EP1192147A1 - Mycolactone et composes et compositions qui y sont lies ainsi que procedes d'utilisation correspondants - Google Patents

Mycolactone et composes et compositions qui y sont lies ainsi que procedes d'utilisation correspondants

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Publication number
EP1192147A1
EP1192147A1 EP00936488A EP00936488A EP1192147A1 EP 1192147 A1 EP1192147 A1 EP 1192147A1 EP 00936488 A EP00936488 A EP 00936488A EP 00936488 A EP00936488 A EP 00936488A EP 1192147 A1 EP1192147 A1 EP 1192147A1
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EP
European Patent Office
Prior art keywords
polyketide
mammal
mixture
ulcerans
aseptic
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Application number
EP00936488A
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German (de)
English (en)
Inventor
Pamela L. C. Small
Kathleen M. George
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US Department of Health and Human Services
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US Department of Health and Human Services
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Publication of EP1192147A1 publication Critical patent/EP1192147A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D313/00—Heterocyclic compounds containing rings of more than six members having one oxygen atom as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/08—Oxygen as only ring hetero atoms containing a hetero ring of at least seven ring members, e.g. zearalenone, macrolide aglycons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/32—Mycobacterium

Definitions

  • the present invention relates to pharmacoactive polyketide macrolides obtained from M. ulcerans, semi-synthetic derivatives thereof, aseptic mixtures of polyketide macrolides obtained from M. ulcerans. and methods of using the same.
  • Mycobacteria ulcerans is the causative agent of buruli ulcers. Buruli ulcers are progressive necrotic skin lesions that can persist for decades without treatment and which are not uncommonly suffered by persons living in tropical climates, including parts of Africa. Buruli ulcers are painless, not accompanied by symptoms of systemic disease, and are generally not accompanied by an initial acute inflammatory response.
  • M. ulcerans produces a toxin, although the identity of this toxin or toxins has heretofore remained unknown. Read et al., Infect. Immun., 9, 1 1 14 (1974), reported that the sterile filtrate of an M.
  • ulcerans liquid culture has cytopathic activity on cultured murine fibroblasts.
  • Pimsler et al., J. Infect. Pis.. 157, 577 (1988) reported that the sterile filtrate of an M. ulcerans has an immunosuppressive property.
  • George et al., Infect. Immun.. 66. 587-593 reported that a lipid toxin that is soluble in acetone could be isolated from M. ulcerans culture supernatant by organic extraction and causes a cytopathic effect by arresting L929 murine fibroblasts in the Gi phase of the cell cycle.
  • the present invention provides a polyketide macrolide that can be isolated from virulent cell cultures of Mycobacterium ulcerans.
  • the inventive polyketide macrolide has the formula of formula 1 :
  • Suitable pharmacologically acceptable esters, ethers and prodrugs of the polyketide macrolide include those of Formula 2:
  • R'-R 3 are the same or different and each is independently selected from the group consisting of hydrogen, R 6 , a C(O)R 7 , a C(S)R 7 , a C(O)NHR 7 , and a C(S)NHR 7 , each occurrence of R is independently selected from the group consisting of a C ⁇ -C 6 alkyl, a C 5 -C1 .2 aryl, and a sugar, each occurrence of R 7 is independently selected from the group consisting of hydrogen, a C ⁇ -C 6 alkyl, and a C 5 -C12 aryl, and wherein R 1 and R 2 , R 2 and R 3 , and/or R 4 and R 3 can be taken together to form a ketal ring.
  • the present invention also provides an aseptic mixture of macrolides comprising (i) a polyketide macrolide isolated from Mycobacteria ulcerans wherein the fraction corresponding to the distance the polyketide macrolide travels from the origin divided by the distance the solvent front travels past the origin (Rf) is greater than 0J5 and less than 0.60 when chromatographically separated by silica-gel thin- layer-chromatography employing a solvent system of 90:10:1 of chloroform:methanol:water and (ii) a pharmaceutically acceptable carrier.
  • the present invention further provides methods of using a polyketide macrolide to inhibit cancer in a mammal and to suppress an inflammatory response in a mammal. These methods comprise administering an effective amount of an isolated polyketide macrolide or an aseptic mixture of macrolides to the mammal so as to inhibit cancer or to suppress an inflammatory response, respectively.
  • the method comprises inoculating a mammal with an immune response-inducing amount of M. ulcerans cells that produce less than about 5% of polyketide macrolides per cell in comparision to a fresh culture of a virulent isolate of M. ulcerans 1615, wherein said polyketide macrolides have an Rf of greater than 0J5 and less than 0.60 when chromatographically separated by SG-TLC employing a solvent system of 90: 10:1 of chlorform: methanol: water.
  • the mammal has an immune response to M. ulcerans and does not develop a buruli ulcer.
  • composition comprising M. ulcerans cells that produce less than about 5% of polyketide macrolides per cell in comparison to a fresh culture of a virulent isolate of Mycobacteria ulcerans, wherein the polyketide macrolides have an Rf of greater than 0.15 and less than 0.60 when chromatographically separated by SG-TLC employing a solvent system of 90:10:1 of chloroform:methanol: water.
  • the present invention provides an isolated polyketide macrolide that can be isolated from virulent isolates of Mycobacterium ulcerans.
  • the polyketide macrolide has the formula of Formula 1 :
  • Suitable pharmacologically acceptable esters, ethers and prodrugs of the polyketide macrolide include those of Formula 2:
  • R'-R 3 are the same or different and each is independently selected from the group consisting of hydrogen, R 6 , a C(O)R 7 , a C(S)R 7 , a C(O)NHR 7 , and a C(S)NHR 7 , each occurence of R is independently selected from the group consisting of a C ⁇ -C 6 alkyl, a C -C 12 aryl, and a sugar, each occurrence of R 7 is independently selected from the group consisting of hydrogen, a C ⁇ -C 6 alkyl, and a C 5 -C ⁇ 2 aryl, and wherein R 1 and R 2 , R 2 and R 3 , and/or R 4 and R ?
  • Suitable sugars in the context of the present invention include, but are not limited tetroses, pentoses, hexoses, heptoses, and disaccharides and polysacchafides comprising tetroses, pentoses, hexoses, and heptoses.
  • Suitable aryl substituents of the present invention include heteroaryl substituents comprising a nitrogen, oxygen, or sulfur heteroatom.
  • the structure of-mycolactone (Formula 1) has been unambiguously identified by multiple analytical methods including, but not limited to, proton NMR (including GMQCOSY, TOCSY, HSQC, and ROESY), and mass spectrometry. Both the observed and calculated m/z ratio by mass spectrometry (HRMS) was 765.4912 ( ⁇ 0J ppm). The long-range correlations between the proton signals at 2.02 and 2.41 ppm (2-CH 2 ) and the carbon signal at 173J ppm confirmed the position of one of the ester carbonyls at Cl .
  • proton NMR including GMQCOSY, TOCSY, HSQC, and ROESY
  • HRMS mass spectrometry
  • the proton signal at 4.71 ppm (5-CH) showed long-range coupling to the carbon signal at 166.9 ppm (Cl ') indicating that the hydroxyl function at C5 is acylated with the hexadecyl moiety.
  • the protons on the remaining 6 hydroxyl-bearing methine functions resonated at 4.9 (Cl 1), 4.28 (C12), 3.99 (C15'), 3.96 (C19), 3.68 (C13'), and 3.5 (C17) ppm. Based on chemical shifts, it is evident that the hydroxyl function at Cl 1 is involved in the formation of the lactone.
  • the macrolides Of Formula 2 can be made by standard, semi-synthetic techniques starting with the polyketide macrolide of Formula 1.
  • the polyketide macrolide of Formula 1 can be isolated directly from M. ulcerans.
  • M. ulcerans is available from at least the Trudeau Collection (Lake Saranac, New York, USA) and the American Type Culture Collection (ATCC), is propagated by a multiplicity of research laboratories, and can be isolated from buruli ulcers.
  • M. ulcerans can be propagated by any suitable means.
  • One suitable means for propagating M. ulcerans comprises growing M. ulcerans 1615 (from the Trudeau Collection) at 32 °C in Middlebrook 7H9 medium supplemented with 0.2% (v/v) glycerol and 10% oleic acid, albumin, dextrose, and catalase enrichment (Difco). If desired, the M. ulcerans can be concentrated out of the growth medium by filtration through a 0J2 micrometer filter.
  • M. ulcerans cells were extracted with an organic solvent and subjected to a suitable purification step that separates molecules on the basis of their hydrophobicity.
  • Any suitable organic solvent can be used for the extraction step, for example, a 2:1 mixture of chloroform and methanol or other solvents with a comparable dielectric constant, effectively extracts mycolactone and other polyketide macrolides of the present invention in about four hours with stirring.
  • Insoluble components and hydrophilic moieties can usefully be separated from the extract by centrifiigation and by adding a small volume of water (e.g., about 0J to about 1.0 volume) in order to facilitate phase separation.
  • any suitable technique for separating molecules based on their hydrophobicity for example, silica-gel based thin layer chromatography or reverse phase liquid chromatography (e.g., reverse phase HPLC) can be used to separate the inventive polyketide macrolides from other lipophilic molecules in the extract.
  • silica-gel based thin layer chromatography or reverse phase liquid chromatography e.g., reverse phase HPLC
  • phospholipids and other co-extracted molecules can selectively be precipitated by transferring the organic extract into a solution consisting essentially of ice-cold (i.e., about 4 °C) acetone.
  • the organic solvent containing the extract can be evaporated until dry, or nearly dry, and resuspended in ice-cold acetone.
  • the hydrophobic separation technique results in isolation of polyketide macrolides that have an Rf of greater than 0J5 and less than 0.60 when chromatographically analyzed by silica-gel thin-layer-chromatography (SG-TLC) employing a solvent system of 90: 10:1 of chloroform:methanol:water.
  • SG-TLC silica-gel thin-layer-chromatography
  • an "Rf ' is a number obtained by dividing the distance an analyte or compound travels from the origin of a TLC divided by the distance the solvent front travels past the origin.
  • the origin is the location on a TLC at which a mixture of compounds to be separated is placed.
  • polyketide macrolide While it may be desirable for many uses of the polyketide macrolide to isolate a composition comprising only one type of polyketide macrolide, for example mycolactone (Formula 1), mixtures polyketide macrolides are also useful in the context of the present inventive methods.
  • Mycolactone. in the TLC system described above has an Rf of about 0.23 (i.e., between about 0J9 and about 0J7).
  • polyketide macrolides of the present invention have observed Rf values of about 0J 1 (i.e., between about 0J8 and about 0J5), of about 0J8 (i.e., between about 0J6 and about 0.42), of about 0.44 (i.e., between about 0.41 and about 0.48), and of about 0.54 (i.e., between about 0.49 and 0.60). All of these polyketide macrolides have 12-membered lactone rings and modifications in the side chains, have a molecular weight that is less than 1 ,000, such as 600-750, and fluoresce yellow at 375 nm of ultraviolet light.
  • Australian isolates of M. ulcerans contain high levels of a mycolactone having an Rf of about 0.40, i.e., between about 0J6 and about 0.42, a 12-membered lactone ring and a different carbon double bond pattern in the lower ester chain from the mycolactones of the formulae set forth herein.
  • the activity level of the mycolactone found in Australian isolates is about 10,000-fold lower than the other mycolactones described herein.
  • Mass spectroscopy data indicate that this mycolactone (designated mycolactone C) has a molecular mass of 728, whereas the mycolactones of formula 1 have a molecular mass of 743.
  • Proton NMR data of mycolactone C indicate that it is identical to mycolactones A and B in the lactone portion of the molecule, with differences occurring in the side chain.
  • the polyketide macrolides of the present invention are susceptible to molecular damage induced by prolonged exposure to light. For example, if a polyketide macrolide is maintained at room temperature for about one week or so, an approximate 50% reduction in activity will be realized. Accordingly, the polyketide macrolide or mixtures thereof can be isolated and provided in a sterile, aseptic solution suitable for administration to humans and to other mammals in order to preserve bioactivity.
  • the mixture can be packaged in light-resistant bottles meeting U.S. Food and Drug Administration standards, or ISO standards, for the shipment of light-sensitive bottles.
  • the bottle or container is labeled with a label indicating the concentration of polyketide macrolides in the mixture and, optionally, a date of preparation or manufacture.
  • the polyketide macrolide can be provided in a pharmaceutically acceptable carrier (described below) in order to facilitate accurate administration of precise quantities of the polyketide macrolide to mammals and to achieve beneficial pharmacodynamics.
  • the present invention provides in vivo and in vitro methods for selectively enriching a mixed population of fibroblasts and epithelial cells for epithelial cells.
  • This embodiment of the present inventive method comprises administering a suitable quantity of a polyketide macrolide to the mixed population of cells and maintaining the cells under normal growth or maintenance conditions.
  • the population is enriched in epithelial cells because fibroblasts are particularly sensitive to the polyketide macrolide, whereas epithelial cells are relatively resistant to the effects of the polyketide macrolide.
  • the present invention also provides a method of inhibiting cancer in a mammal in need thereof, such as a human, including, for example, melanoma, lung cancer, breast cancer, central nervous system cancer, and other types of cancer.
  • inhibiting is meant the inhibition of further growth of an existing tumor, further expansion of cancerous cells, or metastasis.
  • cancer is inhibited by administering to the mammal an effective amount of a polyketide macrolide or an aseptic mixture of polyketide macrolides as described herein, whereupon the cancer in the mammal is inhibited.
  • the present invention also provides a method of suppressing an inflammatory response in a mammal, such as a human.
  • the method comprises administering to a mammal an effective amount of a polyketide macrolide or an aseptic mixture of polyketide macrolides as described herein, whereupon the inflammatory response in the mammal is suppressed.
  • suppressing is meant the suppression of the inflammatory response. While complete suppression is desirable, any degree of suppression is beneficial to the mammal being treated in accordance with the method.
  • the polyketide macrolide or mixture thereof is present in an effective concentration at the site of inflammation.
  • Sites of inflammation typically include injuries or wounds, the loci of inflammatory autoimmune diseases (for example, but not limited to, the joints and connective tissues of mammals afflicted with rheumatoid arthritis), and sites of infection.
  • suppression of the inflammatory response is manifested by a decreased number of polymorphonuclear neutrophils. preferably a substantial decreased number, i.e., at least about 100-fold reduction in the number of polymorphonuclear neutrophils, that accumulate at a site of inflammation in an untreated, but otherwise identical, mammal, such as within the first 24 hours of treatment, when the treatment occurs at approximately the same time as the cause for the inflammatory response.
  • the method reduces the number of polymorphonuclear neutrophils at the site of inflammation by at least 10-fold.
  • the present invention also provides a method of inducing an immune response to Mycobacteria ulcerans without inducing a buruli ulcer.
  • An isolate of M ulcerans that produces less than about 5%. and preferably 0%, per cell of polyketide macrolides, which have an Rf of greater than 0J5 and less than 0.60 when chromatographically separated by SG-TLC employing a solvent system of 90: 10:1 of chlorofor ⁇ umethanol: water, in comparision to a fresh culture of a virulent isolate of M. ulcerans 1615 is propagated.
  • Such an isolate can be obtained in any suitable manner.
  • One suitable method to obtain such an isolate is to propagate M.
  • ulcerans on solid medium and to maintain the culture until colonies containing multiple levels of pigmentation are obtained (i.e.. until the colonies have slightly discolored portions).
  • a small number of discolored cells can be removed with an inoculating needle and propagated in isolation.
  • These isogenic isolates surprisingly do not produce the polyketide macrolides of the present invention and are nonvirulent.
  • the mammal, which is preferably a human, to be protected is inoculated with an immune response- inducing amount of non-virulent M. ulcerans cells such that the mammal has an immune response to M- ulcerans, which may be manifest by the appearance of a granuloma, and does not develop a buruli ulcer.
  • the non-virulent M is preferably a human, to be protected is inoculated with an immune response- inducing amount of non-virulent M. ulcerans cells such that the mammal has an immune response to M- ulcerans, which may be manifest by the appearance of a granuloma, and does not
  • ulcerans can optionally be killed or attenuated prior to inoculation, although the non-lethal pathology of non-attenuated M. ulcerans is mild enough that such attenuation is not required for safety.
  • the determination of an immune response-inducing amount is within the ordinary skill in the art.
  • the mammal treated in accordance with this method is more resistant to the development of a buruli ulcer upon subsequent exposure to M. ulcerans than an untreated, but otherwise identical, mammal.
  • the polyketide macrolide, or aseptic mixture of polyketide macrolides can be administered to a mammal in any suitable manner.
  • the polyketide macrolide can be administered to a mammal orally, via inhalation, parenterally, topically, subcutaneously, intravenously, intramuscularly, intraperitoneally, rectally, and vaginally.
  • the route of administration is selected to maximize the exposure to the afflicted portion e.g., cancerous or inflamed portion, of the mammal's body and to minimize systemic or widespread distribution of the polyketide macrolide to non-affected regions of the mammal's body in the method of inhibiting cancer and in the method of suppressing an inflammatory response.
  • Preferred routes of administration for induction of an immune response are known in the art and include subcutaneous and intradermal routes of administration.
  • the dose administered to a mammal, particularly a human, in the context of the present inventive methods should be sufficient to cause the desired response.
  • the ordinarily skilled artisan will appreciate that the dosage, the route, and the frequency of administration will vary depending upon the age, species, condition, and weight of the mammal, as well as any peculiar sensitivities of the mammal to be treated, and other variables known to those of ordinary skill in the art. Determination and adjustment of these parameters is within the skill of the ordinarily skilled clinician in the art as a matter of routine clinical development.
  • Suitable therapeutic dosages range from about 0J0 nanograms per kilogram of body weight to 10 milligrams per kilogram of body weight, and optionally range from 1 to 100 milligrams per kilogram of body weight.
  • the dosage desirably should achieve greater than 300 pg of the polyketide macrolide per ml of cells. Efficacious results can be achieved at a concentration of polyketide macrolide from about 3 ng/ml to about 3 ⁇ g/ml.
  • the polyketide macrolide can be mixed with any pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier does not consist essentially of acetone, e.g., is less than about 90% acetone.
  • the pharmaceutical carrier comprises an alcohol, an oil, fatty acid, or a glycol.
  • the pharmaceutically acceptable carrier can comprise an aqueous solution (e.g., water) in which the polyketide macrolide is suspended, mixed, or emulsified.
  • aqueous solutions e.g., water
  • the composition preferably comprises a suspending agent.
  • injectable formulations are among those formulations that are suitable in the context of the present inventive methods.
  • the requirements for effective pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art (See. Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company, Philadelphia, PA. Banker and Chalmers, eds., pages 238-250, (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)). It is preferred that such injectable compositions be administered intravenously or locally, e.g., at or near the site of the cancer or the inflammation (e.g., infection or injury).
  • Formulations suitable for parenteral administration include aqueous and non- aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers. thickening agents, stabilizers, and preservatives.
  • the polyketide macrolide can be administered in a physiologically acceptable diluent, such as a sterile liquid, or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals, such as 2J-dimethyl-lJ-dioxolane-4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable suspending agent, such as pectin, carbomers.
  • a physiologically acceptable diluent such as a sterile liquid, or mixture of liquids, including water, saline
  • Oils, which can-be used in parenteral formulations include petroleum and animal, vegetable and synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral.
  • Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.
  • All formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
  • sterile liquid excipient for example, water
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • Topical formulations are well-known to those of ordinary skill in the art and are suitable in the context of the present invention. Such formulations are typically applied to the skin or other body surfaces.
  • Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the polyketide macrolide dissolved or suspended in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions.
  • liquid solutions such as an effective amount of the polyketide macrolide dissolved or suspended in diluents, such as water, saline, or orange juice
  • capsules, sachets, tablets, lozenges, and troches each containing a predetermined amount of the active ingredient, as solids or granules
  • Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable suspending agent or emulsifying agent.
  • diluents such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable suspending agent or emulsifying agent.
  • Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch.
  • Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients.
  • Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
  • a flavor usually sucrose and acacia or tragacanth
  • pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
  • the present invention further provides a composition
  • a composition comprising M. ulcerans cells that produce less than about 5%, preferably 0%, of polyketide macrolides per cell in comparison to a fresh culture of a virulent isolate of Myco bacteria ulcerans, wherein the polyketide macrolides have an Rf of greater than 0J5 and less than 0.60 when chromatographically separated by SG-TLC employing a solvent system of 90:10: 1 of chloroform:methanol:water.
  • M. ulcerans cells that produce low levels of or no amounts of polyketide macrolides can be obtained by streaking a sample of wild-type M. ulcerans. such as that which can be obtained from the American Type Culture Collection, onto an agar plate and selecting for spontaneous mutants.
  • Wild-type colonies will be yellow, whereas mutant colonies will be white.
  • the mutant colonies can be subsequently cultured and the lipids isolated and tested for mycolactone content as exemplified herein.
  • the composition is useful to induce an immune response, and even protection upon challenge, in accordance with the present inventive method of inducing an immune response to M. ulcerans and as exemplified in the Examples set forth herein.
  • Example 1 The following example demonstrates that acetone-soluble lipids partially purified from M. ulcerans are a powerful inhibitor of cancer.
  • M. ulcerans 1615 was grown in liquid culture at 32 °C in Middlebrook 7H9 medium supplemented with 0.2% (v/v) glycerol and 10% oleic acid, albumin, dextrose, and catalase enrichment (Difco). Intact bacteria were harvested by passing the culture through a 0J2 micrometer filter. The bacterial mass was stirred in a 2: 1 mixture of chloroform and methanol to extract the lipophilic molecules. One-fifth volume of water was added to the organic solvent extract and separated by centrifiigation. The organic phase was transferred to a new container and the solvent was evaporated in a rotary evaporator.
  • the residual sample was resuspended in ice- cold acetone to prevent solubilization of phospholipids.
  • the acetone solution was transferred to a new container and the solvent was evaporated.
  • the residual sample was resuspended in a saline solution and tested for the ability to inhibit the growth of cancerous cells in the (U.S.) National Cancer Institute's standardized 60 cell screening activity.
  • the sample was a potent inhibitor of cancerous cell growth. Melanoma cells were extremelyly sensitive to the activity of the sample.
  • the cytotoxic effects of the sample are reversible during the first 48 to 72 hours.
  • an apoptosis inhibitor can be administered in conjunction with the polyketide macrolide, in which case the cells die by necrosis instead of apoptosis.
  • the following example demonstrates that purified mycolactone, a polyketide macrolide of the present invention, is a potent inhibitor of the growth of H460 lung cancer cells, MCF-7 breast cancer cells, and SF-268 central nervous system cancer cells.
  • This example also demonstrates that a polyketide macrolide of the present invention having an Rf of about 0J3 by SG-TLC developed in 90:10: 1 chloroform:methanol:water is a potent inhibitor of cancer cell growth.
  • Acetone-soluble lipids (ASLs) were prepared from intact M. ulcerans cells as in Example 1, except that the acetone resuspension was applied to a preparative TLC plate to allow substantial separation of the ASLs based on relative mobility in the TLC system.
  • each major ASL was a polyketide macrolide of analogous structure to mycolactone.
  • One ASL, a polyketide macrolide of the present invention is structurally related to mycolactone except that it lacks two hydrogen atoms.
  • Another ASL of the present invention is structurally related to mycolactone except that it lacks two hydrogen atoms and an oxygen atom.
  • Loss of the two hydrogen atoms and an oxygen atom allows an epoxide to form between the 12' and 13', between the 13' and 15', or between the 17' and 19' carbon atoms.
  • two R groups and one of the oxygens to which they would be attached are not present in the molecule.
  • cytopathological assays including the ability to inhibit the growth of murine fibroblasts and cause buruli-like skin lesions in guinea pigs, revealed that mycolactone and the other polyketide macrolides of the present invention are structurally and functionally analogous.
  • Mycolactone was purified as in Example 2. Hartley guinea pigs were inoculated with an infectious quantity of M. marinum. When the inoculum was not accompanied by mycolactone, a painful, purulent lesion developed in the guinea pigs, indicative of an inflammatory reaction. In contrast, when mycolactone was injected into the locus, tissue necrosis still developed, but the infiltration of macrophages and polymorphonuclear neutrophils was delayed by 24 to 48 hours, resulting in greater than a 1, 000-fold reduction in the number of neutrophils invading the site of infection 16 hours after infection.
  • M. ulcerans that produce less than about 5% of polyketide macrolides per cell in comparison to a fresh culture of a virulent isolate of M. ulcerans can be used to induce an immune response in a mammal to M. ulcerans without inducing a buruli ulcer.
  • mycolactone-negative isogenic mutants of M. ulcerans namely 1615A, 1615B, 1615D (has a growth defect at stationary phase of cell cycle) and 1615E, were isolated and are available from Dr. Pamela L.C. Small, Rocky Mountain Laboratories, National Institutes of Health, Hamilton, Montana, USA.
  • Guinea pigs were vaccinated with 1615E.
  • the guinea pigs were challenged with the corresponding parental virulent strain by injection into shaved skin on the back of the neck.
  • Non- vaccinated control animals developed buruli ulcers following injection with the virulent parental strain, whereas none of the vaccinated animals developed ulcers.
  • the mycolactone-negative mutant conveyed protective immunity to infection, i.e., protection upon challenge, with M. ulcerans.

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Abstract

La présente invention concerne, entre autres, des macrolides de polycétides comprenant un macrolide de polycétide correspondant à la structure (2) dans laquelle R1-R5 sont identiques ou différents, chacun étant sélectionné indépendamment dans le groupe comprenant l'hydrogène, R6, un C(O)R7, un C(S)R7, un C(O)NHR7 et un C(S)NHR7, chaque R6 étant sélectionné indépendamment dans le groupe constitué d'un alkyle C1-C6, d'un aryle C5-C12 et d'un sucre, R1 et R2, R2 et R3, et/ou R4 et R5 pouvant être pris ensemble pour former un noyau de cétal. L'invention concerne aussi des mélanges aseptiques de macrolides de polycétides isolés à partir de M. ulcerans dans un excipient pharmaceutiquement acceptable, des procédés pour utiliser les macrolides de polycétides et les mélanges aseptiques pour inhiber le cancer et supprimer une réponse inflammatoire chez un mammifère, un procédé pour induire une réponse immune à Mycobacteria ulcerans sans induire d'ulcère de Buruli et une composition comprenant M. ulcerans.
EP00936488A 1999-06-03 2000-06-02 Mycolactone et composes et compositions qui y sont lies ainsi que procedes d'utilisation correspondants Ceased EP1192147A1 (fr)

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PCT/US2000/015428 WO2000075126A1 (fr) 1999-06-03 2000-06-02 Mycolactone et composes et compositions qui y sont lies ainsi que procedes d'utilisation correspondants

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AU2002223149A1 (en) * 2000-11-23 2002-06-03 Biogenia Co., Ltd. Anticancer agent comprising mycolactone
TWI311558B (en) * 2001-02-01 2009-07-01 Mercian Corporatio Novel physiologically active substance
TWI334866B (en) * 2002-05-29 2010-12-21 Mercian Corp Novel physiologically active substances
US7576204B2 (en) 2002-07-31 2009-08-18 Mercian Corporation Heterocyclic macrolide pharmaceutical agent, a method of producing the same and use of the same
CA2507641A1 (fr) 2002-11-29 2004-06-17 Mercian Corporation Procede de production d'un compose macrolide
JP2007511218A (ja) * 2003-11-14 2007-05-10 インスティテュート パストゥール マイコラクトン遺伝子座：新規ポリケチド製造のためのアッセンブリーライン、治療的および防御的使用法
WO2006000923A2 (fr) * 2004-06-03 2006-01-05 Novimmune Sa Compositions a base de macrolide utilisees en tant qu'agent therapeutique
BRPI0805837A2 (pt) 2007-01-29 2011-08-30 Eisai R&D Man Co Ltd forma sólida de um composto, método para preparação dos cristais, composição farmacêutica, método de tratamento de cáncer, e uso do composto sólido

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JP2001523970A (ja) * 1997-05-02 2001-11-27 インテグレイテッド リサーチ テクノロジー，エルエルシー 感受性試験および抗菌治療のアジュバントとしてのベタイン

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