USE OF ANTHOCYANIDIN AND DERIVATIVES FOR TREATMENT OF RETROVIRAL INFECTIONS

Abstract: The invention relates to the use of an anthocyanidin or an anthocyanidin derivative of general formula (I) wherein R1, R2, R3 and R6 independently of each other is H, OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or more acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R4 is OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or more acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R5 is H, OH, and Y is a counterion or a salt, prodrug or complex thereof for the preparation of a pharmaceutical composition for the prevention and/or treatment of retroviral infections in mammals, as well as to novel anthocyanidin derivatives of general formula (I) and methods for preparation of said compounds and novel pharmaceutical compositions.

Description: SE OF ANTHOCYANIDIN AND DERIVATIVES FOR TREATMENT OF RETROVIRAL INFECTIONS The present invention relates to the use of an anthocyanidin or an anthocyanidin derivative of the general formula I or a pharmaceutically acceptable salt, prodrug or complex thereof for the preparation of a pharmaceutical composition for the prevention and/or treatment of a disease caused by a retro- virus in a mammal including a primate such as a human. The composition may be useful in the prevention of infection by HIV, the treatment of infection by HIV and/or the treatment of the resulting acquired immune deficiency syndrome (AIDS) and in the treatment of other retrovirus-related diseases. BACKGROUND OF THE INVENTION A retrovirus designated human immunodeficiency virus (HIV) is the causative agent of the complex disease termed Acquired Immune Deficiency Virus (AIDS) , and is a member of the lenti- virus family of retroviruses. M.A. Gonda, F. Wong-Staal, R.C. Gallo, "Sequence Homology and Morphological Similarity of HTLV III and Visna Virus, A Pathogenic Lentivirus", Science 227, 173 (1985) ; P. Sonigo, N. Alizon et al. , "Nucleotide Sequence of the Visna Lentivirus: Relationship to the AIDS Virus", Cell 42, 369 (1985) . The complex disease AIDS in¬ cludes progressive destruction of the immune system and degeneration of the central and peripheral nervous systems. The HIV virus was previously known or referred to as LAV, HTLV-III or ARV. Other retrovirus-related diseases are described in John M. Coffin, "Retroviridae and Their Replication", pp. 1437-1500 in "Virology", second edition, ed. by B.N. Fields, D.M. Knipe et al., Raven Press, Ltd. New York, 1990, see e.g. Table I. Anthocyanins are the most important group of water-soluble plant pigments visible to the human eye. As the anthocyanins seem to have non-toxic effects on the human being, their possible pharmaceutical use has been further investigated. DETAILED DESCRIPTION OF THE INVENTION The present invention discloses that anthocyanidin and antho¬ cyanidin derivatives at non-cytotoxic concentrations can exhibit antiviral effects in HIV infected cells. The present invention relates to the use of an anthocyanidin or an anthocyanidin derivative of the general formula I wherein R χ , R 2 , R 3 and R 6 independently of each other are H, OH, C^ g - alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or more acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R 4 is OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or one acyl groups, or an -O- glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R 5 is H, OH, and Y is a counterion, or a prodrug or complex thereof for the preparation of a pharmaceutical composition for the prevention and/or treat- ment of a disease caused by a retrovirus in a mammal includ¬ ing a primate such as a human. In particular, the invention relates to the use of a compound wherein at least one of R 3 , R 4 , and R 6 is an -O-glycosyl group, an -O-glycosyl group which is substituted with at least one acyl group, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups. The -O-glycosyl moiety may comprise at least two glycosyl groups and at least one acyl group arranged alternate with one glycosyl followed by one acyl group; an acyl group may also be located at the very end of the moiety. A presently preferred embodiment of the invention is the use of the compound petanin wherein, with reference to formula I, R_ is OCH 3 , R 2 is OH, R 3 is 6-0- (4-O-E-p-coumaroyl-Qf-L-rhamnopyranosyl) -jS-D-gluco- pyranosyl , R 4 is /3-D-glucopyranosyl , R 5 is H , and R 6 is OH . Other presently preferred embodiments are the use of the individual anthocyanins outlined in Tables I and II below, e.g. in compositions wherein the relative quantities of the various anthocyanins are as outlined in Table I or Table II. TABLE I Structures and relative proportions (%) of the individual antho cyanins in the first purified Vaccinium myrtillus sample (sampl II) with reference to formula I, R 4 is OH, R 5 is H and R 6 is OH COMPOUND Proportions (%) -O-galactosyl 10.6 -O-glucosyl 10.7 -O-galactosyl 6.8 -O-arabinosyl 10.6 -O-glucosyl 8.1 -O-galactosyl 4.6 -O-arabinosyl * -O-glucosyl 15.2* -O-galactosyl 1.2 -O-arabinosyl 2.9 -O-glucosyl ** -O-galactosyl 10.3** -O-glucosyl 14.1 -O-arabinosyl 0.9 -O-arabinosyl 4.0 * Pigment 7 and 8 together ** Pigment 11 and 12 together TABLE II Structures and relative proportions (%) of the individual antho¬ cyanins in the second purified Vaccinium myrtillus sample (sample III) with reference to formula I, R 4 is OH, R 5 is H and R 6 is OH COMPOUND Proportions (%) * Pigment 7 and 8 together ** Pigment 10, 11 and 12 together The compound or mixture of compounds may be further defined as an anthocyanidin or an anthocyanidin derivative, which, when dissolved in DMSO at a concentration so that the final concentration of DMSO does not exceed 0.2% v/v DMSO, and tested as described in section 2.3, does not have a cytotoxic effect on the growth of uninfected SupTl cells resulting in a decrease in OD 580 of more than 10% as a result of incubation with the anthocyanidin or the anthocyanidin derivative, and when tested as described in section 3 has an antiviral effect on the growth of Molt 3 IIIB cells infected with HIV-1 defined as a decrease in syncytia formation of more than 10% as a result of 48 hours of incubation with the anthocyanidin or the anthocyanidin derivative. It is contemplated that the anthocyanidin or anthocyanidin derivatives and pharmaceutically acceptable salts thereof inhibits the reverse transcriptase or HIV integrase encoded by human immunodeficiency virus (HIV) type 1 (HIV-1) and type 2 (HIV-2) . The exact mechanism of action is yet unknown, but if desired it can be further analysed by PCR of the various intermediates in the replication cycle in order to ascertain at which stage the replication is inhibited by the antho¬ cyanidin or anthocyanidin derivatives. Based upon the disclosure of the present invention, the person skilled in the art will be able to test the compounds of formula I as outlined above without using inventive skill. Substances which are considered useful may then be tested for cytotoxic effects in other appropriate cell systems such as different fibroblasts (e.g. HeLa) or other uninfected T-cell lines, uninfected T-cell (e.g. cell lines from ATCC) , and in primary human lymphocytes from blood donors, e.g. enriched for CD4+ cells by means of Dynabeads ® . Furthermore, toxicity tests may be performed such as single dose toxicity tests, e.g. LD 50 (i.e. the dosage at which half of the experimental animals die) . In addition to the LD 50 value in rodents it is desirable to determine the highest tolerated dose and/or lowest lethal dose for other species, e.g. dog and rabbit. If the in vi tro test results are promising and the LD 50 is high, clinical experiments using humans may be approved taking into consideration that at present no treatment exists of AIDS. The person skilled in the art would by use of methods described in standard textbooks, guidelines and regulations as well as common general knowledge within the field be able to select the exact dosage regimen to be implemented for any selected compound using merely routine experimentation proce- dures. During the process, the person skilled in the art may decide not to continue studying all the initially selected com¬ pounds, or it may be decided to synthesize and test new com¬ pounds in view of the initial toxicity and biological results obtained. Experiments in progress to further characterize general biological effects and antiviral properties of anthocyanidins or anthocyanidin derivatives To further study the effect of anthocyanidins in relation to antiviral potentials of these compounds and to get a mecha¬ nistic understanding of how these compounds interfere with the replication of retroviruses such as HIV, a number of different experiments have been initiated or designed. The experiments can be divided into groups according to the goals of the studies: I. Cytotoxic effects i) In tissue culture studies ii) In vivo studies in mice II. In vi tro studies with defined enzymatic systems i) Reverse transcriptase (RT) ii) Integrase (IN) III. Antiviral effects of the compounds measured in tissue culture studies i) Testing the effect of the compounds on the labora- tory strain of HIV in different cell lines ii) Analysis of the progress of retroviral replication in the presence of compounds at concentrations giving substantial inhibition of cytopathogenic effects and virus production iii) Testing the effect of the compounds on HIV iso¬ lated from patients in primary cell lines Studies in group I i) includes characterization of effects of the compounds on cell growth of a number of different estab¬ lished cell lines like the CD4+ human cell lines with lympho- cytic phenotypes (Jurkat, CME, H-9, Molt-3, all from ATCC) , the monocytic cell line U937 (also from ATCC) , and a CD4+ HeLa (fibroblast) cell line with the ability to permit repli¬ cation of the laboratory strain of HIV when exposed to this virus. The studies also include the human epithelial cell lines SGHTL-34 (derived from gl. thyroidea) and 293 (derived from kidney) obtained from Professor Johan R. Lillehaug, Department of biochemistry and molecular biology, University of Bergen, Norway) and which cannot be infected with HIV due to the absence of CD4 receptors. Peripheral human lymphocytes are also included in these studies. These cells are isolated from normal healthy blood donors, isolated by standard Ly - phoprep methods (Nycodens) , incubated with the test com¬ pounds, stimulated with phytohemagglutinin or cytokines and tested for their ability to incorporate radioactive thymidine. The aim of these studies is to determine what doses of the test compounds human cells can tolerate without affecting the growth potential of these cells. Furthermore, these studies will be expanded to include long term effects on the cells of low concentrations of the test compounds. At doses where growth is affected, the aim is to study the mechanisms of growth inhibition. To get a general idea of how these com¬ pounds interact with cells at toxic or semitoxic doses, the cells are first characterized after treatment with test compounds using electron microscopy. Based on the results of those studies, different biochemical studies will be designed to further elucidate the mechanism behind the cytotoxic effects Using these tissue culture systems, pharmacokinetic proper¬ ties of the compounds will be studied, the goal being to evaluate the efficiency of uptake as well as the stability of the compounds in human cells. The main goal of the group I ii) studies is to determine LD 50 in mice. As part of these studies, it is also desired to eva¬ luate the clearance of the different compounds by analyzing urine samples from the treated animals. The group II i) and ii) studies include a number of different tests designed to find how the test compounds interact with different activities of IN and RT like substrate interac¬ tions, template interactions and protein-protein interac¬ tions. III i) Based on the results from the group I i) studies, the effect of the test compounds at doses not affecting cell growth on syncytia formation and virus production will be studied in the same cell lines. The aim is to find if there are cell line specificities with respect to the antiviral activities of the compounds studied. At doses giving an inhibition of viral production, the group III ii) studies will be conducted. These experiments involve extraction of viral components from infected cells after treatment with the test compounds.. The analysis of the ex- tracts include different types of PCR analysis of viral nucleic acids (RNA and DNA) to determine at what stage of the replication cycle inhibition occurs. These studies will be complemented with analysis of viral proteins in the extracts. For the protein analysis, the viral proteins will be meta- bolically labelled during infection and treatment, precipi¬ tated with specific antisera and/or antibodies, and analyzed by SDS-PAGE and autoradiography. Since the laboratory strains of HIV do not efficiently infect CD4+ cells from healthy individuals, virus isolated from HIV- positive individuals will be used to infect freshly isolated CD4+ cells from blood donors. The cells will be tested with test compounds at concentrations not influencing the stabi¬ lity of these cells to be stimulated by phytohemagglutinin or cytokines. Examples of "C^g alkoxy" are methoxy, ethoxy, propoxy, iso- propoxy, n-butoxy, isobutoxy, tert.butoxy, pentoxy and hex- oxy. In one embodiment of the invention the alkoxy is selected from the group consisting of methoxy, ethoxy, propoxy, iso- propoxy, and butoxy, such as R lr R 2 , R 3 , and/or R 4 being methoxy. In a presently preferred embodiment of the inven- tion, the anthocyanin or the anthocyanin derivative is derived from an anthocyanidin selected from the group con¬ sisting of pelargonidin, apigeninidin, and aurantinidin. In certain embodiments of the invention at least one of R x and R 2 is H, whereas in other embodiments at least one of R-, and R 2 is OH. In a presently preferred embodiment, the antho¬ cyanin or the anthocyanin derivative is derived from an anthocyanidin selected from the group consisting of cyanidin, delphinidin, luteolinidin, tricetinidin, 6-hydroxy-cyanidin, 6-hydroxy-delphinidin, 5-methyl-cyanidin, and pulchellidin. In still other embodiments, at least one of R-_ and R 2 is alkoxy. It is presently preferred that in this embodiment the anthocyanin or the anthocyanin derivative is derived from an anthocyanidin selected from the group consisting of peonidin, petunidin, alvidin, rosinidin, europinidin, hirsutidin, and capensinidin. The glycosyloxy may be selected from the group consisting of mono-, di-, tri-, oligo-, polysaccharides, and derivatives thereof. In particular, the glycosyloxy may be substituted with one or more acyl groups, or the glycosyl may comprise at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups. In particular, the acyl group may be selected from the group consisting of acyl groups derived from aromatic and aliphatic acyl groups, such as the group consisting of 4-coumaric acid, caffeic acid, ferulic acid, sinapic acid, 4-hydroxybenzoic acid, gallic acid, acetic acid, oxalic acid, alonic acid, malic acid, maleic acid, and succinic acid. In one embodiment of the invention, the glycosyl group is a group derived from a monosaccharide selected from the group consisting of glucose, galactose, rhamnose, arabinose, xylose, and glucuronic acid. In another embodiment, the glycosyl group is a group derived from a disaccharide selected from the group consisting of 1,2-glucosylglucoside (sophorose) , 1,3-glucosylglucoside (laminariobiose) , 1, 6-glucosylglucoside (gentiobiose) , 1,2- xylosylgalactoside (lathyrose) , 1,2-rhamnosylglucoside (neo- hesperidose) , 1,6-rhamnosylglucoside (rutinose) , 1,2-xylosyl- glucoside (sambubiose) , 1,6-arabinosylglucoside, and 1,6- rhamnosylgalactoside. In a third embodiment, the glycosyl group is a group derived from a trisaccharide selected from the group consisting of 1,2-glucosyl-1,6-glucosylglucoside, 1,2-glucosy1-1,6-rhamno¬ sylglucoside, 1,2-xylosyl-1, 6-glucosylglucoside, and 1,2- xylosyl-1, 6-glucosylgalactoside. Although it is contemplated that the compositions may be useful for the treatment of all retrovirus-related diseases, the invention in particular relates to the use of a compound for the prevention or treatment of infection by a retrovirus such as infection by Human Immunodeficiency Virus (HIV) and/or for prevention or treatment of Acquired Immune Defi¬ ciency Syndrome (AIDS) . Some of the compounds within the general formula I are known, see e.g. "The Flavonoids", ed. J. B. Harborne, T.J. Mabry and H. Mabry, Chapman & Hall, 1975, "The Flavonoids. Advances in Research", ed. J.B. Harborne and T.J. Mabry, Chapman & Hall, 1982, "The Flavonoids. Advances in Research since 1980", ed. J.B. Harborne, Chapman & Hall, 1988, "The Flavonoids. Advances in Research since 1986", ed. J.B. Harborne, Chapman & Hall, 1994 and references in Chemical Abstract, Vol. 119 to 123 under the General Subject Index entry Anthocyanins. However, the invention in a further aspect relates to novel anthocyanin derivatives of the general formula I wherein R l7 R 2 , R 3 and R 6 independently of each other are H, OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or more acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R 4 is OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or one acyl groups, or an -0- glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R 5 is H, OH , and Y is a counterion, or a prodrug or complex thereof with the exception of the compounds mentioned above. Furthermore, the invention relates to a method for the prepa¬ ration of a novel anthocyanidin or an anthocyanidin deriva¬ tive of the general formula I as defined above, the method comprising isolation and purification of the anthocyanidin or an anthocyanidin derivative essentially by the method out- lined in Example 1. The man skilled in the art will be aware that in isolation and purification of known or novel antho¬ cyanidin and anthocyanidin derivatives, the method described in Example 1 may be amended as appropriate e.g. by use of other extraction procedures and chromatographic techniques. Alternatively, the compounds which are to be used according to the invention or novel compounds according to the inven¬ tion may be synthesized e.g. as described in Iacobucci. G.A. and Sweeny, J. G. (1983) , "The chemistry of anthocyanins, anthocyanidins and related flavylium salts", Tetrahedron, 39, pp. 3005-3038 or as described in Elhabiri, M. et al. (1995), "Anthocyanin chemical synthesis: an important access to natural and synthetic pigments", Polyphenols Actuali tes , No. 13, pp. 11-13. Chemical synthesis of the anthocyanidins and the anthocyanidin derivatives may give appropriate amendments to stabilize the compounds. In general, anthocyanins from blueberries are rather simple anthocyanins. Compared to other anthocyanins, in particular those acylated with aromatic acids like petanin (Sample I) , they are more unstable and may therefore be less useful for pharmaceutical purposes. Thus, forms of anthocyanins involv¬ ing co-pigmentation of anthocyanins and intra- and inter¬ molecular association states of anthocyanins are within the scope of the present invention. Each anthocyanin may exist on an extraordinary number of equilibrium forms. Together with the variation of building blocks of each anthocyanin and the possibility of existing in several association states (including association with metal ions such as Mg 2+ , Fe 2+ , Fe 3+ and Al 3+ , other phenolics such as cinnamic acids and other flavonoids, and polymeric mate¬ rial) this allows quite a number of structural modifications which may influence effects/activity. As a consequence of asymmetric centres, the compounds of the present invention can occur as mixtures of diastereomers, racemic mixtures and as individual enantiomers. All asymme¬ tric forms, individual isomers and combinations thereof are within the scope of the present invention. Pharmaceutical compositions comprising mixtures of antho- cyanins derived from e.g. blueberries such as Myrtocyan ® { Vaccinium myrtillus anthocyanosides corresponding to 25% as anthocyanidines) as well as topical medicinal compositions containing fruit juice or fermented fruit juice as described in CA 1086651, a topical composition consisting of an isopro- panol extraction of mountain ash berries as described in US 4,132,782, alcoholic extracts of anthocyanosides described in FR 2456747, compositions comprising bilberry anthocyanidines, grape anthocyanidines or elder anthocyanidines described in GB 1,589,294 and anthocyanidin glycosides extracted from bilberries, black currents and blackberries described in US 3,546,337 are known. However, these compositions are based upon partially purified products from fruit or berries and, in addition to the anthocyanin, do also contain other com¬ pounds with a potential pharmaceutical activity such as flavonoids. In contrast, the present invention is based upon much more purified anthocyanins. A further aspect of the invention thus relates to a pharma¬ ceutical composition comprising an anthocyanidin or antho¬ cyanidin derivative of the general formula I wherein R R 3 and R 6 independently of each other are H, OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or more acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R 4 is OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or one acyl groups, or an -0- glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R 5 is H, OH, and Y is a counterion, or a prodrug or complex thereof with the exception of the above mentioned compositions. The cytotoxic and antiviral effects in HIV infected cells have been examined for one anthocyanin sample isolated from blue potatoes ( Solanu tuberosum) (Sample I) and two antho¬ cyanin samples isolated from blueberries { Vaccinium myrtillus (Samples II and III) . Sample I contains one clean anthocyanin (called petanin) comprising an aglycone, three monosaccharide moieties and one aromatic acyl group. Samples II and III both contain a mixture of anthocyanins. Each anthocyanin in these mixtures are built from only one aglycone and one monosaccha- ride. Sample III contains the same, however, a reduced number of anthocyanin compared to Sample II. Sample I which contains only one, rather complex anthocyanin, shows the best test results. For sample II at concentrations above 20 μg/ml, an antiviral effect is observed (Figure 7) . When studying the figure, it is noted that an antiviral effect can also be observed at a concentration of 1 μg/ml, however, this effect is not clearly reproduced at concentrations between 1 and 20 μg/ml. In this respect, it should be emphasized that Sample III is a puri¬ fied sample of Sample II which may include compounds with no antiviral effect as well as compounds which do have the desired antiviral effect. This fact may explain that no dose- response curve can be obtained for this sample. Further studies may be performed with respect to various con¬ centrations of the compounds and mixtures of compounds accor¬ ding to the invention in order to titrate the exact concen- tration at which a cytotoxic effect or an antiviral effect is obtained. A particular preferred embodiment of the invention relates to a pharmaceutical composition comprising petanin in combina¬ tion with a pharmaceutically acceptable excipient. Other preferred embodiments are pharmaceutical compositions comprising a mixture of individual anthocyanins as outlined in Table I or in Table II in combination with a pharmaceuti¬ cally acceptable excipient. Also pharmaceutical compositions comprising a novel anthocyanin derivative in combination with a pharmaceutically acceptable excipient are within the con¬ cept of the present invention. With respect to the counterion Y, it should be recognized that the particular counterion forming part of the salt of this invention is noc of a critical nature, as long as it is compatible with the anthocyanidin or anthocyanidin derivative cation. The counterion is in particular a pharmacologically acceptable anion. The counterion may be organic as well as inorganic in nature. The term "pharmaceutically acceptable anion" as used herein refers to anions in the salts of the above formula which are substantially non-toxic to living organisms. Typical pharma¬ ceutically acceptable anions include those derived from a mineral or organic acid. Examples of such inorganic acids are hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid and the like, and examples of the organic acids are p- toluenesulfonic acid, methanesulfonic acid, oxalic acid, p- bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid and the like. Examples of the anions are sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, di- hydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, proprionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, pro¬ pionate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-l,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylproprionate, phenyl- butyrate, citrate, lactate, γ-hydroxybutyrate, glycollate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1- sulfonate, naphthalene-2-sulfonate, and mandelate anions, and the like. Preferred anions are those derived from mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as maleic acid and methanesulfonic acid. The compositions of the present invention are useful in the prevention or treatment of infection by the human immunodefi¬ ciency virus (HIV) and the treatment of consequent pathologi¬ cal conditions such as AIDS. Treating AIDS or preventing or treating infection by HIV is defined as including, but not being limited to, treating a wide range of states of HIV infection: AIDS, ARC (AIDS related complex) , both symptomatic and asymptomatic, and actual or potential exposure to HIV. For example, the compositions of this invention are useful in treating infection by HIV after suspected past exposure to HIV by, e.g., blood transfusion, organ transplant, exchange of body fluids, bites, accidental needle stick, or exposure to patient blood during surgery. For these purposes, the compounds of the present invention may be administered orally, parenterally (including subcuta¬ neous injections, intravenous, intramuscular, intrasternal injection or infusion techniques) , by inhalation spray, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. Thus, in accordance with the present invention there is fur¬ ther provided a method for the prevention and/or treatment of a disease caused by a retrovirus, the method comprising admi¬ nistering to a mammal in need thereof an effective amount of an anthocyanin derivative of the general formula I wherein R 1# R 2 , R 3 and R 6 independently of each other are H, OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or more acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups R 4 is OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or one acyl groups, or an -0- glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R 5 is H, OH, and Y is a counterion, or a prodrug or complex thereof. The treatment involves administering to a patient in need of such treatment a pharmaceutical composition comprising a pharmaceutical carrier and a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof. These pharmaceutical compositions may be in the form of orally administrable suspensions or tablets; nasal sprays; sterile injectable preparations, for example, as sterile injectable aqueous or oleaginous suspensions or supposito- ries. When administered orally as a suspension, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhan¬ cer, and sweeteners/flavouring agents known in the art. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, mag¬ nesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art. When administered by nasal aerosol or inhalation, these com¬ positions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other sui¬ table preservatives, absorption promoters to enhance bio- availability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenteral¬ ly acceptable diluents or solvents, such as mannitol, 1,3- butanediol, water, Ringer's solution or isotonic sodium chlo¬ ride solution, or suitable dispersing or wetting and suspend¬ ing agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. When rectally administered in the form of suppositories, these compositions may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, syn¬ thetic glyceride esters or polyethylene glycols, which are solid at ordinary temperature but liquidity and/or dissolve in the rectal cavity to release the drug. Dosage levels of the order of 0.02 to 5.0 or 10.0 g per day are useful in the treatment or prevention of the above-indi¬ cated conditions, with oral doses two to five times higher. For example, infection by HIV is effectively treated by the administration of from 1.0 to 50 mg of the compound per kg of body weight from one to four times per day. In one preferred regimen, dosages of 100-400 mg every six hours are admini¬ stered orally to each patient. It will be understood, how¬ ever, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of ac¬ tion of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excre¬ tion, drug combination, the severity of the particular con¬ dition, and the host undergoing therapy. The anthocyanidin or anthocyanidin derivatives may be useful either as compounds or mixtures of compounds, pharmaceuti- cally acceptable salts, pharmaceutical composition ingredi¬ ents, either solely anthocyanidin or anthocyanidin deriva¬ tives or in combination with other anti-viral agents, immuno¬ modulators, antibiotics or vaccines. For example, the com¬ pounds of this invention may be effectively administered, whether at periods of pre-exposure and/or post-exposure, in combination with effective amounts of other antiviral agents, immunomodulators, anti-infectives, or vaccines known to those of ordinary skill in the art. LEGEND TO FIGURES Figure 1 shows the relationship between cell number and staining by MTT. Figure 2 shows the effect of DMSO on cell growth and that 0.33% DMSO can be used as a solvent for the compounds without affecting cell growth. Figure 3 shows the effect of petanin in different concentra¬ tions dissolved in DMSO on the growth of SupTl cells measured after five days of incubation. Figure 4 shows the effect of the first purified Vacciπium myrtillus sample (Sample II) in different concentrations dissolved in DMSO on the growth of SupTl cells measured after 48 hours of incubation. Figure 5 shows the effect of the second purified Vaccinium myrtillus sample (Sample III) in different concentrations dissolved in DMSO on the growth of SupTl cells measured after five days of incubation. Figure 6 shows the effect of petanin in different concentra¬ tions on the inhibition of formation of syncytia. The effect is shown as a percentage of the formation of syncytia in cells incubated with only DMSO. Figure 7 shows the effect of the first purified Vaccinium myrtillus sample (Sample II) in different concentrations on the inhibition of formation of syncytia. The effect is shown as a percentage of the formation of syncytia in cells incu¬ bated with only DMSO. Figure 8 shows the effect of the second purified Vaccinium myrtillus sample (Sample III) in different concentrations on the inhibition of formation of syncytia. The effect is shown as a percentage of the formation of syncytia in cells incu¬ bated with only DMSO. Figure 9 shows the high performance liquid chromatography pro- files of the anthocyanin content of Solanum tuberosu during the purification procedure. A, crude extract; B, after parti¬ tion against ethyl acetate and treatment with Amberlite XAD- 7; C, after droplet-current chromatography; D, after Sephadex LH-20 gel filtration. The different samples are monitored simultaneously at two different spectral areas (i and ii) . Figure 10 shows the structure of petanin, which is the antho¬ cyanin isolated from Solanum tuberosum. Figure 11 shows the anthocyanin content of the first purified Vaccinium myrtillus sample (Sample II) detected at 520 ± 20 n . The peaks are labelled according to the numbers given in Figure 12. Figure 12 shows a) the structures and b) the relative propor¬ tions (%) of the individual anthocyanins in the first puri¬ fied Vaccinium myrtillus sample (Sample II) . Figure 13 shows the anthocyanin content of the second puri- fied Vaccinium myrtillus sample (Sample III) detected at 520 ± 20 nm. The peaks are labelled according to the numbers given in Figure 14. Figure 14 shows a) the structures and b) the relative propor¬ tions (%) of the individual anthocyanins in the second puri- fied Vaccinium myrtillus sample (Sample III) . EXAMPLES TEST METHODS Determination of cytotoxic and antiviral effects in HIV infected cells of compounds or mixtures of compounds accord- ing to the invention 1. Cultivation of cells The human CD4+ lymphocyte cell line Sup Tl derived from a Non-Hodgkin's T-cell lymphoma patient (Smith et al. (1984), Cancer Research 44, 5657) was a gift from Dr. J. Sodroski at the Division of Human Retroviruses, Dana Farber Cancer Insti¬ tute, Harvard Medical School, Boston, U.S.A., and was chosen for these studies due to its high content of CD4+ receptors and ability to form large syncytia following infection with HIV-1. The cells were cultivated as suspension cultures in plastic flasks (NUNC, Copenhagen, Denmark - T25 flasks or T125 flasks) in RPMI 1640 medium (Bio Whittaker, Walkers- ville, MD, USA) supplemented with 5% v/v fetal calf serum, 2 mM glutamine (both from Bio Whittaker) and ABAM (Cat.No. A 9909, Sigma Chem. Company, an 0.IM antibiotic and antimycotic solution containing penicillin and fungizone) in 1 mM final concentration and gentamicine (Bio Whittaker) to a final concentration of 50 μg/ml at 37°C and 5% C0 2 in an incubator (Assab Kebo BioMed) . Counting of cell numbers was performed the same day the experiments started using the Trypan blue exclusion method (Tissue Culture Chemicals, a catalogue from Sigma, 1994) and a Burker counting chamber ("ASSISTENT", Germany) at a magni¬ fication of 400x. The ratio between the living and dead cells was at least 95/5 in all experiments determined as described in John Paul, "Cell & Tissue Culture", p. 368, Fifth Edition, Churchill Livingstone, 1975) . Prior to the experiments the medium was half-changed in order to add new growth compo¬ nents. The cell density was adjusted to approximately 5 x 10 5 cells/ml and kept at this concentration throughout the experiment by counting the cell number and adding new medium as appropriate or, if necessary, by centrifugation of the cell suspension and resuspension of the cell pellet in an appropriate amount of RPMI 1640 medium. HIV virus producing Molt 3 IIIB cell line The cell line was established by infecting Molt 3 cells (American Type Culture Collection, ATCC CRL 1552) with the HIV-1 strain HTLV IIIB obtained from Dr. W. A. Haseltine at the Division of Human Retroviruses, Dana Farber Cancer Insti¬ tute, Harvard Medical School, Boston, U.S.A. The Molt 3 IIIB cell line is producing virus particles constitutively. The cells were cultivated as suspension cultures in plastic flasks (NUNC, Copenhagen, Denmark - T25 flasks or T125 flasks) in RPMI 1640 medium (Bio Whittaker, Walkersville, MD, USA) supplemented with 5% v/v fetal calf serum, 2 mM glutamine (both from Bio Whittaker) and ABAM (Cat. o. A 9909, Sigma Chem. Company, an 0.1M antibiotic and antimycotic solution containing penicillin and fungizone) in 1 mM final concentration and gentamicine (Bio Whittaker) to a final concentration of 50 μg/ml at 37°C and 5% C0 2 in an incubator (Assab Kebo BioMed) . 2. Cvtotoxicitv of the compounds or mixture of compounds tested 2.1. The MTT assay method for determining the number of viable cells The principle of this assay is based on the cleavage of the yellow tetrazolium salt MTT (3- (4,5-dimethylthiazol-2-yl) - 2,5-diphenyltetrazolium bromide (Thiazolyl blue, Product No. M 5655, Sigma Chemical Company) to form formazan crystal due to the dehydrogenase activity in the living cells (Mosman, T. et al. J. Immunol. Methods, 6_5_, 55) . A standard curve for the MTT assay was established (Fig. 1) by diluting exponentially growing SupTl cells at known cell numbers in a standard medium (RPMI 1640) into a 96 wells tissue plate (NUNC) at a total volume of 100 μl followed by adding 50 μl of MTT rea- gent (3 mg/ml in phosphate buffer solution (PBS), pH 7.20) to each well. After addition of the MTT reagent, the plate was incubated at 37°C and 5% C0 2 for 3 hours in an incubator (Assab Kebo BioMed) . Then the cells were centrifuged at 2,000 rpm (800 x g) for 10 minutes in a centrifuge equipped with micro-titer plate holders (Beckman centrifuge, GS-6) . After centrifugation 100 μl of supernatant was removed from the wells. For this purpose a multichannel micro-pipette was used (Finnpipettes, Finland) . The pelleted cells were resuspended in 100 μl DMSO (dimethyl sulfoxide, Merck) and the plates were gently shaken by hand for about 10 minutes at room temperature before the absorption was read in an ELISA reader (Titerek ® Multiskan Plus MK II photometer equipped with a 580 nm light filter (Flow Laboratories, USA) . The standard curve of the relationship between cell number and staining by MTT is shown in Figure 1 with a ranging from 10 3 cells/well to 5 x 10 4 cells/well corresponding to OD 580 = 0.01 and OD 580 = 0.50, respectively. As shown in Figure 1, within the amount of cells used, there is a linear relationship between the number of living cells and the intensity of staining between cell numbers of 20.000 and 60.000. A new standard curve is established as appropriate e.g. when a new series of experi¬ ments are started by a hitherto unexperienced person. The reproducibility of the standard curve is good. 2.2. Determination of the effect of DMSO on cell growth Since the water solubility of the compounds to be tested varies, the compound or mixture of compounds to be tested are dissolved in DMSO prior to addition to the cell cultures. The effect of DMSO on the cell growth was therefore tested. The cells were added to a 96 wells micro-titer plate; each well containing 1 x 10 4 cells in 100 μl of RPMI 1640 medium. To the suspension of cells was then added DMSO at different concentrations ranging from 0.01% v/v DMSO to 1.0% v/v DMSO. Following incubation in an incubator (Assab Kebo BioMed) at 37°C with 5% C0 2 , the amount of living cells as a function of the DMSO concentration was evaluated after 1, 2, and 5 days of incubation by applying the MTT assay as described above. From the results of the experiments (Figure 2) it can be deducted that the maximum concentration of DMSO that could be used without affecting cell growth was 0.2% v/v. Above that concentration DMSO has a significant effect on the growth of SupTl cells. At 0.2% v/v concentration or lower of DMSO, practically no difference between cells with or without DMSO could be observed. For this reason compounds to be tested in SupTl cultures in the presence of DMSO have to be kept in solutions at concentrations so that the final concentration of DMSO does not exceed 0.2% v/v DMSO. 2.3. Effect of test substances on the growth of uninfected SupTl cells For each substance to be tested or for each mixture of sub¬ stances to be tested, 3 parallel experiments were done. Survival of cells were tested after 1, 2, 5, and 7 days, respectively, after starting treatment of the cells with the substances. The cells were maintained in a 96 wells micro- titer plate. To each well 1 x 10 4 cells in 100 μl RPMI 1640 medium were added. To the suspension of cells was then added 10 μl of the test substance in DMSO and RPMI 1640 medium in order to ensure that the final concentration of DMSO did not exceed 0.2% v/v. As control, cells, with or without DMSO were used. At the end of incubation at 37°C and 5% C0 2 for 3 hours in an incubator (Assab Kebo BioMed)) with the compounds, survival of the cells was measured by adding the MTT reagent and the samples were processed as described above in section 2.2. The results of the 3 extracts containing 3 different com¬ pounds or mixture of compounds dissolved in DMSO after five days of incubation are shown in Figures 3-5. 3. Testing compounds or mixture of compounds for antiviral effects The screening of antiviral effect of different compounds or mixtures of compounds was based on measuring the formation of syncytia as the exact number of syncytia present after infec¬ tion of cells with HIV-1 can easily be counted by use of an inverse microscope and thereby an effect obtained by the compound or mixture of compounds added can be measured. HIV-1 containing supernatant from Molt 3 IIIB cell super- natant was prepared by centrifugation of the Molt 3 IIIB cell culture at 1,000 rpm in a Beckmann GS-6 centrifuge equipped with a GH-3.7 rotor for 5 minutes. In order to standardize the supernatant with respect to the amount of virus, p24 Ag was measured using an ELISA based technique (Sundqvist et al. (1989), J. Medical Virology 2,9:170-175) . Each virus supernatant used in the experiment had a p24 Ag concentration of 1.5 - 2 ng/10 5 cells. Each T25 (NUNC) flask was filled with 1 x 10 4 cells/ml in a total volume of 5 ml. The test substances was added 30 minutes prior to the addi- tion of the virus containing supernatant and during this pre- incubation the flasks were kept at 37°C and 5% C0 2 in an incubator (Assab Kebo BioMed) . After preincubation, 500 μl of virus supernatant was added. The number of syncytia was counted after 24 and 48 hours of incubation at 37°C and 5% CO in an incubator (Assab Kebo BioMed) (this time was found to be the standard times for optimal syncytia formation for this cell line at the concentration of virus used) . For each test substance 2 flasks were used and the syncytia were counted by counting the number of syncytia at 5 diffe- rent places on each flask in an inverse microscope (Olympus CK 2) using a magnification of lOx, thus giving 10 indepen¬ dent countings for each test substance. The parallels ob¬ tained were within +- 10% for each experiment. The results of the 3 extracts containing 3 different com¬ pounds or mixture of compounds are shown in Figures 6-8. For each compound or mixture of compounds, the inhibition of formation of syncytia is shown as a percentage of the forma¬ tion of syncytia in untreated cells. EXAMPLES EXAMPLE 1 Isolation and purification of the anthocyanin, petanin, from Sample I, blue potatoes (Solanum tuberosum L.) and a mixture of anthocyanins from Samples II and III, blueberries ( Vacci ¬ nium myrtillus L.) Samples Sample I: Tubers (297 g) of Solanum tuberosum L. (anthocyanin pigmentation in skin and flesh) from cultivation at the Agri- cultural University of Norway, NLH-As, Norway, were collected in October 1994, cut with a pair of scissors and extracted for 3 hours (three times) with methanol containing 0.1% v/v concentrated hydrochloric acid. Sample II and Sample III: Ripe berries of Vaccinium myrtillus L. were collected in Asane near Bergen on the west coast of Norway in August 1992. The frozen berries (100 g) were extracted for 5 hours (twice) with 500 ml of methanol con¬ taining 0.05% v/v concentrated hydrochloric acid. For all three samples: The filtered extracts were combined and concentrated under reduced pressure at 28°C. Procedure for purification of the samples For sample III, the lower layer of n-butanol-acetic acid- water (4:1:5, v/v) was used as mobile phase. A flow rate of 9 ml/hour was used throughout the experiment. Some stationary phase (110 ml) was displaced prior to elution of the first drop of mobile phase. Then 150 fractions, each of 4 ml, were collected. Fractions 13-15 were collected and concentrated under reduced pressure at 28°C before the sample was sub¬ jected to gel filtration. The concentrated solution (ca. 100 ml) was washed twice with 100 ml ethyl acetate, and the lower layer was further concen¬ trated under reduced pressure at 28°C before it was passed through an 18 x 2.6 cm Amberlite ® XAD-7 column (an ion exchange resin from BDH Chemicals Ltd.) which had been washed in advance with distilled water. The XAD-7 column (with the adsorbed anthocyanins) was washed with 2 1 of distilled water. To elute the anthocyanins, 300 ml each of 50 % aqueous methanol and anhydrous methanol (both containing 0.5% v/v CF 3 COOH) were used successively. Droplet counter-current chromatography (DCCC) was carried out using a Tokyo Rikakikai Eyela Model DCC-300 chromatograph fitted with 300 glass capillaries (40 cm x 2 mm i.d.) . For Sample I, the upper layer of n-butanol-acetic acid-water (4:1:5, v/v) was used as mobile phase. A flow rate of 10 ml/hour was used throughout the experiment. Some statio¬ nary phase (100 ml) was displaced prior to elution of the first drop of mobile phase. Then 45 fractions, each of 7 ml, were collected. Fractions 12-35 were collected and concen¬ trated under reduced pressure at 2.8°C before the sample was subjected to gel filtration. For Sample II, the lower layer of n-butanol-acetic acid-water (4:1:5, v/v) was used as mobile phase. A flow rate of 10 ml/hour were used throughout the experiment. Some statio¬ nary phase (150 ml) was displaced prior to elution of the first drop of mobile phase. Then 160 fractions, each of 4 ml, were collected. Fractions 20-100 were collected and concen- trated under reduced pressure at 28°C before the sample was subjected to gel filtration. For Sample III, the lower layer of n-butanol-acetic acid- water (4:1:5, v/v) was used as mobile phase. A flow rate of 9 ml/hour was used throughout the experiment. Some stationary phase (110 ml) was displaced prior to elution of the first drop of mobile phase. Then 150 fractions, each of 4 ml, were collected. Fractions 13-15 were collected and concentrated under reduced pressure at 28°C before the sample was subject- ed to gel filtration. Gel filtration was performed on a 100 x 3 cm Sephadex ® LH-20 column using 40% v/v aqueous methanol containing 1% v/v CF 3 COOH as eluent. All the anthocyanin fractions belonging to each sample were put together and evaporated to dryness under reduced pressure. Monitoring of fractions Thin-layer chromatography (TLC) analyses were performed on 0. mm cellulose layers (Schleicher and Schϋll, F1440) in the following solvent systems: A. Formic acid-concentrated hydrochloric acid-water (5:1:5, v/v) B. n-Butanol-acetic acid-water (4:1:5, v/v, upper phase) . High performance liquid chromatography (HPLC) was carried out using a slurry packed ODS-Hypersil column 20 x 0.5 cm, 5 μm) . Two solvents were used for elution (A: formic acid-water (1:9, v/v) and B: formic-acid-water-methanol (1:4:5, v/v). Several slightly different elution profiles were used: A typical elution profile was composed of isocratic elution (90% v/v A, 10% B) over 4 min, linear gradient from 10% v/v B to 100% B over the next 17 min, followed by linear gradient from 100% B to 10% v/v B over 1 min. The flow rate was 1.5 ml min -1 , and aliquots of 10 μl were injected. UV/Vis absorption spectra were recorded using a photodiode array detector (HP 1050, Hewlett-Packard) , and spectral measurements were made over the wavelength range 210-600 nm. The relative quantities of the individual anthocyanins in the purified Vaccinium myrtillus sample (Sample II) were based on integration of the different peaks in the HPLC chromatogram (Figure 11) of the purified sample. This chromatogram was recorded by measuring the absorption values on every second nm between 500 and 540 nm simultaneously, and do not take into account the different molar absorption coefficients of the individual anthocyanins. In a similar manner, the relative quantities of the indi- vidual anthocyanins in the second purified Vaccinium myr¬ tillus sample (Sample III) were based on integration of the different peaks in the HPLC chromatogram (Figure 13) of the purified sample. Contents of Sample I Figure 9 shows the high performance liquid chromatography profiles of the anthocyanin content of Solanum tuberosum during the purification procedure. A, crude extract; B, after partition against ethyl acetate and treatment with Amberlite XAD-7; C, after drople -current chromatography; D, after Sephadex LH-20 gel filtration. The.different samples are monitored simultaneously at two different spectral areas (i and ii) . Figure 10 shows the structure of petanin, which is the antho¬ cyanin isolated from Solanum tuberosum. A sample of 10 mg of petanin was tested for biological acti¬ vity as described in Example 2.1. Contents of Sample II Figure 11 shows the anthocyanin content of the first purified Vaccinium myrtillus sample detected at 520 ± 20 nm. The peaks are labelled according to the numbers given in Figure 12. Figure 12 shows the structures and b) the relative propor¬ tions (%) of the individual anthocyanins in the first puri¬ fied Vaccinium myrtillus sample. A sample (600 mg) of this purified anthocyanin mixture of Vaccinium myrtillus was tested for biological activity as described in Example 2.2. Contents of Sample III Figure 13 shows the anthocyanin content of the second puri¬ fied Vaccinium myrtillus sample detected at 520 ± 20 nm. The peaks are labelled according to the numbers given in Figure 14. Figure 14 shows a) the structures and b) the relative propor¬ tions (%) of the individual anthocyanins in the purified Vaccinium myrtillus sample. A sample (16 mg) of this purified anthocyanin mixture of Vaccinium myrtillus was tested for biological activity as described in Example 2.3. EXAMPLE 2 Determination of cytotoxic and antiviral effects in HIV in¬ fected cells in three different extracts containing compounds or mixtures of compounds according to the invention The cytotoxic effect and the antiviral effect in HIV infected cells of the three extracts obtained according to Example 1 were tested as described in Test Methods, sections 2 and 3. Cytotoxic effect of a compound or mixture of compounds is defined here as the concentration of the compound or mixture of compounds which effects the growth rate of the cells tested. Here, a cytotoxic effect of a compound or mixture of compounds is considered present if a decrease in OD 580 of more than 10% is observed as a result of incubation with the compound or mixture of compounds. With respect to the cytotoxic effect, the results are shown in Figures 3-5. An antiviral effect is here considered present if a decrease in syncytia formation of more than 10% is observed as a result of incubation with the compound or mixture of com¬ pounds. With respect to the antiviral effect, the results are shown in Figures 6-8 wherein for each compound or mixture of compounds the inhibition of formation of syncytia is shown as a percentage of the formation of syncytia in untreated cells. In Figures 6-8 are shown the results after 24 hours and/or 48 hours. At 48 hours the same pattern is observed although the total amount of syncytia is higher. All three compounds or mixture of compounds have a clear inhibitory effect on the cytopathogenic effect of HIV although complete inhibition of syncytia formation cannot be obtained at the experimental conditions used. I Cytotoxic and antiviral effect of Sample I At concentrations between 0.02 and 0.2 mg/ml, a cytotoxic effect on cell growth rate is observed (Figure 3) . At concentrations above 1 μg/ml, an antiviral effect is observed (Figure 6) . II Cytotoxic and antiviral effect of Sample II At concentrations above 40 μg/ml, a cytotoxic effect on cell growth rate is observed (Figure 4) . At concentrations above 20 μg/ml, an antiviral effect is observed (Figure 7) . When studying the figure, it is noted that an antiviral effect can also be observed at a concentra¬ tion of 1 μg/ml, however, this effect is not clearly repro- duced at concentrations between 1 and 20 μg/ml. Ill Cytotoxic and antiviral effect of Sample III At concentrations above 0,03 mg/ml, a cytotoxic effect on cell growth rate is observed (Figure 5) . At concentrations above 0.1 μg/ml, an antiviral effect is ob- served (Figure 8) .

Claims: CLAIMS 1. The use of an anthocyanidin or an anthocyanidin derivative of the general formula I wherein R-, R 2 , R 3 and R 6 independently of each other are H, OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or more acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R 4 is OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or one acyl groups, or an -O- glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R 5 is H, OH, and Y is a counterion, or a salt, prodrug or complex thereof for the preparation of a pharmaceutical composition for the prevention and/or treat¬ ment of a disease caused by a retrovirus in a mammal includ¬ ing a primate such as a human. 2. The use according to claim 1, wherein the anthocyanidin or the anthocyanidin derivative, when dissolved in DMSO at a concentration so that the final concentration of DMSO does not exceed 0.2% v/v DMSO, a) when tested as described in section 2.3, the anthocyani¬ din or the anthocyanidin derivative does not have a cytotoxic effect on the growth of uninfected SupTl cells resulting in a decrease in OD 580 of more than 10% as a result of incubation with the anthocyanidin or the antho- cyanidin derivative, and b) when tested as described in section 3, the anthocyanidin or the anthocyanidin derivative has an antiviral effect on the growth of Molt 3 IIIB cells infected with HIV-1 defined as a decrease in syncytia formation of more than 10% as a result of 48 hours of incubation with the antho¬ cyanidin or the anthocyanidin derivative. 3. The use according to claim 1 or 2, wherein alkoxy is selected from the group consisting of methoxy, ethoxy, pro¬ poxy, isopropoxy, and butoxy. 4. The use according to claim 3, wherein R 1# R 2 , R 3 , and/or R 4 is methoxy. 5. The use according to any of claims 1-4, wherein at least one of R λ and R 2 is H. 6. The use according to any of claims 1-4, wherein at least one of R x and R 2 is OH. 7. The use according to any of claims 1-3, wherein at least one of R and R 2 is alkoxy. 8. The use according to claim 5, wherein the anthocyanin or the anthocyanin derivative is derived from an anthocyanidin selected from the group consisting of pelargonidin, apigeni- nidin, and aurantinidin. 9. The use according to claim 6, wherein the anthocyanin or the anthocyanin derivative is derived from an anthocyanidin selected from the group consisting of cyanidin, delphinidin, luteolinidin, tricetinidin, 6-hydroxy-cyanidin, 6-hydroxy- delphinidin, 5-methyl-cyanidin, and pulchellidin. 10. The use according to claim 7, wherein the anthocyanin or the anthocyanin derivative is derived from an anthocyanidin selected from the group consisting of peonidin, petunidin, malvidin, rosinidin, europinidin, hirsutidin, and capensini- din. 11. The use according to any of the preceding claims, wherein the glycosyloxy is selected from the group consisting of mono-, di-, tri-, oligo-, polysaccharides, and derivatives thereof. 12. The use according to claim 11, wherein the glycosyl group is substituted with one or more acyl groups, or the glycosyl moiety comprises at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups. 13. The use according to claim 12, wherein the acyl group is selected from the group consisting of aromatic and aliphatic acyl groups. 14. The use according to claim 13, wherein the acyl group is selected from the group consisting of acyl groups derived from 4-coumaric acid, caffeic acid, ferulic acid, sinapic acid, 4-hydroxybenzoic acid, gallic acid, acetic acid, oxalic acid, malonic acid, malic acid, maleic acid, and succinic acid. 15. The use according to any one of claims 11-14, wherein the glycosyl group is a group derived from a monosaccharide selected from the group consisting of glucose, galactose, rhamnose, arabinose, xylose, and glucuronic acid. 16. The use according to any one of claims 11-14, wherein the glycosyl group is a group derived from a disaccharide selected from the group consisting of 1,2-glucosylglucoside (sophorose) , 1,3-glucosylglucoside (laminariobiose) , 1,6- glucosylglucoside (gentiobiose) , 1,2-xylosylgalactoside (lathyrose) , 1,2-rhamnosylglucoside (neohesperidose) , 1,6- rhamnosylglucoside (rutinose) , 1,2-xylosylglucoside (sambu- biose) , 1,6-arabinosylglucoside, and 1,6-rhamnosylgalacto- side. 17. The use according to any one of claims 11-14, wherein the glycosyl group is a group derived from a trisaccharide selected from the group consisting of 1,2-glucosyl-l,6-gluco¬ sylglucoside, 1,2-glucosyl-l,6-rhamnosylglucoside, 1,2-xylo- syl-1, 6-glucosylglucoside, and 1,2-xylosyl-l,6-glucosylgalac- toside. 18. The use according to claim 1, wherein at least one of R 3 , R 4 , and R 6 is an -O-glycosyl, an -O-glycosyl group which is substituted with at least one acyl group, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups. 19. The use according to claim 1, wherein R x is OCH 3 , R 2 is OH, R 3 is 6-0- (4-O-E-p-coumaroyl-α-L-rhaτnnopyranosyl) -0-D-gluco- pyranosyl, R 4 is (3-D-glucopyranosyl, R 5 is H, and R 6 is OH. 20. The use according to claim 1, wherein the composition con¬ sists of the individual anthocyanins outlined in Table I, preferably in the relative quantities outlined in the table. 21. The use according to claim 1, wherein the composition consists of the individual anthocyanins outlined in Table II, preferably in the relative quantities outlined in the table. 22. The use according to any of the preceding claims for the prevention or treatment of infection by a retrovirus such as infection by Human Immunodeficiency Virus (HIV) and/or for prevention or treatment of Acquired Immune Deficiency Syn¬ drome (AIDS) . 23. A novel anthocyanin derivative of the general formula I wherein R χ , R 2 , R 3 and R 6 independently of each other are H, OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or more acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R 4 is OH, alkoxy, an -O-glycosyl, an -O-glycosyl group which is substituted with one or one acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R 5 is H , OH , and Y is a counterion, or a prodrug or complex thereof with the exception of the compounds mentioned in "The Flavonoids", ed. J. B. Harborne, T.J. Mabry and H. Mabry, Chapman & Hall, 1975, "The Flavonoids. Advances in Research", ed. J.B. Harborne and T.J. Mabry, Chapman & Hall, 1982, "The Flavonoids. Advances in Research since 1980", ed. J.B. Harborne, Chapman & Hall, 1988, "The Flavonoids. Advances in Research since 1986", ed. J.B. Harborne, Chapman & Hall, 1994 and in references in Chemical Abstract, Vol. 119 and 123 under the General Subject Index entry Anthocyanins. 24. A pharmaceutical composition comprising an anthocyanidin or anthocyanidin derivative of the general formula I wherein Rx, R 2 , R 3 and R 6 independently of each other are H, OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or more acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R 4 is OH, alkoxy, an -O-glycosyl, an -O-glycosyl group which is substituted with one or one acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R 5 is H, OH, and Y is a counterion, or a prodrug or complex thereof with the exception of Myrto- cyan ® ( Vaccinium myrtillus anthocyanosides corresponding to 25% as anthocyanidines) , topical medicinal compositions containing fruit juice or fermented fruit juice as described in CA 1086651, a topical composition consisting of an isopro- panol extraction of mountain ash berries as described in US 4,132,782, the alcoholic extracts of anthocyanosides described in FR 2456747, the compositions comprising bilberry anthocyanidines, grape anthocyanidines or elder anthocyani¬ dines described in GB 1,589,294 and the anthocyanidin glycosides extracted from bilberries, black currents and blackberries described in US 3,546,337. 25. A pharmaceutical composition comprising petanin in combi¬ nation with a pharmaceutically acceptable excipient. 26. A pharmaceutical composition comprising a mixture of individual anthocyanins as outlined in Table I in combination with a pharmaceutically acceptable excipient. 27. A pharmaceutical composition comprising a mixture of individual anthocyanins as outlined in Table II in combina¬ tion with a pharmaceutically acceptable excipient. 28. A pharmaceutical composition comprising a novel antho¬ cyanin derivative according to claim 23 in combination with a pharmaceutically acceptable excipient. 29. A method for the preparation of an anthocyanidin or an anthocyanidin derivative of the general formula I as defined in claim 23, the method comprising isolation and purification of the anthocyanidin or an anthocyanidin derivative by the methods outlined in Example 1. 30. A method for the prevention and/or treatment of a disease caused by a retrovirus, the method comprising administering to a mammal in need thereof an effective amount of an antho¬ cyanin derivative of the general formula I wherein ^1' R. R 3 and R 6 independently of each other are H, OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or more acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R 4 is OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or one acyl groups, or an -O- glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R 5 is H, OH, and Y is a counterion, or a prodrug or complex thereof.

Inventors: ANDERSEN OEYVIND M, HELLAND DAG E, ANDERSEN, OEYVIND, M., HELLAND, DAG, E.
Applicants: ANDERSEN OEYVIND M, HELLAND DAG E, UNIFOB STIFTELSEN UNIVERSITETS, ANDERSEN, OEYVIND, M., HELLAND, DAG, E., UNIFOB STIFTELSEN UNIVERSITETSFORSKNING BERGEN
Application year: 1995
Main classification: A61K31
IPC codes: A61K31