EP0362359A1 - Composition antivirale contenant des diones aromatiques polycycliques et des analogues de nucleosides, et procede de traitement d'infections retrovirales - Google Patents

Composition antivirale contenant des diones aromatiques polycycliques et des analogues de nucleosides, et procede de traitement d'infections retrovirales

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Publication number
EP0362359A1
EP0362359A1 EP89904668A EP89904668A EP0362359A1 EP 0362359 A1 EP0362359 A1 EP 0362359A1 EP 89904668 A EP89904668 A EP 89904668A EP 89904668 A EP89904668 A EP 89904668A EP 0362359 A1 EP0362359 A1 EP 0362359A1
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European Patent Office
Prior art keywords
hypericin
pharmaceutical formulation
dione compound
micrograms
virus
Prior art date
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EP89904668A
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German (de)
English (en)
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EP0362359A4 (en
Inventor
Daniel Meruelo
Gad Lavie
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New York University NYU
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New York University NYU
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Publication of EP0362359A1 publication Critical patent/EP0362359A1/fr
Publication of EP0362359A4 publication Critical patent/EP0362359A4/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals

Definitions

  • 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.
  • 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) .
  • HIV human immunodeficiency virus
  • HIV-l and HIV-2 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.
  • 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.
  • AZA -azido-3'- deoxythymidine
  • cDNA premature complementary DNA chain termination
  • AZT is currently used to treat certain patients suffering from Acquired Immunodeficiency Syndrome (AIDS) caused by HIV.
  • AIDS Acquired Immunodeficiency Syndrome
  • 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.
  • a dose-dependent suppression of bone marrow, resulting in anemia and leukopenia an abnormally low number of leukocytes in the circulating blood
  • This has limited the effectiveness of AZT for the treatment of AIDS.
  • 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.
  • 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.
  • 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.
  • nucleoside analogs such as AZT
  • 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.
  • 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.
  • 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.
  • 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.
  • nucleoside analogs typified by AZT
  • 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.
  • antiretroviral-effective nucleoside analog treatments utilizing drugs such as AZT
  • 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.
  • 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.
  • AZT and related nucleo- sides analogs work by causing premature chain termination of growing viral cDNA.
  • 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) .
  • 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.
  • Protein above 60% by weight 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
  • Morphology spherical enveloped virions 80-120-nm diameter
  • variable surface projections 8-nm diameter
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • the minimum dosage of the aromatic polycyclic dione component of this regimen may be decreased if desired or appropriate.
  • the minimum effective dosages will be even smaller.
  • 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.
  • an ⁇ tiviral activity of hypericin is a function of the frequency of treatment.
  • a single dose of ten micrograms per mouse was less effective than a single dose of 100 micrograms per mouse, as expected.
  • administration of 10 micrograms every day for ten days was less effective than even a single 10-microgram dose.
  • 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.
  • mice may not be applicable to other mammals or humans
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • PSEUDOHYPERICIN FROM ST JOHN'S WORT Hypericin, (CsoHi ⁇ Og, 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 Schenta ⁇ . 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.
  • hypericin HY
  • Rf 0.45 a yield of 0.19 grams
  • PS pseudohypericin
  • Rf 0.35 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.
  • Friend Leukemia Virus 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) .
  • SFFV spleen focus forming virus
  • F-MuLv Murine Friend Leukemia Helper Virus
  • 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.
  • spleen size (splenomegaly) of BALB/c mice (Jackson Labs, Bar Harbor, ME) was examined.
  • 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.
  • mice treated with 10 micrograms of PS 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.
  • AZT dose-dependent AZT
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ) 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.
  • 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,
  • 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
  • yeast tRNA Sigma Chemical, St. Louis, MO
  • 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.

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Abstract

Procédé de traitement d'infections rétrovirales chez les mammifères par lequel on administre des quantités efficaces anti-rétrovirales d'un analogue de nucléoside et d'un dione polycyclique aromatique choisi dans le groupe formé d'hypéricine, de pseudohypéricine, de sels et de mélanges de ces substances. On décrit également des compositions permettant de traiter des infections rétrovirales chez des mammifères, comportant des quantités efficaces anti-rétrovirales d'un analogue de nucléoside et d'un dione polycyclique aromatique choisi dans le groupe formé d'hypéricine, de pseudohypéricine, de sels et de mélanges de ces substances.
EP19890904668 1988-03-23 1989-03-22 Antiviral composition containing aromatic polycyclic diones and nucleoside analogs and method for treating retrovirus infections Withdrawn EP0362359A4 (en)

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US17206488A 1988-03-23 1988-03-23
US172064 1988-03-23
US32417789A 1989-03-16 1989-03-16
US324177 1989-03-16
US32639289A 1989-03-20 1989-03-20
US326392 1989-03-20

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DE332679T1 (de) * 1987-08-10 1990-04-12 New York University, New York, N.Y. Antivirale zusammensetzungen mit aromatischen polycyclischen dionen sowie methode zur behandlung retroviraler infektionen.
CZ285232B6 (cs) * 1991-05-16 1999-06-16 Glaxo Group Limited Protivirové směsi
DE19547317A1 (de) * 1995-12-18 1998-12-24 Dreluso Pharm Dr Elten & Sohn Antivirales Medikament
US6607754B1 (en) * 1997-07-11 2003-08-19 Upsher-Smith Laboratories, Inc. Delivery of Hypericum perforatum (St. John's Wort) in tablet form

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DK586989A (da) 1990-01-19
EP0362359A4 (en) 1991-11-21
WO1989009056A1 (fr) 1989-10-05
DK586989D0 (da) 1989-11-22

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