence of Antiviral Effect of Acyclovir Monophosphate in HSV Strains (DM.21) TK Mutants, Resistant to Acyclovir Cultures separately from Wi-38 fibroblasts were infected with either wild type strains of herpes simplex virus (HSV) , or with a mutant strain of HSV (DM.21) and were treated individually with acyclovir or acyclovir monophosphate. The mutant DM.21 lacks the enzyme thymidine kinase, which normally transforms the ACV into ACV-PM and, therefore, is resistant to acyclovir. The results for HSV-1 are shown in Figure 1 and those of HSV-DM.21 are shown in Figure 2. An IC50 is that concentration of antiviral agent that inhibits 50% of the formation of viral plaques. In wild type isolates and in laboratory strains of herpes simplex virus (HSV-1), acyclovir and acyclovir monophosphate have IC50 of 0. 1 μM (figure 1). In contrast, both acyclovir and acyclovir monophosphate had IC 50 exceeding 100 μM against mutant HSV strains in this assay (Figure 2). Based on these in vitro results, acyclovir monophosphate would not be expected to exhibit significant activity when administered topically in animals infected with a defective thymidine kinase strain or other HSV mutant. EXAMPLE 12 Antiviral Effect of Acyclovir Phosphate Esters in Mice Infected with Acyclovir-Resistant HSV Strains HRS / J type mice were infected by the cutaneous route using the snoring method, as described by Ellis, M. et al, Antimicrobial Agents and Chemotherapy 33 (3): 304-310 (1989). Briefly, groups of 10 mice under light anesthesia with ether, were inoculated in the snout by scarification with a 25-gauge needle, followed by 10 seconds of rubbing with a cotton-tipped applicator soaked with the diluted virus. The virus used for the infection was the TK-deficient strain referred in Ellis M. et al, (TK). Three hours after infection, the animals were treated topically, three times a day for four days, with acyclovir or acyclovir phosphate formulations in the form of an aqueous cream (CA), in accordance with the aforementioned Ellis reference. The results are presented in Figure 3. A formulation comprising 5 g% acyclovir was active. In contrast, a formulation comprising 5 g% of a mixture of 80% acyclovir monophosphate together with 20% of other acyclovir phosphate esters (acyclovir diphosphate and acyclovir glycerol diphosphate), demonstrated superior activity, where only about how many mice developed herpetic lesions. All injuries healed at 8 days in all groups. The above procedure was repeated with a continuous treatment for 5 days, using the TK-altered HSV-1 virus (TK, Ellis, reference above), which is a more virulent strain. Unlike the TK virus, the T? T ^ virus is deadly in untreated mice. Treatment with 5 g% of acyclovir moderately reduced the score of the lesions, where most of the animals survived and improved substantially by day 14. However, with the same concentration of phosphate esters, there was a drastic improvement in the injury ratings, where all lesions resolved after 9 days and all animals survived, as demonstrated in Figure 4. EXAMPLE 13 Antiviral effect of acyclovir onophosphate in mice infected with a wild-type strain of HSV -1 Acyclovir Resistant The procedure of Example 12 was repeated using a wild-type acyclovir-sensitive strain HSV-1 and a formulation containing only acyclovir monophosphate (ACV-MP) as the acyclovir derivative. Two creams were formulated, one having ACD-MP in the aqueous cream at a concentration of 14.5 millimoles / 100 mL and the other containing acyclovir at a concentration of 22.2 millimoles / 100 mL (both with 5 g%, however, due to the addition of the phosphate group, the number of moles of acyclovir present in the monophosphate is reduced in relation to the net content of acyclovir). The treatment was started 24 hours after infection and continued four times a day for four days. The 10 untreated mice developed stage 4 lesions by the fifth day and all had died by day 14 (Figure 5). Animals treated with acyclovir monophosphate did not develop lesions and 10 of the 10 animals survived (Figure 5). In the group treated with acyclovir, several animals developed mild lesions on days 7 to 9, which were cured; 9 of the 10 animals survived. This study demonstrates that ACV-MP at a lower dose (14.5 mmol / 100 mL) was more effective than acyclovir (22.2 mmol / 100 mL) to prevent lesions in infection by wild-type HSV-1. EXAMPLE 14 Antiviral Effect of Acyclovir Monophosphate in Mice Infected with Acyclovir Resistant HSV-1 Strain The procedure of Example 12 was repeated using a formulation having only acyclovir monophosphate (ACV-MP) as an acyclovir derivative. The treatment started three hours after infection, applying the treatments twice on the day of infection and then three times a day until day 4. Now with reference to Figure 6, the ACV-MP at a dose of 14.5 mmol / 100 mL, clearly is more effective than Acyclovir at 22.2 mmol / 100 mL to reduce lesion scores in animals infected with an acyclovir-resistant strain of HSV-1 (TK-altered). In the control and acyclovir treated groups, 8 of the 10 animals survived on day 14 of the experiment, compared to 10 of 10 survivors in the acyclovir monophosphate treatment group. EXAMPLE 15 Antiviral effect of acyclovir monophosphate in guinea pigs infected with an acyclovir-resistant HSV-2 strain Acyclovir monophosphate (ACV-MP) was tested in the form of an aqueous cream (CA) to determine whether it was more effective than acyclovir 5% polyethylene glycol (ACV-PEG), for the treatment of primary genital herpes. In particular, an infection of genital herpes was studied in guinea pigs caused by a strain of HSV-2 resistant to ACV.

Additionally, the treatments with acyclovir were compared in two delivery systems: Aqueous cream (CA) and polyethylene glycol (PEG). The experiments were controlled with a placebo and the uninfected animals were treated with an ACV preparation to evaluate skin and vaginal irritation. Intravaginal inoculation of guinea pigs weaned with HSV-2 results in a primary genital infection characterized by the initial replication of the virus in the vaginal tract, followed by the development of external vesicular lesions. Virus titers reach a peak on days 1 to 3 in the vaginal tract and gradually decrease on days 7 to 10. External genital lesions appear for the first time on day 4, the peak of severity of the lesions occurs in days 6 to 8 and the lesions usually heal for days 15 to 18. The animals were inoculated with strain HSV-2 12247, which is an altered strain in thymidine kinase and resistant in vitro to treatment with acyclovir. Female guinea pigs of the Hartley breed (Charles River, Kingston, NY), weaned from 250 to 300 g of weight, were cleansed of the vaginal cavity with an isopo to remove the secretions. After one hour, the animals were inoculated intravaginally with 2.4 x 4 10 plaque forming units (pfu). The inoculation was completed by inserting an isopo soaked with the virus into the vaginal tract and rotating it approximately six times.

Groups of 10 guinea pigs were treated intravaginally and on the external genital skin with 0.1 mL (total volume of 0.2 mL per animal per treatment) of each preparation. The animals were treated three times a day for 7 days, starting 24 hours after the viral inoculation. Three uninfected animals were tested with each preparation with the same administration protocol, to assess local toxicity and irritation. To determine the efficacy of the various treatments on the replication of HSV-2 in the vaginal tract, vaginal exudates were obtained during the primary infection on days 1, 3, 5, 7 and 10 after inoculation of HSV-2. The isopos were placed in tubes containing 2.0 mL of culture medium, vortexed and frozen at -70 ° C until HSV titration. When all the samples were collected, they were thawed, serially diluted and the HSV-2 titers were determined using rabbit kidney cells in a microplate CPE assay. The development and severity of external injuries were also measured to determine the efficacy of the treatment. The severity of the injuries was rated on a scale of 0 to 5+. The presence or absence and the severity of the lesions were recorded during 19 days after the viral inoculation. The infection rates, the peak lesion scores, the virus peak titers, the areas under the virus curves on the day of titration and the day-rating curves were compared between animals treated with a SRF placebo and the animals treated with PEG-drug or with aqueous cream with placebo and with aqueous cream with drug, using the Mann-Whitney U-rank sum test. A p-value of 0.05 or less was considered significant. The effect of topical treatment with ACV preparations on vaginal viral replication is shown in Table I. only the treatment with preparations of ACV-MP (5% ACV-MP-PEG or 5% ACV-MP-AC) significantly reduced the area under the curve (ABC) of the virus on the day of titration and the average peak virus titers .

TABLE I EFFECT OF TREATMENT WITH ACICLOVR MONOPHOSPHATE ON VAGINAL TITLES OF VIRUSES IN INOCULATED COBAYS I- ^ TRAVAGINALLY WITH HSV-2 RESISTANT TO ACICLOVIR A. Topical and intravaginal treatment was started 24 hours after viral inoculation and continued three times a day for 7 days. The content of acyclovir on a molar basis was lower in the tests performed with acyclovir monophosphate (14.5 mmol / 100 mL) versus those carried out with acyclovir alone (22.2 mmol / 100 mL). B. NS = Not statistically significant in comparison with the appropriate group treated with placebo.

The effect of topical treatment with ACV preparations on the development of the lesions is illustrated in Table II. The ACV and ACV-MP preparations significantly altered the score of the ABC-lesions of the day, compared with the appropriate group treated with placebo. However, only the treatment with 5% ACV-MP-PEG significantly reduced the average of the peak scores of the lesions.

TABLE II EFFECT OF THE TREATMENT WITH ACICLOVTR MONOPHOSPHATE ON THE DEVELOPMENT OF EXTERNAL INJURIES IN COBAYS WITH GENITAL INFECTION BY HSV-2 RESISTANT TO ACICLOVIR A. Topical and intravaginal treatment was started 24 hours after viral inoculation and continued three times a day for 7 days. The content of acyclovir on a molar basis was lower in the tests performed with acyclovir monophosphate (14.5 mmol / 100 mL) versus those performed with acyclovir alone (22.2 mmol / 100 mL). B. NS = Not statistically significant in comparison with the appropriate group treated with placebo.

In the guinea pig model of genital herpes infection by HSV-2 resistant to ACV, only the ACV-MP significantly reduced the viral viral replication.

Likewise, the group treated with ACV-MP-PEG had the lowest average peak and title values per day. While the ACV-MP and the ACV altered the development of the lesions, the drugs in PEG had lower ratings than those in aqueous cream (CA) Additionally, the animals that received ACV-MP-PEG presented the injury-day scores and the average peak scores of the lowest injuries. In addition, throughout the study there were no signs of any irritation of the genital area or any other toxicity in the non-infected animals treated with an ACV preparation. These results demonstrate the strong activity of acyclovir monophosphate in the treatment of genital herpes caused by HSV-2. In addition, it is interesting to note that acyclovir monophosphate dispersed in polyethylene glycol demonstrated the best efficacy in the treatment of lesions. EXAMPLE 16 Acyclovir diphosphate activity The procedure of Example 12 was repeated using a formulation containing only acyclovir diphosphate (ACV-DP) as the acyclovir derivative. An efficacy superior to that of the ACV alone was observed. EXAMPLE 17 Acyclovir glycerol monophosphate activity The procedure of Example 12 was repeated using a formulation containing only acyclovir glycerol monophosphate (ACV-MP-G) as the acyclovir derivative. An efficacy superior to that of the ACV alone was observed. EXAMPLE 18 Acyclovir glycerol diphosphate activity The procedure of Example 12 was repeated using a formulation containing only acyclovir glycerol diphosphate (ACV-DP-G) as the acyclovir derivative. An efficacy superior to that of the ACV alone was observed. EXAMPLE 19 Aciclovir Morpholidate Monophosphate Activity The procedure of Example 12 was repeated using a formulation containing only acyclovir morpholidate monophosphate (ACV-MP-morfolidate) as the acyclovir derivative. An efficacy superior to that of the ACV alone was observed. EXAMPLE 20 Activity of isopropylidene acrylovir glycerol monophosphate The procedure of Example 12 was repeated using a formulation containing only acyclovir glycerol monophosphate isopropylidene (ACV-MP-isoP-G) as the acyclovir derivative. An efficacy superior to that of the ACV alone was observed. EXAMPLE 21 Acyclovir isopropylidene glycerol diphosphate activity The procedure of Example 12 was repeated using a formulation containing only acyclovir isopropylidene glycerol diphosphate (ACV-DP-isoP-G) as the acyclovir derivative. An efficacy superior to that of the ACV alone was observed. EXAMPLE 22 Acyclovir Phosphi Ethylene Diphosphonate Activity and Acyclovir Triphosphate The procedure of Example 12 was repeated using a formulation containing only ACV phosphomethyl diphosphonate and acyclovir triphosphate, independently, as the acyclovir derivative. An efficacy superior to that of the ACV alone was observed. EXAMPLE 23 Solubility of acyclovir monophosphate and its salts Several salts of acyclovir monophosphate were tested for their solubility, as follows: 2 mL of distilled water was placed in each of three 10 mL beakers, where each beaker had a magnetic stirring bar. In each individual vessel was added an acyclovir monophosphate salt, which was selected from the group consisting of potassium, sodium, sodium / ammonium salt and H + (free acid), until a saturated solution was formed. Each saturated solution was filtered by gravity. The sodium / ammonium salt solutions of acyclovir monophosphate and the free acid salt solution were filtered through a No. 4 Whatman filter and produced clear solutions. The potassium salt and sodium salt solutions were filtered through a No. 1 Whatman filter paper and each produced a slightly opaque solution. 1 mL of each of the saturated solutions was transferred, by pipetting, to a previously weighed balloon flask and the solutions allowed to dry. After all the liquid had evaporated, the balloon flasks were reweighed and the number of milligrams of acyclovir monophosphate salt present per milliliter was easily determined. The following table establishes the solubility of the various salts prepared in the manner described above, relative to acyclovir: TABLE III In view of the data shown in Table III, it will be noted that through the formation of an acyclovir monophosphate salt, the solubility can be dramatically increased. It is expected that other nucleoside monophosphates, similarly, exhibit higher solubilities. In this way, it is possible to formulate topical compositions containing large amounts of acyclovir monophosphate, due to the higher solubility of their salts. It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates. Having described the invention as above, property is claimed as contained in the following

Claims (23)

1. CLAIMS 1. An antiviral nucleoside analog phosphate ester, characterized in that it is selected from the group consisting of antiviral nucleoside analogue phosphorothioates, nucleoside analog phosphoramidates and nucleoside analog phosphofluoridates.
2. 2. An antiviral nucleoside analog phosphate ester, characterized in that it is selected from the group consisting of diphosphates of 1,2-μ-methylene-nucleódside analogs; triphosphates of 2, 3-μ-methylene-nucleoside analogues; diphosphates of 1,2-μ-thio-nucleoside analogues; and 2,3-μ-thio-nucleoside analog triphosphates.
3. 3. A compound according to any of claims 1 6 2, characterized in that the antiviral nucleoside analogue is 9- (2-hydroxyethoxymethyl) -guanine of acyclovir.
4. 4. A pharmaceutical composition characterized in that it comprises an effective antiviral amount of a nucleoside analogue phosphate ester against herpes according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, or mixtures thereof, in a pharmaceutically acceptable vehicle for topical application.
5. The composition according to claim 4, characterized in that the nucleoside analog phosphate ester is selected from the group consisting of nucleoside analogue phosphorothioates, nucleoside analog phosphoramidates and nucleoside analog phosphofluoridates.
6. 6. The composition according to claim 4, characterized in that the nucleoside analog phosphate ester is selected from the group consisting of 1,2-μ-methylene nucleoside diphosphates; triphosphates of 2, 3-μ-methylene-nucleoside analogues; diphosphates of 1, 2-μ-thio-nucleoside analogues; and 2,3-μ-thio-nucleoside analog triphosphates.
7. The composition according to any of claims 4 to 6, characterized in that the nucleoside analogue is acyclovir.
8. 8. The composition according to any of claims 4 to 6, characterized in that the nucleoside analog phosphate ester is acyclovir monophosphate.
9. 9. The composition according to any of claims 4 to 6, characterized in that the nucleoside analog phosphate ester is acyclovir diphosphate.
10. 10. The composition according to any of claims 4 to 6, characterized in that the nucleoside analog phosphate ester is acyclovir triphosphate.
11. 11. A formulation according to any of claims 4 to 6, characterized in that the nucleoside analogue against herpes is selected from the group consisting of l-beta-D-arabinofuranosyl-E-5 (2-bromovinyl) -uracil; 2'-fluorocarbocyclo-2'-deoxyguanosine; 6'-fluorocarbocyclo-2'-deoxyguanosine; 1- (beta-D-arabinofuranosyl) -5 (E) - (2-iodovinyl) -uracil; (lr-la, 2ß, 3a) -2-amino-9- (2, 3-bis- (hydroxymethyl) -cyclobutyl) -6H-purin-6-one: 9 - ((2- (1-methylethoxy) - 1- ((1-Methylethoxy) -methyl) -ethoxy) -methyl) - (9C1) of 9H-purin-2-amine; trifluorothymidine; 9- [(1,3-dihydroxy-2-propoxy) -methyl] -guanine; 5-ethyl-2'-deoxyuridine; E-5- (2-bromovinyl) -2'-deoxyuridine; 5- (2-chloroethyl) -2'-deoxyuridine; 1- (2-deoxy-2-fluoro-beta-D-arabinofuranosyl) -5-iodocytosine (FIAC); 1- (2-deoxy-2-fluoro-beta-D-arabinofuranosyl) -5-yodouridine (FIAU); buciclovir; 6-deoxyacyclovir; 9- (4-hydroxy-3-hydroxymethylbut-1-yl) -guanine; E-5- (2-iodovinyl) -2'-deoxyuridine; 5-vinyl-l-beta-D-arabinofuranosyl-uracil; 1-beta-D-arabinofuranosyl-thymine; 2 '-nor-2' -deoxyguanosine; 9- (4-hydroxy-3-hydroxymethylbut-1-yl) -guanine; 1-beta-D-arabinofuranosyl-adenine.
12. 12. A composition according to any of claims 4 to 6, characterized in that the nucleoside analog phosphate ester is in the form of a pharmaceutically acceptable salt.
13. 13. A composition according to claim 11, characterized in that the salt is selected from the group consisting of sodium, potassium, ammonium and hydrogen salt.
14. 14. A composition according to any of claims 4 to 6, characterized in that the pharmaceutical carrier is selected from the group consisting of an aqueous cream and polyethylene glycol.
15. 15. A method for the topical treatment of skin or mucosal lesions of an animal infected with the herpes virus, characterized in that it comprises applying to the affected lesions an effective amount of a composition containing an effective amount of an antiviral nucleoside analog in accordance with the invention. with any of claims 4 to 6.
16. 16. A method according to claim 15, characterized in that the nucleoside analog phosphate ester is acyclovir monophosphate.
17. 17. A method according to claim 15, characterized in that the nucleoside analog phosphate ester is acyclovir diphosphate.
18. 18. A method according to claim 15, characterized in that the nucleoside analogue phosphate ester is acyclovir triphosphate.
19. 19. A method for treating a herpes virus infection, wherein the herpes virus has developed a resistance to one or more antiviral components due to an alteration or defect in the viral gene coding for thymidine kinase, characterized in that it comprises applying an effective amount of a nucleoside analogue phosphate ester against the herpes virus, a nucleoside analogue phosphate ester salt thereof against the pharmaceutically acceptable herpes virus or a mixture thereof, in a pharmaceutical carrier suitable for topical application, the cutaneous or mucosal tissues of an animal infected by the virus.
20. 20. A method according to claim 15, characterized in that the animal is a human being.
21. 21. A method according to claim 15, characterized in that the infection is caused by a strain of herpes virus that is resistant to acyclovir.
22. 22. A method according to claim 15, characterized in that the resistance to acyclovir of the virus is caused by an alteration or defect in the viral gene that codes for the thymidine kinase. A method for the treatment of an infection caused by the herpes virus in an animal, characterized in that it comprises the topical application of a composition containing an effective antiviral amount of an acyclovir triphosphate, to the mucosal or cutaneous lesions of the affected animal .