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WO1996011692A9 - Utilisation d'anthocyanidine et de ses derives dans le traitement des infections retrovirales - Google Patents

Utilisation d'anthocyanidine et de ses derives dans le traitement des infections retrovirales

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Publication number
WO1996011692A9
WO1996011692A9 PCT/NO1995/000185 NO9500185W WO9611692A9 WO 1996011692 A9 WO1996011692 A9 WO 1996011692A9 NO 9500185 W NO9500185 W NO 9500185W WO 9611692 A9 WO9611692 A9 WO 9611692A9
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WO
WIPO (PCT)
Prior art keywords
glycosyl
group
groups
anthocyanidin
acyl
Prior art date
Application number
PCT/NO1995/000185
Other languages
English (en)
Other versions
WO1996011692A1 (fr
Filing date
Publication date
Priority claimed from NO943860A external-priority patent/NO943860D0/no
Application filed filed Critical
Priority to AU37569/95A priority Critical patent/AU3756995A/en
Priority to EP95935625A priority patent/EP0785790A1/fr
Publication of WO1996011692A1 publication Critical patent/WO1996011692A1/fr
Publication of WO1996011692A9 publication Critical patent/WO1996011692A9/fr
Priority to NO971573A priority patent/NO971573L/no
Priority to FI971459A priority patent/FI971459A7/fi

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Definitions

  • 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 retrovirus 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.
  • AIDS acquired immune deficiency syndrome
  • a retrovirus designated human immunodeficiency virus 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 .
  • the complex disease AIDS includes 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.
  • the present invention discloses that anthocyanidin and anthocyanidin derivatives at rion-cytotoxic concentrations can exhibit antiviral effaces in HIV infected cells.
  • the present invention relates to the use of an anthocyanidin or an anthocyanidin derivative of the general formula I
  • R 1 , R 2 , R 3 and R 6 independently of each other are H, OH, C 1-6 -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 including a primate such as a human.
  • 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
  • the -O-glycosyl moiety may comprise at least two glycosyl groups and at least 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 1 is OCH 3 ,
  • R 2 is OH
  • R 3 is 6-O-(4-O-E-p-coumaroyl- ⁇ -L-rhamnopyranosyl)- ⁇ -D-glucopyranosyl,
  • R 4 is ⁇ -D-glucopyranosyl
  • R 5 is H
  • R 6 is OH.
  • 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.
  • 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).
  • HIV human immunodeficiency virus
  • HAV-1 human immunodeficiency virus
  • HIV-2 type 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 anthocyanidin or anthocyanidin derivatives.
  • 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).
  • 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 Lymphoprep methods (Nycodens), incubated with the test compounds, stimulated with phytohemagglutinin or cytokines and tested for their ability to incorporate radioactive
  • 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 compounds 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
  • tissue culture systems Using these tissue culture systems, pharmacokinetic properties 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 evaluate 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 interactions, template interactions and protein-protein interactions.
  • 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.
  • 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 extracts include different types of PCR analysis of viral
  • RNA and DNA nucleic acids
  • RNA and DNA nucleic acids
  • 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 stability of these cells to be stimulated by phytohemagglutinin or cytokines.
  • C 1-6 alkoxy are methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, tert.butoxy, pentoxy and hexoxy .
  • the alkoxy is selected from the group consisting of methoxy, ethoxy, propoxy, isopropoxy, and butoxy, such as R 1 , R 2 , R 3 , and/or R 4 being methoxy.
  • the anthocyanin or the anthocyanin derivative is derived from an anthocyanidin selected from the group consisting of pelargonidin, apigeninidin, and aurantinidin.
  • At least one of R 1 and R 2 is H, whereas in other embodiments at least one of R 1 and R 2 is OH.
  • 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.
  • at least one of R 1 and R 2 is alkoxy.
  • the anthocyanin or the anthocyanin derivative is derived from an anthocyanidin selected from the group consisting of peonidin, petunidin, malvidin, rosinidin, europinidin, hirsutidin, and capensinidin.
  • the glycosyloxy may be selected from the group consisting of mono-, di-, tri-, oligo-, polysaccharides, and derivatives thereof.
  • 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
  • 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, malonic acid, malic acid, maleic acid, and succinic acid.
  • the glycosyl group is a group derived from a monosaccharide selected from the group consisting of glucose, galactose, rhamnose, arabinose, xylose, and glucuronic acid.
  • 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 (sambubiose), 1,6-arabinosylglucoside, and 1,6-rhamnosylgalactoside.
  • the glycosyl group is a group derived from a trisaccharide selected from the group consisting of 1,2-glucosyl-1,6-glucosylglucoside, 1,2-glucosyl-1,6-rhamnosylglucoside, 1,2-xylosyl-1,6-glucosylglucoside, and 1,2-xylosyl-1,6-glucosylgalactoside.
  • 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 Deficiency Syndrome (AIDS)
  • HIV Human Immunodeficiency Virus
  • AIDS Acquired Immune Deficiency Syndrome
  • 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 arranced 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 with the exception of the compounds mentioned above.
  • the invention relates to a method for the preparation of a novel anthocyanidin or an anthocyanidin derivative 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 outlined in Example 1.
  • the man skilled in the art will be aware that in isolation and purification of known or novel anthocyanidin 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.
  • the compounds which are to be used according to the invention or novel compounds according to the invention 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.
  • 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 involving co-pigmentation of anthocyanins and intra- and intermolecular association states of anthocyanins are within the scope of the present invention. Each anthocyanin may exist on an extraordinary number of equilibrium forms.
  • the compounds of the present invention can occur as mixtures of diastereomers, racemic mixtures and as individual enantiomers. All asymmetric forms, individual isomers and combinations thereof are within the scope of the present invention.
  • compositions comprising mixtures of anthocyanins 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 isopropanol extraction of mountain ash berries as described in US
  • 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.
  • these compositions are based upon partially purified products from fruit or berries and, in addition to the anthocyanin, do also contain other compounds with a potential pharmaceutical activity such as flavonoids.
  • the present invention is based upon much more purified anthocyanins.
  • a further aspect of the invention thus relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an anthocyanidin or anthocyanidin derivative of the general formula I
  • 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 -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 above mentioned comoositions.
  • sample I contains one clean anthocyanin (called petanin) comprising an aglycone, three monosaccharide moieties and one arcmaaic 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 monosaccharide.
  • 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.
  • Sample III is a purified 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 doseresponse curve can be obtained for this sample.
  • a particular preferred embodiment of the invention relates to a pharmaceutical composition comprising petanin in combination 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 pharmaceutically acceptable excipient.
  • pharmaceutical compositions comprising a novel anthocyanin derivative in combination with a pharmaceutically acceptable excipient are within the concept of the present invention.
  • the particular counterion forming part of the salt of this invention is not 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.
  • pharmaceutically acceptable anion refers to anions in the salts of the above formula which are substantially non-toxic to living organisms.
  • Typical pharmaceutically acceptable anions include those derived from a mineral or organic acid.
  • inorganic acids examples include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid and the like
  • organic acids examples include p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid and the like.
  • anions are sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, proprionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propionate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylproprionate, phenylbutyrate, cit
  • compositions of the present invention are useful in the prevention or treatment of infection by the human immunodeficiency virus (HIV) and the treatment of consequent pathological conditions such as AIDS.
  • HIV human immunodeficiency virus
  • 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.
  • 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.
  • the compounds of the present invention may be administered orally, parenteraily (including subcutaneous 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.
  • a method for the prevention and/or treatment of a disease caused by a retrovirus comprising administering to a mammal in need thereof an effective amount of an anthocyanin derivative of the general formula I
  • 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 -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.
  • 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.
  • 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 suppositories.
  • these compositions When administered orally as a suspension, these compositions are prepared according to techniques well-known in the art of pharmaceutical, formulation and may contain macrocrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners/flavouring agents known in the art.
  • macrocrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners/flavouring agents known in the art.
  • these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.
  • compositions When administered by nasal aerosol or inhalation, these compositions 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 suitable 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, parenterally acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • suitable non-toxic, parenterally acceptable diluents or solvents such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • compositions 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, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperature but liquidity and/or dissolve in the rectal cavity to release the drug.
  • a suitable non-irritating excipient such as cocoa butter, synthetic 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-indicated conditions, with oral doses two to five times higher.
  • 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.
  • dosages of 100-400 mg every six hours are administered orally to each patient.
  • the anthocyanidin or anthocyanidin derivatives may be useful either as compounds or mixtures of compounds, pharmaceutically acceptable salts, pharmaceutical composition ingredients, either solely anthocyanidin or anthocyanidin derivatives or in combination with other anti-viral agents, immunomodulators, antibiotics or vaccines.
  • the compounds 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.
  • 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 concentrations dissolved in DMSO on the growth of SupTl cells measured after five days of incubation.
  • Figure 4 shows the effect of the first purified Vaccinium myrtillus sample (Sample II) in different concentrations dissolved in DMSO on the growth of SupTl cells measured after
  • 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 concentrations 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 incubated 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 incubated with only DMSO.
  • 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 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 anthocyanin isolated from Solanum tuberosum .
  • Figure 11 shows the anthocyanin content of the first purified Vaccinium myrtillus sample (Sample II) detected at 520 +
  • Figure 12 shows a) the structures and b) the relative proportions (%) of the individual anthocyanins in the first purified Vaccinium myrtillus sample (Sample II).
  • Figure 13 shows the anthocyanin content of the second purified Vaccinium myrtillus sample (Sample III) detected at
  • Figure 14 shows a) the structures and b) the relative proportions (%) of the individual anthocyanins in the second purified Vaccinium myrtillus sample (Sample III).
  • the human CD4+ lymphocyte cell line Sup Tl derived from a Non-Hodgkin's T-cell lymphoma patient was a gift from Dr. J. Sodroski at the Division of Human Retroviruses, Dana Farber Cancer Institute, 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, Walkersville, 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 0.1M antibiotic and antimycotic solution containing penicillin and fungizone) in 1 mM final concentration and gentamicine (Bio Whittaker) to a final concentration of 50 ⁇ g/ml at 37°C and 5% CO 2 in an incubator (Assab Kebo BioMed).
  • the cell line was established by infecting Molt 3 cells
  • 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
  • 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, 65, 55). A standard curve for the MTT assay was established (Fig.
  • 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 experiments are started by a hitherto unexperienced person. The reproducibility of the standard curve is good.
  • 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 infection 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 supernatant 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.
  • p24 Ag was measured using an ELISA based technique (Sundqvist et al. (1989), J. Medical Virology 21:170-175).
  • 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 ⁇ 10 4 cells/ml in a total volume of 5 ml. The test substances was added 30 minutes prior to the addition of the virus containing supernatant and during this preincubation the flasks were kept at 37°C and 5% CO 2 in an incubator (Assab Kebo BioMed). After preincubation, 500 ⁇ l 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).
  • Sample I Tubers (297 g) of Solanum tuberosum L. (anthocyanin pigmentation in skin and flesh) from cultivation at the Agricultural 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 containing 0.05% v/v concentrated hydrochloric acid.
  • 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 subjected to gel filtration.
  • the concentrated solution (ca. 100 ml) was washed twice with 100 ml ethyl acetate, and the lower layer was further concentrated under reduced pressure at 28°C before it was passed through an 18 ⁇ 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.
  • 300 ml each of 50 % aqueous methanol and anhydrous methanol (both containing 0.5% v/v CF 3 COOH) were used successively.
  • DCCC Droplet counter-current chromatography
  • High performance liquid chromatography was carried out using a slurry packed ODS-Hypersil column 20 ⁇ 0.5 cm, 5 ⁇ m). 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).
  • 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 ⁇ l 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 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 .
  • FIG. 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 droplet-current chromatography
  • D after Sephadex LH-20 gel filtration.
  • the different samples are monitored simultaneously at two different spectral areas (i and ii).
  • FIG. 10 shows the structure of petanin, which is the anthocyanin isolated from Solanum tuberosum. A sample of 10 mg of petanin was tested for biological activity 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 proportions (%) of the individual anthocyanins in the first purified Vaccinium myrtillus sample.
  • Figure 13 shows the anthocyanin content of the second purified 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 proportions (%) of the individual anthocyanins in the purified Vaccinium myrtillus sample.
  • 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.
  • 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.
  • 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 compounds.
  • 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.
  • 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.

Abstract

Utilisation d'une anthocyanidine ou d'un dérivé d'anthocyanidine répondant à la formule générale (I), dans laquelle R1, R2, R3 et R6 indépendamment les uns des autres, représentent H, OH, alcoxy, un groupe -O-glycosyle, un groupe -O-glycosyle substitué par un ou plusieurs groupes acyles, ou une fraction -O-glycosyle comportant au moins deux groupes glycosyles et au moins un groupe acyle disposés de telle sorte qu'au moins un groupe acyle se situe entre deux groupes glycosyles; R4 représente OH, alcoxy, un groupe -O-glycosyle substitué par un ou plusieurs groupes acyles, ou une fraction -O-glycosyle comportant au moins deux groupes glycosyles et au moins un groupe acyle disposés de telle sorte qu'au moins un groupe acyle se situe entre deux groupes glycosyles; R5 représente H ou OH; et Y représente un contre-ion ou un sel, promédicament ou complexe de celui-ci; dans la préparation d'une composition pharmaceutique destinée à la prophylaxie et/ou au traitement des infections rétrovirales chez les mammifères. On a également prévu de nouveaux dérivés d'anthocyanidine répondant à la formule générale (I), et des procédés de préparation de ces composés et de nouvelles compositions pharmaceutiques.
PCT/NO1995/000185 1994-10-13 1995-10-10 Utilisation d'anthocyanidine et de ses derives dans le traitement des infections retrovirales WO1996011692A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU37569/95A AU3756995A (en) 1994-10-13 1995-10-10 Use of anthocyanidin and derivatives for treatment of retroviral infections
EP95935625A EP0785790A1 (fr) 1994-10-13 1995-10-10 Utilisation d'anthocyanidine et de ses derives dans le traitement des infections retrovirales
NO971573A NO971573L (no) 1994-10-13 1997-04-07 Anvendelse av antocyaner samt derivater derav for behandling av retrovirale infeksjoner
FI971459A FI971459A7 (fi) 1994-10-13 1997-04-08 Antosyanidiinin ja sen johdannaisten käyttö retroviraalisten infektioiden hoitoon

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NO943860 1994-10-13
NO943860A NO943860D0 (no) 1994-10-13 1994-10-13 En kjemisk forbindelse og et preparat til bruk som terapeutikum

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WO1996011692A9 true WO1996011692A9 (fr) 1996-06-20

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WO1997041137A1 (fr) * 1996-04-17 1997-11-06 Unifob Utilisation d'anthocyanidine et de derives d'anthocyanidine
US7737121B2 (en) * 2004-07-29 2010-06-15 Board Of Trustees Of Michigan State University Insulin secretion by anthocyanins and anthocyanidins
EP1882473A1 (fr) 2006-07-28 2008-01-30 Indena S.P.A. Utilistation d'anthocyanosides pour la préparation de formulations pour le traitement de la mucosite induite par les médicaments antitumoraux

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EP0312222B1 (fr) * 1987-10-14 1992-08-05 Kabushiki Kaisha Ueno Seiyaku Oyo Kenkyujo Traitement de maladies virales
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