Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses
  • A61P31/22—Antivirals for DNA viruses for herpes viruses

Definitions

• the present invention generally relates to the technical field of compounds and methods for the treatment or prophylaxis of herpes-type virus infections.
• the present invention more specifically relates to the technical field of the use of sophorolipids as antiviral agents for the use in treating herpes-related viruses and virus infections.
• herpes family of viruses are known to cause a variety of disorders throughout the world. It has been shown that these viruses can create cellular disorders and are known to cause genetic and cellular anomalies. Although there are several methods and agents for treating herpes-related viruses, the inventors are not aware of any uses of sophorolipids for treating such viruses.
• Sophorolipids are microbial extracellular glycolipids that can be produced by resting cells of Candida bombicola.
• the chemical composition of sophorolipids is constituted by a disaccharide sugar, namely, sophorose, and a fatty acid or an ester group.
• Candida bombicola can produce the sophorolipids as a mixture of macrolactones and free acid structures that are acetylated to various extents at the primary hydroxyl sophorose ring positions (see FIG. 1 ). See Bisht, K.S. et al., J. Org. Chem., vol. 64, pp. 780-789 (1999); Gorin, P.A. et al., Can. J. Chem., vol. 39, p.
• sophorolipids are produced during fermentation. Davila, A.M. et al., J. Chromatogr., vol. 648, p. 139 (1993). The main component of the sophorolipid molecule is 17-hydroxyoctadecanoic acid and its corresponding lactone. Tulloch, A.P. et al., Can. J. Chem., vol. 40, p. 1326 (1962) and Tulloch, A.P. et al., Can J. Chem., vol. 46, p. 3337 (1968).
• glycolipids may be useful in treating very severe immune disorders.
• glycolipids have been reported to be of interest for in vivo cancer treatment/anti-tumor cell activity, treatment of autoimmune disorders, in vivo and in vitro anti-endotoxic (septic) shock activity, regulation of angiogenesis, and apoptosis induction, all by cytokine activity.
• U.S. Patent No. 5,597,573 to Massey U.S. Patent No. 5,514,661 to Piljac
• U.S. Patent No. 5,648,343 to Carlson and the references cited in notes 9-13 of Bisht, K.S. et al., J. Org. Chem, vol. 64, pp. 780-789 (1999).
• the inventors are not aware of any work other than the inventors' work that has been carried out regarding using sophorolipids to treat herpes-related viruses.
• sophorolipids for the treatment of herpes-related viral infections in humans, and other similar and related purposes, that the present invention is directed.
• Ethyl 17-L-[(2 ' -O- ⁇ -D-glucopyranosyl- ⁇ -D-glucopyranosyl)-oxy]-cis-9- octadecenoate then was synthesized and then further treated to obtain Ethyl 17- L[(2'-0- ⁇ -D glucopyranosyl- ⁇ -D-glucopyranosyl)-oxy]-cis-9-octadecenoate 6',6"- diacetate.
• Methyl 17-L-[(2 ' -0- ⁇ -[>glucopyranosyl- ⁇ -D-glucopyranosyl)-oxy]-cis-9- octadecenoate-6"-acetate also was synthesized by adding Lipase to a solution of methyl ester, (325.4 mg) and vinyl acetate (230.9 ⁇ l) in dry THF (5 ml), and further treatment.
• Epstein-Barr Virus was used as a model organism to test the anti- herpes virus activity of sophorolipids.
• P3HR-1 EBV were adsorbed on Daudi lymphoid cell lines and sophorolipids added. After incubation the supernatant of the medium was assayed using ELISA method for viroid capsid antigen (VCA).
• VCA viroid capsid antigen
• Table 1 five tested compounds displayed some degree of anti-herpes virus activity. The best activity was displayed by Ethyl 17-L-[(2 ' -O- ⁇ - D-glucopyranosyl- ⁇ -D-glucopyranosyl)-oxy]-cis-9-octadecenoate, which showed high activity.
• results of the assay show that natural mixtures of lactonic, non-lactonic, ethyl, methyl esters of sophorolipids and 6',6"-diacetate ethyl sophorolipids can act as an anti-herpes virus agents.
• sophorolipids in field of medicine would be tremendous to treat various infections, apart from other applications. Further, while sophorolipids can find applications in diversified fields, the present invention indicates that sophorolipids could be used as anti viral agents.
• FIG. 1 are representative structures of sophorolipids produced by Candida bombicola, with FIG. 1A showing lactonic sophorolipid and FIG. 1 B showing open ring sophorolipid.
• General embodiments of the present invention are sophorolipids mixtures and methods of using such mixtures for the prophylaxis or treatment of humans and animals for herpes-related virus infections.
• One exemplary embodiment of this invention includes a method for treating a viral infection caused in a subject comprising the step of administering an effective amount of one or more sophorolipid.
• the term "effective amount”, as used herein, includes a prophylactically effective amount and refers to an amount effective in treating or preventing a viral infection in a subject (e.g. human or animal) either as monotherapy or in combination with other agents.
• the sophorolipid is Ethyl 17-L-[(2 ' -0- ⁇ -D-glucopyranosyl- ⁇ -D-glucopyranosyl)-oxy]-cis- 9-octadecenoate.
• the sophorolipids were synthesized by fermentation using Candida bombicola.
• the fermentation media was composed of glucose 100 g, yeast extract 10 g, urea 1 g and oleic acid 40 g in 1000 ml of water.
• sophorolipids were extracted thrice using ethyl acetate. The extracts were pooled and the solvent therein removed. The obtained product was then washed with hexane to remove the residual fatty acids. In this embodiment, this product is considered a "crude mixture" of sophorolipid.
• the sophorolipid obtained then was dried in a vacuum desiccator. 2.
• a crude mixture of sophorolipids was synthesized by fermentation of Candida bombicola as disclosed above. Lactonic sophorolipid was separated from the crude mixture and all other fractions were mixed together to form a non- lactonic sophorolipid mixture. Ethyl 17-L-[(2 ' -O- ⁇ -D-glucopyranosyl- ⁇ -D- glucopyranosyl)-oxy]-cis-9-octadecenoate then was synthesized and then further treated to obtain Ethyl 17-L[(2'-O- ⁇ -D glucopyranosyl- ⁇ -D-glucopyranosyl)-oxy]- cis-9-octadecenoate 6',6"-diacetate.
• Methyl 17-L-[(2 ' -O- ⁇ -D-glucopyranosyl- ⁇ -D- glucopyranosyl)-oxy]-cis-9-octadecenoate-6"-acetate also was synthesized by adding Lipase to a solution of methyl ester, (325.4 mg) and vinyl acetate (230.9 ⁇ l) in dry THF (5 ml), and further treatment.
• the reaction mixture was then acidified using glacial acetic acid, concentrated by rotoevaporation and poured with stirring into 100 mL of ice-cold water that resulted in the precipitation of the sophorolipid ethylester as a white solid.
• the precipitate was filtered, washed with ice-water, and lyophilized.
• the synthesized ethyl 17-L-[(2 ' -O- ⁇ -D-glucopyranosyl- ⁇ -D- glucopyranosyl)-oxy]-cis-9-octadecenoate 500 mg was dissolved in 20 mL of dry tetrahydrofuran (THF).
• sophorolipid methylester and sophorolipid butylester can be accomplished by substituting sodium methoxide or sodium butoxide, respectively, for the sodium ethoxide, resulting in sophorolipid methylester and sophorolipid butylester, respectively.
• the amount of dry crude sophorolipid and the amount and normality of the sodium (CH 2 ) n oxide can be varied appropriately by those of ordinary skill in the art without undue experimentation.
• Other types of suitable sophorolipids also can be synthesized by those of ordinary skill in the art without undue experimentation.
• Sophorolipids appear to exhibit superior antiviral activity and acceptable cytotoxicity for use as therapeutic agents for preventing or treating viral infections.
• the sophorolipids can be effective against viruses of the herpes family, which includes herpes simplex virus types 1 and 2 among other viruses.
• viruses contemplated to be treatable with the compounds of the present invention includes human cytomegalovirus; human herpesvirus 6; varicella-zoster virus; Epstein-Barr virus; herpesvirus simiae; equine herpesvirus-
• neurolymphomatosis Marek's disease
• influenza viruses A, B and C parainfluenza viruses-1 ,2,3 and 4
• adenovirus reovirus
• respiratory syncytial virus reovirus
• rhinovirus respiratory syncytial virus
• rhinovirus coxsackle virus
• echo virus rubeola virus
• hepatitis viruses of the types B and C papovavirus; and others.
• sophorolipids also may be used to treat animal viruses.
• Animal viruses contemplated to be treatable include bovine rhinotracheitis virus, bovine mammillitis virus, and cercopithecine herpesvirus 1 (B-virus), which are all simplex viruses; pseudorabies virus (PRV, of swine); equine rhinopneumonitis; coital exanthema viruses (varicellaviruses); baboon herpesvirus; pongine (chimpanzee) herpesvirus (lymphocryptovirus); Marek's disease virus (of fowl); turkey herpesvirus; herpesvirus ateles; and herpesvirus saimiri (rhadinovirus); among others.
• B-virus Bacillus virus
• a preferred embodiment of this invention can be used in the pharmaceutical industries as a therapeutic agent for the treatment or prophylaxis for diseases caused by herpes-related viruses. It is appreciated that sophorolipids may exhibit antiviral activity and, in some cases, can exhibit cytotoxicity. The sophorolipids that exhibit a relatively high antiviral activity and a relatively low cytotoxicity can be optimal for such treatment or prophylaxis options. Further, it is understood that a treatment with sophorolipids can include both a prophylactic treatment and/or a disease treatment.
• Preferred embodiments of the compounds and compositions of the present invention can be used in the manufacture of medicaments and in antiviral treatment of humans and other animals by administration in accordance with conventional procedures, such as an active ingredient in pharmaceutical compositions.
• Preferred compounds of the present invention can be provided as pharmaceutically acceptable formulations and/or "prodrugs," and pharmaceutical salts.
• compositions can be administered topically, orally, or parentally and may take the form of tablets, lozenges, granules, capsules, pills, ampoules or suppositories. They may also take the form of ointments, gels, pastes, creams, sprays, lotions, suspensions, solutions and emulsions of the active ingredient in aqueous or non-aqueous diluents, syrups, granulates or powders.
• the pharmaceutical compositions can also contain other pharmaceutically active compounds or a plurality of compounds of the invention. Results
• EBV Epstein-Bar Virus
• the cells were maintained at 37°C in a humidified atmosphere with 5% CO 2 in culture with RPMI- 1640 medium. The cells were transferred twice weekly and cell concentration adjusted to 2 X 10 6 AnI for use.
• the P3HR-1 strain of EBV was used in the screening assays.
• the viruses were cultured at concentration of 2 x 10 5 /ml for two weeks in medium containing 2% FCS at 34°C in a humidified atmosphere with 5% CO2. Concentrated virus then was prepared from the supernatant of the culture by centrifugation at 12,000 g for 90 min in a sorvall centrifuge. The pellets were resuspended in RPMM 640 medium at 1/100 of the original volume and stored at -70 0 C.
• Murine monoclonal antibody to EBV viral capsid antigen (VCA), was used for ELISA assay.
• the assay to be used to determine antiviral activity against EBV was through the VCA production in Daudi cells using an ELISA assay. Superinfection was initiated by the incubation of 0.5 ml of an appropriate concentration of EBV with 10 6 cells/tube in a total of 1 ml/tube. In most cases this amounts to a multiplicity of infection (MOI) of 0.1-0.2 based on VCA induction in Daudi cells. After adsorption at 37°C for 1 hr, 3 ml of RPM 1-1640 medium was added. The cells were pelleted by centrifugation and supematants discarded. Sophorolipids in varying concentration in 4 ml of RPMM 640 was added to the appropriate tubes.
• MOI multiplicity of infection
• RPM M 640 was added to positive and negative control tubes. Daudi cells infected with P3HR-1 virus and treated with the drug were harvested by centrifugation and washed three times with PBS. The cells were pelleted and suspended to a concentration of 4 x 10 6 cells/ml in PBS. 100 ⁇ l of each suspension was dispensed in triplicate into a 96-well plate, air-dried and fixed with 95% Ethanol and 5% Acetic Acid. Uninfected cells were prepared in the same manner and used as controls. After washing the plate, primary and secondary antibodies diluted in 1 % bovine serum albumin containing 0.05% Tween-20 were added sequentially to each well and incubated at room temperature.
• O-phenyldiamine (OPD) substrate was added and the reaction stopped with 3N H 2 SO 4 after ⁇ 10 min. The optical density was measured at 492 nm and the EC50 extrapolated using a computer software program.
• results of the assay show that natural mixtures of lactonic, non-lactonic, ethyl, methyl esters of sophorolipids and 6',6"-diacetate ethyl sophorolipids can act as an anti-herpes virus agents.
• the invention is a method for treating a herpes-related viral infection in a subject comprising the step of administering an effective amount of at least one sophorolipid to the subject.
• the sophorolipid is 17-L-[(2'-O- ⁇ -D- glucopyranosyl- ⁇ -D-glucopyranosyl)-oxy]-cis-9-octadecenoate based and is produced by Candida bombicola.
• the at least one sophorolipid is administered to the subject in a pharmaceutically acceptable carrier.
• the administration is by a method selected from the group consisting of intraperitoneal administration, intraarterial administration, and intravenous administration.
• the sophorolipid is administered in a dose of between about 2 mg of the mixture per kilogram of the human or animal and about 30 mg of the mixture per kilogram of the human or animal.
• the invention also is method for treating a herpes-related viral infection in a subject comprising the step of administering an effective amount of at least one sophorolipid to the subject, wherein the sophorolipid is produced by a process comprising the step of synthesizing the sophorolipid by fermentation of Candida bombicola in a fermentation media to form a natural mixture of lactonic sophorolipids and non-lactonic sophorolipids, and then utilizing the natural mixture for treatment of sepsis and septic shock in a human or animal.
• the invention also is method for treating a herpes-related viral infection in a subject comprising the step of administering an effective amount of at least one sophorolipid to the subject, wherein the sophorolipid is produced by a process comprising the steps of: a. synthesizing the sophorolipid by fermentation of Candida bombicola in a fermentation media to form a natural mixture of lactonic sophorolipids and non-lactonic sophorolipids; b. separating the lactonic sophorolipids from the natural mixture to form a lactonic fraction and mixing all remaining fractions to form a non-lactonic fraction; and c. utilizing the lactonic fraction for treatment of sepsis and septic shock in a human or animal.
• the invention also is method for treating a herpes-related viral infection in a subject comprising the step of administering an effective amount of at least one sophorolipid to the subject, wherein the sophorolipid is produced by a process comprising the steps of: a. synthesizing the sophorolipid by fermentation of Candida bombicola in a fermentation media to form a natural mixture of lactonic sophorolipids and non-lactonic sophorolipids; b. separating the lactonic sophorolipids from the natural mixture to form a lactonic fraction and mixing all remaining fractions to form a non-lactonic fraction; and c. utilizing the non-lactonic fraction for treatment of sepsis and septic shock in a human or animal.
• the 17-L-[(2 ' -O- ⁇ -D-glucopyranosyl- ⁇ -D-glucopyranosyl)-oxy]- cis-9-octadecenoate is selected from the group consisting of 17-L-[(2 ' -O- ⁇ -D- glucopyranosyl- ⁇ -D-glucopyranosyl)-oxy]-cis-9-octadecenoate-6',6"-diacetate, Hexyl 17-L[(2'-O- ⁇ -D glucopyranosyl- ⁇ -D-glucopyranosylJ-oxyJ-cis- ⁇ - octadecenoate, Ethyl 17-L[(2'-0- ⁇ -D glucopyranosyl- ⁇ -D-glucopyranosyl)-oxy]-cis- 9-octadecenoate, Butyl 17-L[
• the invention also is a composition for prophylaxis or treatment of herpes- related viral infection in a human or animal comprising a mixture of sophorolipids.
• the composition has the formula Ethyl 17-L-[(2 ' -O- ⁇ -D- glucopyranosyl- ⁇ -D-glucopyranosyl)-oxy]-cis-9-octadecenoate.
• the composition preferably is mixed with a pharmaceutically acceptable carrier.
• a preferred pharmaceutically acceptable carrier is selected from the group consisting of physiologically compatible buffers, physiological saline, a mixture consisting of saline and glucose, and heparinized sodium-citrate- citric acid-dextrose solution.
• the composition is a pharmaceutically acceptable salt of the mixture of sophorolipids.

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• 2007
  • 2007-03-09 JP JP2008558555A patent/JP5394073B2/ja not_active Expired - Fee Related
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