ANTIVIRAL COMPOSITION CONTAINING AROMATIC POLYCYCLIC DIONES AND NUCLEOSIDE ANALOGS AND METHOD FOR TREATING RETROVIRUS INFECTIONS

Abstract: A method for treating retroviral infections in mammals comprising administering anti-retroviral effective amounts of a nucleoside analog and an aromatic polycyclic dione selected from the group consisting of hypericin, pseudohypericin, salts and mixtures thereof is provided. Also provided are compositions for treating retroviral infections in mammals comprising anti-retroviral effective amounts of a nucleoside analog and an aromatic polycyclic dione selected from the group consisting of hypericin, pseudohypericin, salts and mixtures thereof.

Description: ANTIVIRAL COMPOSITION CONTAINING AROMATIC POLYCYCLIC DIONES AND NUCLEOSIDE ANALOGS AND METHOD FOR TREATING RETROVIRUS INFECTIONS BACKGROUND OF THE INVENTION This invention is related to pharmaceutical formula¬ tions for treating retroviral infections in mammals comprising antiviral effective amounts of aromatic polycyclic dione compounds in combination with azidothymidine, dideoxycytidine or related nucleoside analogs and methods for use thereof. The pathogenicity of retroviruses ranges from the slowly progressive encephalitic lentiviruses, to retroviruses causing oncogenic transformations which elicit a wide variety of leu emias, sarcomas, and carcinomas to infections by the recently identified human immunodeficiency virus (HIV) . Limited therapeutic agents exist which can be used to combat retroviral infections in mammals (especially HIV) and are confined to reagents which can interfere with the different stages of the viral replication cycle. The inability of the host immune system to deal effectively with many retroviruses is due to the inherent properties of the infectious retroviruses, such as their capacity to spread directly by cell-to-cell contact, their high recombinational activity, and their ability to integrate in the host genome and remain latent for extended periods of time. In addition, it is now known that HIV causes immunosuppression characterized by a depletion of CD4+ T cells in infected individuals. It has also been recently determined that two related but distinct viruses cause AIDS, designated HIV-l and HIV-2. The genomes of HIV-1 and HIV-2 are only about 50% homologous at the nucleotide level but the two viruses contain the same complement of genes and appear to attack and kill the same human cells by much the same mechanisms. In the discussion below, HIV will be used to refer to these viruses in a generic sense. The most effective antiretroviral agents to date are the nucleoside analogs 2', 3' dideoxycytidine and 3- * -azido-3'- deoxythymidine (AZT) , which become phosphorylated in the cell and are incorporated into viral DNA by the viral reverse transcriptase, leading to premature complementary DNA (cDNA) chain termination. However, the use of these drugs is limited due to their severe toxicity to the treated host. AZT is currently used to treat certain patients suffering from Acquired Immunodeficiency Syndrome (AIDS) caused by HIV. AZT has been shown to improve immunologic functions, to reverse, at least partially, HIV-induced neurological disfunction in some patients, and to improve certain other clinical abnormalities associated with AIDS. However, a dose- dependent suppression of bone marrow, resulting in anemia and leukopenia (an abnormally low number of leukocytes in the circulating blood) has been found to occur with its use. This has limited the effectiveness of AZT for the treatment of AIDS. Nevertheless, AZT is the only drug available for the treatment of AIDS patients. Dideoxycytidine has also been tested in patients with AIDS but has proven even more toxic than AZT. Therefore, there is a need in the art for compositions for treating infections caused by retroviruses, especially HIV, utilizing nucleoside analogs whereby such compositions will enable a significant reduction in the dose of the nucleoside analog to levels which do not cause any measurable toxicity in the afflicted individuals and/or provide an increased antiviral effect. It is an object of the present invention to provide compositions and methods for the treatment of retroviral infections in mammals. Another object of the present invention is to provide compositions and methods for treating retroviral infections in mammals employing nucleoside analogs such as AZT whereby the toxic effects of the nucleoside analog are minimized. SUMMARY OF THE INVENTION The present inventors have unexpectedly discovered that nucleoside analogs, such as AZT, and the polycyclic dione compounds hypericin and pseudohypericin interact synergistical- ly and provide a more effective anti-retroviral inhibitory effect. Therefore, such a combination of antiviral drugs may provide an increased benefit to those individuals afflicted with AIDS or with other retroviral infections sensitive to these drugs. This is a most important finding since AZT is the only drug currently in use which has been proven effective for the treatment of AIDS patients. In one aspect, the present invention provides a method for treating retroviral infections in mammals comprising administering to mammals in need of such treatment an anti- retroviral-effective amount of a nucleoside analog and a compound selected from the group consisting of hypericin, pseudohypericin, salts and mixtures thereof and physiologically acceptable salts and diluents. In another aspect, the present invention provides a composition for treating mammals suffering from retroviral infections comprising an anti-retroviral-effective amount of a nucleoside analog and a compound selected from the group consisting of hypericin, pseudohypericin, salts and mixtures thereof and physiologically acceptable salts and diluents. These and other aspects of the present invention will be apparent to those of ordinary skill in the art in view of the present description, accompanying claims, and appended figures. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the survival of mice infected with Friend Leukemia Virus treated with various concentrations of pseudohypericin. Figure 2 is a graph showing the survival of mice infected with Friend Leukemia Virus treated with various concentrations of hypericin. Figure 3 is a graph showing the survival of mice infected with Friend Leukemia Virus treated with various concentrations of AZT plus either hypericin or pseudohypericin. Figure 4 is a graph showing the survival of mice infected with Friend Leukemia Virus treated with various doses of hypericin and pseudohypericin. DETAILED DESCRIPTION OF THE INVENTION The present inventors have unexpectedly discovered that nucleoside analogs, typified by AZT, and the aromatic polycyclic dione compounds hypericin and pseudohypericin interact synergistically so that antiretroviral-effective nucleoside analog treatments (utilizing drugs such as AZT) can be employed at concentrations which are non-toxic for the treated mammals and in which the nucleoside analog alone is ineffective. This is a most important finding because AZT treatment cannot be tolerated by some patients with AIDS and others must stop treatment when the toxic effects of AZT are manifested. In addition, the use of the two drug antiviral therapy for treatment of patients with retrovirus infections such as AIDS may lead to an increased antiviral effect in such patients. The nucleoside analogs and the polycyclic dione compounds inhibit retroviral replication via different mechan¬ isms of action. As mentioned above, AZT and related nucleo- sides analogs work by causing premature chain termination of growing viral cDNA. However, the polycyclic diones of the present invention may inhibit late stages of viral replication such as the shedding or assembly of viral particles from infected cells or may directly inactivate the virus particles. The polycyclic dione compounds did not effect the levels of total viral mRNA or the expression of viral antigens within the cultures of mouse cells infected with Friend Leukemia virus in vitro at dosages which inhibited viral replication (data not shown) . Moreover, incubation of the compounds with purified reverse transcriptase alone had no effect on the activity of the enzyme (data not shown) . However, incubation of the polycyclic diones directly with the Friend Leukemia Virus totally abrogates its infectivity. Without wishing to be bound by theory, it is believed that PS and HY work by directly inactivating the virions and limiting the spread of viral infection. The present inventors have tested over 800 mice with PS and HY with concentrations ranging between about O.lng and about 300 microgram per mouse administered parenterally and have neither observed any serious toxic effects of the com¬ pounds on the treated animals nor felt that an experiment should be terminated due to the discomfort of the animals. As used herein, the term retrovirus refers to viruses containing an RNA genome and RNA-dependent DNA poly erase (reverse transcriptase) enzymatic activity. All retroviruses have common morphological, biochemical and physical properties that justify their inclusion into a single virus, family. These parameters are summarized in Table I below. TABLE I GENERAL PHYSICAL PROPERTIES OF KNOWN RETROVIRUSES Nucleic acid linear positive-sense single-stranded RNA (60S-70S) composed of identical subunits (30S-35S) ; 5' structure (m 7 G 5 ppp 5 NmpNp) ; polyadenylated 3' end; repeated sequences at 3' and 5* * ends; tRNA base-paired to genome complex Protein above 60% by weight; grag * , internal structural proteins; pol - reverse transcriptase; env, envelope proteins Lipid about 35% by weight; derived from cell membrane Carbohydrate about 4% by weight; associated with envelope proteins Physiochemical density 1.16-1.18 g/ml in sucrose, 1.16-1.21 properties g/ml in cesium chloride; sensitive to lipid solvents, detergents, and heat inactivation (56*C, 30 min) ; highly resistant to UV- and X-irradiation Morphology spherical enveloped virions (80-120-nm diameter) , variable surface projections (8-nm diameter) , icosahedral capsid containing a ribonucleoprotein complex with a core shell (nucleoid) In addition, the genome of HIV encodes at least 5 other proteins, designated TAT, ART/TRS, 3 , -0RF, SOR and R whereas HTLV I contains the pX gene which may encode up to four proteins. All retroviruses have similar overall chemical composi¬ tions. In general, they comprise about 60-70% protein, 30-40% lipid, 2-4% carbohydrate, and about 1% RNA. The envelope of retroviral particles is derived from the cell-surface membrane, and most, if not all, of the lipids in viral particles are located in the unit-membrane envelope of the virion. Non- limiting examples of retroviruses include Friend Leukemia Virus (FV) , Radiation Leukemia Virus (RadLV) , Feline Leukemia virus, Avian Myeoblastosis Virus, and the human T-cell lymphotropic virus family (HTLV I, II, III and IV; HTLV III is also known as Human Immunodeficiency Virus or HIV) . HTLV I has been shown to cause adult T cell leukemia and HTLV II hairy cell leukemia. HTLV IV is related to simian immunodeficiency virus and has been found in African natives suffering from AIDS; its re¬ lationship to HTLV III is currently under investigation. The composition of the present invention comprising the aromatic, polycyclic diones and nucleoside analogs can be utilized for the treatment of mammals suffering from diseases caused by retroviruses such as HIV. Due to the fact that nucleoside analogs can be employed at sub-toxic concentrations, and that the polycyclic diones lack cellular toxicity at the doses administered, the compositions of the present invention may be particularly useful as specific antiretroviral therapeutic agents for these disorders. The effectiveness of the composition of the present invention in treating retroviral infections is shown in Example 2 below wherein 100% of Friend Leukemia Virus-infected mice survived 65 days post-infection when treated with a composition comprising non-toxic concentra¬ tions of AZT and HY. In contrast, 100% of animals treated with the same concentrations of AZT alone were dead by day 65. It is known that HIV can infect cells of the brain and central nervous system causing neurological dysfunction and dementia. AZT has been shown to cross the blood-brain barrier; hypericin and pseudohypericin have been previously administered to humans with positive effects as anti-depressant agents, indicating an action on the central nervous system. Therefore, the use of the polycyclic dione compounds in combination with nucleoside analogs may prove highly efficacious when adminis¬ tered to humans suffering from the neurological effects of AIDS. Another advantage of the composition of the present invention is the fact that both the polycyclic diones and the nucleoside analogs are effective when administered orally. In addition, the efficacy of PS or HY treatment on Friend Leukemis Virus infection is shown in Figure 4, where single treatments with nanogram amounts of either of the polycyclic dione compounds inhibited virally-induced splenomegaly when administered intraperitoneally 24 hours after infection. About 1 nanogram of PS or 100 nanograms of HY inhibited FV-induced splenomegaly 50% in infected Balb/c mice. These low doses of HY and PS are surprisingly effective in inhibiting an aggressive retrovirus when administered as a single intraperitoneal dose 24 hours post-infection. Hypericin (HY) and pseudohypericin (PS) are aromatic polycyclic dione compounds which are present in plants of the family Hypericum (St. John's Wort) . U.S. Patent Application Serial No. 084,000 of D. Lavie et al. filed August 10, 1987 (incorporated by reference herein) discloses that HY and PS are potent inhibitors of retroviral replication in vivo and in vitro. HY and PS may be obtained by extraction from the St. John's Wort plant, as detailed below in Example 1, or alterna- tively may be chemically synthesized using the methods of Brockmann, H. et al, U.S. Patent No. 2,707,704, issued May 3, 1955 and of Brockmann, H. , Tetrahedron Letters 23; 1991-1994, 1974, both incorporated herein by reference. Due to the wide distribution and availability of the St. John's Wort plant throughout the world and the relatively convenient and inexpen¬ sive procedure for the extraction and purification of HY and PS (as detailed in Example 1 below) , the extraction procedure is preferred when small amounts (i.e. grams) are desired. However, for the production of large scale amounts (kilograms or greater) , chemical synthesis is preferred. The composition of the present invention further includes salts or other derivatives of hypericin and pseudo¬ hypericin which retain their antiviral activity. Salts in which the base of the alkaline or a ine type are particularly comprehended within the scope of the present application. Nucleoside analogs envisioned for use in the present invention include but are not limited to 2', 3' dideoxycyti¬ dine, 2', 3" dideoxythymidine, 2', 3' dideoxyadenosine, 2 ' , 3' dideoxyinosine and preferably AZT. AZT for practicing the present invention is commercially available from Burroughs Welcome (Research Triangle Park, NC) , 2', 3' dideoxycytidine and 2', 3' dideoxyadensine are commercially available from Calbiochem-Behring (San Diego, CA) , 2', 3' dideoxythymidine is available from Pharmacia Fine Chemicals (Piscataway, NJ) . The present invention also provides a method for treating mammals suffering from infections caused by retroviruses comprising administering to mammals in need of such treatment an antiretroviral effective amount of a nucleoside analog plus an aromatic polycyclic dione selected from the group consisting of hypericin, pseudohypericin, salts and mixtures thereof. When treating mammals suffering from infections caused by retroviruses according to the present invention, the deter¬ mination of the most effective type and mixture of the nucleos¬ ide analog plus polycyclic dione compound for treatment of the particular retrovirus responsible for the infection can be ascertained by routine experimentation using suitable systems, such as that described in Example 4 for HIV in vitro or in Example 2 for Friend Leukemia Virus in experimental animals, and 2', 3' dideoxyadensine are commercially available from When employed in vivo to treat AIDS, viremia (i.e. the presence of virus in the blood stream) or sepsis (viral con¬ tamination of bodily fluids) caused by viruses, the composi¬ tions of the present invention may be administered orally, topically or preferably parenterally, and most preferably intravenously at dosages which can be broadly defined by reference to hypericin as follows: Antiretroviral compositions containing aromatic polycyclic diones as one of the active ingredients can be used at dosages containing from about 0.001 to about 100,000 micrograms per kilogram bodyweight per treatment, preferably between about 2 micrograms and about 5 x 10 4 micrograms per kilogram bodyweight per treatment, and most preferably between about 200 micrograms and 5 x 10 4 micrograms per kilogram bodyweight per treatment of said diones. When one or more other aromatic polycyclic dione compounds are used as the active ingredient, the broad dosages will generally be the same as with hypericin. It is under¬ stood, however, that if a given aromatic polycyclic dione compound or mixture has e.g. twice the activity of hypericin, the minimum effective dosage will be one-half that of hyper- icin. Moreover, when more than one active (antiviral) in¬ gredient (i.e., at least one non-aromatic polycyclic dione antiviral agent is included) is used in a therapeutic or prophylactic regimen according to the invention, the minimum dosage of the aromatic polycyclic dione component of this regimen may be decreased if desired or appropriate. Finally, when more than one active ingredient is used and there is synergism between the aromatic polycyclic dione component and the other antiviral ingredient or ingredients (or between two or more polycyclic dione compounds and the nucleoside analogs) , the minimum effective dosages will be even smaller. To illustrate the foregoing, consider a therapeutic or prophylactic regimen that involves administration of an aromatic polycyclic dione compound in conjunction with an antivirally active nucleoside analog. (It should be understood that "in conjunction'* means coadministered or administered sequentially but as part of the same treatment regimen) . When one or more nucleoside analogs are used in combination with the compounds of the present invention, the nucleoside analog may be administered in conjunction with the aromatic polycyclic dione compound(s) at doses broadly ranging between about 100 and about 50,000 micrograms/kg body weight of said mammal per treatment. The duration and number of doses or treatments required to control the disease will vary from subject to subject, depending upon the severity and stage of the illness and the subject's general condition and will also depend on the specific antiviral activity of each composition, as well as its toxicity (if any) . The total dose required for each treatment may be administered in divided doses or in a single dose. The antiviral treatment may be administered daily, more than once daily, one or two times, a week, or as determined by the subject's condition and the stage of the disease. The present inventors have also discovered that the an¬ tiviral activity of hypericin is a function of the frequency of treatment. For example, in mouse studies, a single dose of ten micrograms per mouse was less effective than a single dose of 100 micrograms per mouse, as expected. However, administration of 10 micrograms every day for ten days was less effective than even a single 10-microgram dose. By contrast, administration of 10 micrograms once a week was as effective as the single 10- microgram dose. This indicates that the frequency of treatment affects its efficacy. Therefore, this should be taken into account when administering the compositions of the present invention as well. While the foregoing observations in mice may not be applicable to other mammals or humans, those skilled in the art will appreciate that the frequency of treatment is subject to optimization, which can be determined by routine experimentation according to methods well known in the art, e.g. by establishing a matrix of dosage and frequency and assigning a group of experimental subjects to each point of the matrix. Design of this experiment should preferably also take into account the tissue accumulation properties of the com¬ pounds of the present invention. The present invention also provides pharmaceutical compositions and formulations for treating retroviral infec- tions. The aromatic polycyclic diones and/or nucleoside analogs of the present invention can be incorporated in conventional, solid and liquid pharmaceutical formulations (e.g. tablets, capsules, caplets, injectable and orally administrable solutions) for use in treating mammals that are afflicted with retroviral infections. The pharmaceutical formulations of the invention comprise an effective amount of the aromatic polycyclic dione compounds and nucleoside analogs of the present invention (as disclosed above) as the active in¬ gredients. For example, a parenteral therapeutic composition may comprise a sterile isotonic saline solution containing between about 0.001 micrograms and about 100,000 micrograms of the aromatic polycyclic dione compounds of the present inven¬ tion and between about 100 and 50,000 micrograms of the nucleoside analogs as described above. It will be appreciated that the unit content of active ingredients contained in an individual dose of each dosage form need not in itself con¬ stitute an effective amount since the necessary effective amount can be reached by administration of a plurality of capsules, tablets, injections or combinations thereof. Each formulation according to the present invention may additionally comprise inert constituents including pharmaceuti¬ cally-acceptable carriers, diluents, fillers, salts, and other materials well-known in the art the selection of which depends upon the dosage form utilized and the particular purpose to be achieved according to the determination of the ordinarily skilled artisan in the field. For example, tablets may be formulated in accordance with conventional procedures employing solid carriers well known in the art. Examples of solid carriers include, starch, sugar, bentonite, silica and other commonly used carriers. Propylene glycol, benzyl alcohol, isopropanol, ethanol, dimethylsulfoxide (DMSO) dimethylacetam¬ ide or other biologically acceptable organic solvents or aqueous solutions (e.g. water with a pH higher than 7 and preferably about 8) may be used as diluents, carriers or solvents in the preparation of solid and liquid pharmaceutical formulations containing the anti-retroviral compositions of the present invention. Further nonlimiting examples of carriers and diluents include carbohydrates, albumin and/or other plasma protein components such as low density lipoproteins, high density lipoproteins and the lipids with which these serum proteins are associated. Such lipids include phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine and neutral lipids such as triglycerides. Additional lipid carriers include without limitation tocopherol, retinoic acid and cyclodextranes. Semisolid formulations such as those well- known in the art (e.g. suppositories) are also contemplated. Preferred parenteral dosage forms may comprise for exa ple an isotonic saline solution, containing between about 0.1 micrograms and about 100,000 micrograms of the aromatic polycyclic dione compounds and between about 100 micrograms and 50,000 micrograms of the nucleoside analogs of the present invention. Capsules employed in the present invention may be made from any pharmaceutically acceptable material, such as gelatin or cellulose derivatives. Sustained release oral and transder al delivery systems are also contemplated. The compounds of the present invention may additionally be incorporated into liposomes for use as specific drug carriers. Such liposomes may also comprise other active agents e.g., specific anti-HIV antibodies directed against viral proteins expressed by virally infected cells such as HIV pl20, p41 and p24 (as well as glycosylated forms thereof) to act as specific targeting agents. The present invention is described below in specific working examples which are intended to illustrate the invention without limiting the scope thereof. EXAMPLE 1; EXTRACTION OF HYPERICIN AND PSEUDOHYPERICIN FROM ST JOHN'S WORT Hypericin, (CsoHiβOg, molecular weight 504.43, referred to herein as HY) , and pseudohypericin (C3QHI6 0 9 molecular weight 520.43, referred to herein as PS) were obtained as detailed below. The herb of the whole St. John's Wort plant was harvested at its flowering time, (July through October in the Eastern hemisphere), dried at 55*C, cut and milled, and then extracted with acetone (about 5-10 liters per kg) . One kilogram of the material was placed in a soxhlet (Kimax, available from Fisher Scientific, New Brunswick, NJ) and extracted until the extracting solvent was colorless (about five to ten hours) . The solution containing the aromatic polycyclic diones had a red fluorescent color with absorption and fluorescence spectra as described in Scheibe Schentaσ. Ber. 75: 2019, 1942, Brockmann, M. , Natureweiss 28.: 47, 1951, both incorporated herein by reference. The solvent (containing the aromatic polycyclic diones) was evaporated under reduced pressure to complete dryness of the residue, yielding 95 grams. This residue was then further fractionated on a chromatographic column, packed with silica gel 60 (0.06-0.20mm, Malinckrodt, American Scientific Products, McGaw Park, IL) . A dry chromatographic procedure was utilized whereby 25 grams of the above obtained residue was dissolved in about 500 ml acetone, added to an equal amount of silica gel 60, and evaporated on a rotavapor (Buchi, American Scientific Products) with swirling until the mixture was homogeneous and dry. The mixture was then placed on top of a column containing one kilogram of silica gel 60 and eluted with chloroform until the solvent reached the bottom of the column. This was followed by washing the column with a solvent mixture compris¬ ing chloroform-acetone-methanol , 75:15:10 (Vol/Vol/Vol) . When the red color became 1/5 of the original intensity, the concentration of chloroform was reduced and the column eluted with a solvent mixture comprising chloroform-acetone-methanol in the ratio of 55:15:10 respectively as above. The chromatog- raphy was completed in about two days. Further purification and separation of the two main components was obtained by two rounds of flash chromatography using a silica gel 60 (mesh 0.04-0.06) under pressure as described in Gas Chromatography. Principles. Techniques and Applications. A.B. Littlewood, ed. Academic Press. New York 1970, incorporated herein by reference. Two main components were identified: hypericin (HY) , Rf 0.45, in a yield of 0.19 grams; and pseudohypericin (PS), Rf 0.35, in a yield of 0.73 grams. The NMR spectrum analysis of the two components were the same as those previously reported (Brockmann, H, et al, Tetrahedron Letters A:37, 1975, incor¬ porated by reference) . The compounds were stored in the dark, dry or at 4°C in absolute ethanol, until use. EXAMPLE 2: ANTIRETROVIRAL EFFECT OF THE COMPOSITION OF THE PRESENT INVENTION The effects of the composition of the present invention on the infection of mammals with Friend Leukemia Virus (FV) was examined. Friend Leukemia Virus (FV) is an aggressive retrovirus which induces an acute erythroleukemia in sensitive strains of mice such as BALB/c and NIH Swiss mice as described in Friend, C.J. E E. Med. 105: 307-324, 1957; Friend, C. et al Proc. Natl. Acad. Sci. U.S.A. 68: 378-383, 1971; Friend, C. et al. Nat. Cancer Inst. Monogr. 2 % 505-522, 1966, (all incorporated by reference herein) . The malignant transformation is the result of the combined activities of the spleen focus forming virus (SFFV) and the ecotropic Murine Friend Leukemia Helper Virus (F-MuLv) . The acute erythroleukemia is characterized by hepatosplenomegaly (a marked increase in the size of the spleen and liver) and a severe anemia. Friend Leukemia Virus was prepared by homogenizing the enlarged spleen of a mouse previously infected with FV, ten days after intravenous virus injection. The spleen was homogenized in phosphate buffered saline in a volume equal to ten times the weight of the isolated spleen. The effects of HY and PS or mixtures thereof plus AZT on the increase in spleen size (splenomegaly) of BALB/c mice (Jackson Labs, Bar Harbor, ME) was examined. In these experiments, the virus (10 6 focus forming units-FFU) was inoculated intravenously and the indicated doses of the antiretroviral composition of the present invention was administered to the BALB/c mice in- traperitoneally 24 hours later. The animals were then sacrificed 10 days later and their spleens weighed. The long-term effects of single doses of PS and HY compounds administered intravenously at the time of virus inoculation (mixed with the virus) was studied and contrasted with the effects of AZT. In order to circumvent the high toxicity of AZT and to obtain modes of treatment similar to those used for the polycyclic dione compounds described in U.S. Patent Application Serial No. 084,000, filed August 10, 1987, AZT was applied in only three intraperitoneal injections at 2, 5, and 16 hours after virus inoculation. Groups of mice (5 per group) were treated with AZT (3 intraperitoneal injections) and PS or HY (1 intravenous injection) alone and in combination and the survival of the animals was followed. The results are presented below in Table II and are graphically shown in Figures 1-3. TAS E II Addition % Survival at day 65 None 0 10 ug PS 40 50 ug PS 80 150 ug PS 100 10 ug HY 100 50 ug HY 80 150 ug HY 100 0.2 ug AZT 0 2 ug AZT 0 20 ug AZT 40 0.2 ug AZT + 50 PS 100 2 ug AZT + 50 PS 100 20 ug AZT + 50 PS 100 0.2 ug AZT + 50 HY 100 ug AZT + 50 HY 100 20 ug AZT + 50 HY 100 As shown in Figure 1 and Table II, while all FV- infected mice were dead by 30 days after virus inoculation, 40% of mice treated with 10 micrograms of PS, 80% of mice treated with 50 micrograms per mouse of PS and 100% of mice treated with 150 micrograms of PS were alive by day 65. In addition as shown in Figure 2 and Table II, 100% of the mice treated with 150 micrograms of HY and 80% of those treated with 10 or 50 micrograms of HY were alive at day 65. By contrast, AZT at doses ranging from 0.2 to 20 micrograms per mouse increased overall survival of FV inoculated mice only minimally. Combinations of AZT and HY or PS were synergistic (Figure 3 and Table II) , even at the lowest doses of AZT used. For example, when used at a concentration of 0.2 micrograms AZT per mouse, 100% of FV inoculated mice were dead by day 30, and at 50 micrograms per mouse of PS, 20% of mice are dead by day 30, but 100% of mice treated with 0.2 micrograms of AZT plus 50 micrograms of PS were alive at day 65 after FV inoculation. It should be noted that as of day 65 following FV inoculation, no mouse receiving a combination of any dose of AZT with any dose of PS or HY had died. These findings demonstrate the use of AZT plus PS/HY combination therapy in enabling the reduction of the frequency and concentrations of administered AZT, minimizing the side effects of the latter drug, without reducing its effectiveness. EXAMPLE 3: EFFECTS OF PS ON HIV REPLICATION IN VITRO 5, The effects of PS on HIV replication were studied in HUT-78 cells infected with HIV and contrasted with those of AZT. Infection was monitored by indirect immunofluorescence using antisera to the entire HIV (inactivated sera from HIV positive individuals and AIDS patients) , or to purified virus 0 components prepared in rabbits. The effect of pseudohypericin on the infection of cells by HIV was monitored by simultaneously mixing various dilutions of the compound with the virus and with uninfected cells and incubating the cultures in medium RPMI-1640 (GIBCO, Grand * 5 * Island, NY) supplemented with 10% heat inactivated fetal calf serum for 24, 48, 72 and 144 hours. The cells are then washed-, fixed and incubated with the anti-HIV antibody followed by the fluorescein labeled anti-immunoglobulin and the percentage of infected cells was determined. Since hypericin and 0 psuedohypericin must initially be solubilized in ethanol, the final alcohol concentrations did not exceed 1-2% and the controls of virus with cells contained the equivalent amounts of alcohol, since alcohol has been found to increase the percentage of cells which become infected with HIV. 5 Spread of infection with HIV was followed by co-cul¬ tivation of 1% of infected cells with 99% of uninfected cells in the presence of the appropriate concentrations of pseudo¬ hypericin. Samples of the cultures were collected after 24, 48, 72 and 144 hours after the onset of the experiment for 0 determination of percentage of infected cells by the indirect immunofluorescence techniques mentioned above. The results are present below in Table III. TAB fi III EFFECTS OF PS AND AZT ON HIV INFECTION Experiment No. _l: Percent Positive Cells Sample 24 hr 72 hr 144 hr CONTROL 2.7 26.7 62.9 CONTROL + EtOH 2.9 30.9 76.8 1 uM AZT 1.8 0.75 0.6 10 uM AZT 1 0.4 0.3 2 ug PS 3 52.8 77.9 10 ug PS 3.3 38 90.1 50 ug PS 0.2 1.2 18.8 Experiment No. 2: Sample 24 hr 48 hr 72 hr CONTROL 2.7 26 68 1 uM AZT 2 3 3 20 ug PS 2 22 58 40 ug PS 2 24 47 80 ug PS 2 5 29 The above experiments show that PS was effective in limiting the spread of HIV in HUT-78 cells in culture. Although AZT at 1 and 10 micromolar concentrations lead to a greater than 95% inhibition of the spread of HIV to uninfected cells (Table III) , PS at 50 micrograms decreased the spread of HIV infection about 80% at 144 hours post-infection (Table III, Experiment 1) and 80 micrograms of PS decreased the spread of HIV infection about 70% (Table III, Experiment 2). EXAMPLE 4: INHIBITION OF HIV BY THE COMPOSITION OF THE PRESENT INVENTION The activity of PS, HY, salts or mixtures thereof plus AZT or 2 ' , 2' didexoycytidine against human immunodeficiency virus (HIV) may be investigated in the following manner. HIV- infected, OKT4+ lymphoblastoid cells, such as clone H9 (described in Popovic, M. , et al. Science 224:497-500. 1984, incorporated by reference) or HUT 78 cells (Gazdar, AF et al. Blood 5_j5:409, 1980, incorporated by reference) or Molt-78 (available as ATCC CRL 1582 from the American Type Culture Collection, Rdckville, MD) are maintained in RPMI-1640 medium (GIBCO, Grand Island, New York) containing 20% fetal calf serum (Flow Laboratories, Inglewood, CA) . Triplicate cultures of cells, seeded at a concentration of about 4X10 5 cells per ml, are exposed to polybrene (2 micrograms per ml, Sigma Chemical Co., St. Louis, MO), infected with 2X10 8 HIV particles per 4X10 5 cells, and cultured in the presence or absence of the composition of the present invention as in Example 2 above. The antiviral activity of the composition of the present invention is determined by monitoring the reverse transcriptase activity and the expression of HIV proteins p24 and pl7, as described in Sarin, P.S. et al, _T. Nat. Cancer Inst. 211:663-665, 1987, incorporated herein by reference and is described below. EXPRESSION OF HIV GAG PROTEINS P24 AND P17. HUT-78, Molt-4 or H9 cells (2X10 5 ) , either uninfected or HIV infected, are continuously exposed to various concentra¬ tions of PS, HY and mixtures thereof at concentrations between 5 and 100 micrograms per ml plus 2', 3'-dideoxycytidine or AZT at 0.2 to 20 micrograms per ml for 4 days. The percentage of cells expressing p24 and pl7 gag proteins of HIV is determined by indirect immunofluorescence microscopy with the use of mouse monoclonal antibodies to HIV pl7 and p24 (available in numerous commercial sources such as those in HIV serum antigen detection kits from Abbott Labs, North Chicago, IL, and from DuPont, Wilmington, DE) . The positive cells are visualized by treat¬ ment with fluorescein-labeled goat anti-mouse IgG (Cappell Laboratories, Cochranville, PA) . The experiments are performed in duplicate and repeated at least three times. DETERMINATION OF REVERSE TRANSCRIPTASE ACTIVITY H9, HUT-78 or MOLT-4 cells infected with HIV (500 virus particles/cell) are exposed to various concentrations of PS, HY, and mixtures thereof plus AZT or 2', 3'-dideoxycytidine as above. At day 4, supernatants of the cultures are collected and virus particles are precipitated with polyethylene glycol and obtained by centrifugation as described above in Example 2 for FV. The virus pellet is suspended in 300 microliters of buffer containing 50 mM Tris-HCl (pH 7.5), 5mM dithiothreitol, 250 mM KC1, and 0.25% Triton X-100. Reverse transcriptase activity in these samples is analyzed in a 50 microliter reaction mixture containing 50 mM Tris-HCl (pH 7.5), 5mM dithiothreitol, 100 mM KC1, 0.01% Triton X-100, 10 microliters dTi5rA n as template primer, 10 mM MgCl2, 15 micro olar [ 3 H]dTTP (New England Nuclear, Boston, MA) , and 10 microliters of disrupted virus suspension. After incubation for 1 hour at 37"C and subsequent addition of 50 micrograms of yeast tRNA (Sigma Chemical, St. Louis, MO), the incorporation into the cold trichloroacetic acid-insoluble fraction is assayed. Assays are performed in duplicate and repeated three times. EXAMPLE 5: THE EFFECT OF THE COMPOSITION OF THE PRESENT INVENTION ON THE REPLICATION OF FELINE LEUKEMIA VIRUS Cats which test positive for feline leukemia virus (FeLV) viremia will be inoculated with PS or HY at doses of 5- 20mg/kg. Additional cats will be treated with a combination therapy consisting of a single dose of PS/HY and 4-40 doses administrations of AZT or 2', 3'-dideoxycytidine (Img/kg) , twice a day for various intervals of time. Serum levels of FeLV will then be followed and treatment will be resumed at the same regimens or adjusted with respect to the levels of viremia suppression obtained. (A control group of cats will receive the deoxynucleoside analog alone at identical concentrations) . The length of follow up will be determined by experimental considerations. A minimum of six months follow-up will be undertaken.

Claims: WHAT IS CLAIMED IS: 1. A method for treating a mammal afflicted with a disease caused by a retrovirus which comprises administering to a mammal in need of such treatment an effective antiviral amount of a nucleoside analog and a polycyclic dione compound selected from the group consisting of hypericin, pseudohyper- icin, salts and mixtures thereof. 2. The method of claim 1 wherein said nucleoside analog is selected from the group consisting of 2', 3' dideoxycytidine, 2', 3' dideoxyadenosine, 2', 3' dideoxythymidine, 2', 3' dideoxyguanosine and 3'-azido-3'- deoxythymidine. 3. The method of claim 1 which comprises administer- ing said effective amount orally. 4. The method of claim 1 which comprises administer- ing said effective amount parenterally. 5. The method of claim 1 which comprises administer- ing said effective amount intravenously. 6. The method of claim 1 wherein said polycyclic dione compound comprises hypericin. 7. The method of claim 1 wherein said polycyclic dione compound comprises pseudohypericin. 8. The method of claim 1 wherein said polycyclic dione compound comprises a mixture of hypericin and pseudo- hypericin. 9. The method of claim 1 wherein said effective amount comprises between about 1,000 and about 50,000 micrograms per kilogram body weight of said mammal of said nucleoside analog and between about 0.001 and about 100,000 micrograms of said polycyclic dione compound per kilogram body weight of said mammal. 10. A pharmaceutical formulation for treating mammals suffering from an infection caused by a retrovirus comprising an effective amount for treating retrovirus infection in mammals of a nucleoside analog and a polycyclic dione compound selected from the group consisting of hypericin, pseudo- hypericin, salts and mixtures thereof. 11. The pharmaceutical formulation of claim 10 wherein said nucleoside analog is selected from the group consisting of 2', 3' dideoxycytidine, 2', 3' dideoxyadenosine, 2', 3' dideoxythymidine, 2', 3' dideoxyguanosine and 3'-azido-3'- deoxythymidine. 12. The pharmaceutical formulation of claim 10 wherein said polycyclic dione compound comprises hypericin. 13. The pharmaceutical formulation of claim 10 wherein said polycyclic dione compound comprises pseudohypericin. 14. The pharmaceutical formulation of claim 10 wherein said polycyclic dione compound comprises a mixture of hypericin and pseudohypericin. 15. The pharmaceutical formulation of claim 10 comprising a parenteral dosage form. 16. The pharmaceutical formulation of claim 10 comprising an oral dosage form selected from the group consist- ing of a tablet and a capsule. 17. The pharmaceutical formulation of claim 10 wherein said effective amount comprises between about 1,000 and about 50,000 micrograms of said nucleoside analog and between about 0.001 and about 100,000 micrograms of said polycyclic dione compound. 18. The pharmaceutical formulation of claim 9 compris- ing a pharmaceutically-acceptable carrier or diluent. 19. The pharmaceutical formulation of claim 9 which comprises an i jectable solution. 20. The method of claim 1 wherein said retrovirus com- prises Human Immunodeficiency Virus. 21. The method of claim 1 wherein said virus comprises Friend Leukemia virus. 22. The method of claim 1 wherein said virus comprises Feline Leukemia Virus.

Inventors: LAVIE GAD, MERUELO DANIEL, LAVIE, GAD, MERUELO, DANIEL
Applicants: UNIV NEW YORK, NEW YORK UNIVERSITY
Application year: 1989
Main classification: A61K31
IPC codes: A61K31, C07H19