US20020183267A1 - Vancoresmycin, a process for its production and its use as a pharmaceutical - Google Patents
Vancoresmycin, a process for its production and its use as a pharmaceutical Download PDFInfo
- Publication number
- US20020183267A1 US20020183267A1 US10/103,880 US10388002A US2002183267A1 US 20020183267 A1 US20020183267 A1 US 20020183267A1 US 10388002 A US10388002 A US 10388002A US 2002183267 A1 US2002183267 A1 US 2002183267A1
- Authority
- US
- United States
- Prior art keywords
- vancoresmycin
- pharmaceutically acceptable
- compound
- effective amount
- hil
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- YUHMKCQHJBTRHJ-AWNSINMNSA-N C/C(=C\CC(O)CC(O)CCC(C)C(O)C(C)C(O)CCCC(O)C(C)C(O)/C(C)=C/C(C)C(=O)/C(C)=C/C(O)C(O)CC(O)CC(OC1OC(C)C(O)C(N)C1O)C(C)CCC(O)CC(O)CCC(C)C(O)C(C)C(O)C(C)C)C(=O)C1=C(O)/C(=C/C(C)C)N(C)C1=O Chemical compound C/C(=C\CC(O)CC(O)CCC(C)C(O)C(C)C(O)CCCC(O)C(C)C(O)/C(C)=C/C(C)C(=O)/C(C)=C/C(O)C(O)CC(O)CC(OC1OC(C)C(O)C(N)C1O)C(C)CCC(O)CC(O)CCC(C)C(O)C(C)C(O)C(C)C)C(=O)C1=C(O)/C(=C/C(C)C)N(C)C1=O YUHMKCQHJBTRHJ-AWNSINMNSA-N 0.000 description 3
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/02—Acyclic radicals, not substituted by cyclic structures
- C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- 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
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
-
- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
Definitions
- Methicillin resistant Staphylococcus aureus (MRSA) infections are known to be predominant in several infectious conditions, for example, wounds and burns.
- Vancomycin and teicoplanin, belonging to the glycopeptide class are the only two antibiotics clinically used for the treatment of MRSA infections. Due to the recent emergence of vancomycin- and teicoplanin-resistant strains, however, these infections are reported to have become menacing and fatal.
- an intensive search for a structurally different class of compounds active against these vancomycin- and teicoplanin-resistant strains has been initiated. For instance, methylsulfomycin I, a cyclic thiopeptide, has been described earlier (European Patent Publication No. 0818539 filed Jul. 11, 1996) as an antibiotic active against vancomycin- and teicoplanin-resistant strains.
- Vancoresmycin may be obtained by cultivating a microorganism referred to as culture No. HIL-006734 (henceforth referred to as HIL-006734). This microorganism was isolated from a soil sample collected from National Park, Borivli, Mumbai, India. The microorganism HIL-006734 belongs to the order of Actinomycetales, genus Amycolatopsis. It was deposited on Jun. 4, 1998 with the German Collection of Microorganisms and Cell Cultures (DSMZ—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH), Braunschweig, Germany, and has been given the accession number DSM No. 12216.
- DSMZ German Collection of Microorganisms and Cell Cultures
- the nutrient inorganic salts are, for example, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, calcium chloride, calcium carbonate, potassium nitrate, ammonium sulphate, and magnesium sulphate. Calcium carbonate, sodium chloride, and magnesium sulphate are typical.
- the compound Vancoresmycin may be converted into pharmaceutically acceptable salts and derivatives, like esters and ethers, and other obvious chemical equivalents, which are all covered by the present invention.
- the salts and derivatives can be prepared by standard procedures known to one skilled in the art. Salts like sodium and potassium salts, for example, may be prepared by treating Vancoresmycin with suitable sodium or potassium bases.
- Esters and ethers may be prepared by the methods given in the literature, for example, in Advanced Organic Synthesis, 4 th Edition, J. March, John Wiley & Sons., 1992.
- Vancoresmycin has antibacterial activity. Minimum inhibitory concentrations of Vancoresmycin against a wide range of bacterial are given in Table 3 below. Vancoresmycin and its pharmaceutically acceptable salts and derivatives can be administered to animals, such as mammals, including humans, as pharmaceuticals on their own, in mixtures with one another, and in the form of pharmaceutical compositions that permit parenteral administration. Accordingly, the present invention also relates to Vancoresmycin and its pharmaceutically acceptable salts and derivatives for use as pharmaceuticals and to the use of Vancoresmycin and its pharmaceutically acceptable salts and derivatives for the production of medicaments having antibacterial activity. The present invention further relates to pharmaceutical compositions which contain an effective amount of Vancoresmycin and/or one or more pharmaceutically acceptable salts and/or derivatives thereof, together with a pharmaceutically acceptable carrier.
- auxiliaries and/or excipients which may be mentioned are tragacanth, lactose, talc, agar-agar, polyglycols, ethanol, and water. Suitable and preferred for parenteral administration are suspensions or solutions in water. It is also possible to administer the active substances as such, without vehicles or diluents, in a suitable form, for example, in capsules.
- the mixture of soil suspension and medium was allowed to settle and incubated at 28° C. ( ⁇ 1° C.) for 7 days.
- the petri plate was periodically observed and HIL-006734 (culture No. Y-9439786) was isolated from amongst the growing microorganisms.
- the above medium was distributed in 100 ml amounts in 300 ml Erlenmeyer flasks and autoclaved at 121° C. for 20 minutes. The flasks were cooled to room temperature and inoculated with the abovementioned agar slant. The incubation was carried out for five days on a rotary shaker at 180 rpm and 28° C. 1.5 ml of this culture was mixed with 1.5 ml glycerol (99%) and stored at ⁇ 20° C.
- composition of production medium Glucose 20.0 g Soybean meal 10.0 g CaCO 3 0.2 g CoCl 2 .6H 2 O 0.001 g Demineralized water 1 liter pH 7.0 or Starch 10.0 g Glucose 10.0 g Glycerol 99% 10.0 g Corn steep liquor 2.5 g Peptone 5.0 g Yeast extract 2.0 g NaCl 1.0 g CaCO 3 3.0 g Demineralized water 1 liter pH 7.2 (before sterilization)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Communicable Diseases (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Oncology (AREA)
- Epidemiology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Saccharide Compounds (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The present invention relates to a compound named Vancoresmycin which is obtainable by cultivation of the microorganism HIL-006734 (DSM 12216), and to its pharmaceutically acceptable salts. The present invention further relates to a process for the production of Vancoresmycin, to the microorganism HIL-006734 (DSM 12216), to the use of Vancoresmycin and its pharmaceutically acceptable salts as pharmaceuticals, and in particular to their use as antibiotics, and to pharmaceutical compositions comprising Vancoresmycin or a pharmaceutically acceptable salt thereof.
Description
- Under the provisions of Section 119 of 35 U.S.C., Applicants hereby claim the benefit of the filing date of European Patent Application Number 98121299.6, filed Nov. 9, 1998, which Application is hereby incorporated by reference.
- This invention relates to a compound named Vancoresmycin, which is obtainable by cultivation of the microorganism HIL-006734 (DSM 12216), and to its pharmaceutically acceptable salts and derivatives. The present invention further relates to a process for the production of Vancoresmycin, to the microorganism HIL-006734 (DSM 12216), to the use of Vancoresmycin and its pharmaceutically acceptable salts and derivatives as pharmaceuticals, including their use as antibiotics, and to pharmaceutical compositions comprising Vancoresmycin or a pharmaceutically acceptable salt or derivative thereof.
- Methicillin resistantStaphylococcus aureus (MRSA) infections are known to be predominant in several infectious conditions, for example, wounds and burns. Vancomycin and teicoplanin, belonging to the glycopeptide class, are the only two antibiotics clinically used for the treatment of MRSA infections. Due to the recent emergence of vancomycin- and teicoplanin-resistant strains, however, these infections are reported to have become menacing and fatal. In response, an intensive search for a structurally different class of compounds active against these vancomycin- and teicoplanin-resistant strains has been initiated. For instance, methylsulfomycin I, a cyclic thiopeptide, has been described earlier (European Patent Publication No. 0818539 filed Jul. 11, 1996) as an antibiotic active against vancomycin- and teicoplanin-resistant strains.
- It has now been found that a novel compound named Vancoresmycin has antibiotic activity.
-
- and to its pharmaceutically acceptable salts and derivatives, such as esters, ethers, and obvious chemical equivalents, including all stereoisomeric forms and all tautomeric forms.
- Vancoresmycin has the molecular formula C71H126N2O21 (MW1343.80) and may be characterized by any one or more of its physico-chemical and spectral properties given below, such as its 1H NMR spectroscopic data and its 13C NMR spectroscopic data, both provided in Table 2.
- Vancoresmycin is a new antibiotic active against vancomycin- and teicoplanin-resistant strains. It has a hitherto unreported structure with a tetramic acid moiety bearing an acyl substituent at the 3-position. This acyl substituent has a highly oxygenated long alkyl chain substituted with an amino sugar. A chemical abstract literature search established Vancoresmycin to be a new compound.
- Vancoresmycin may be obtained by cultivating a microorganism referred to as culture No. HIL-006734 (henceforth referred to as HIL-006734). This microorganism was isolated from a soil sample collected from National Park, Borivli, Mumbai, India. The microorganism HIL-006734 belongs to the order of Actinomycetales, genus Amycolatopsis. It was deposited on Jun. 4, 1998 with the German Collection of Microorganisms and Cell Cultures (DSMZ—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH), Braunschweig, Germany, and has been given the accession number DSM No. 12216.
- The present invention further provides a process for the production of Vancoresmycin from amycolatopsis species HIL-006734, its mutants and variants, comprising the steps of: growing the amycolatopsis species HIL-006734 under aerobic conditions in a nutrient medium containing one or more sources of carbon and one or more sources of nitrogen and optionally nutrient inorganic salts and/or trace elements; isolating the Vancoresmycin compound; and purifying the Vancoresmycin compound in a customary manner.
- Mutants and variants of the microorganism HIL-006734 may also be able to synthesize the compound according to the present invention. Such mutants may be produced in a known manner by physical means, for example irradiation such as with ultraviolet- or X-rays, or chemical mutagens, such as ethylmethylansulfonate (EMS), 2-hydroxy-4-methoxy-benzophenone (MOB) or N-methyl-N′-nitro-N-nitrosoguanidine (MNNG).
- The screening for suitable mutants and variants which can produce the compound according to the invention can be confirmed by determination of the biological activity of the active compounds accumulated in the culture broth, for example by testing the antibacterial action.
- The nutrient medium preferably contains sources of carbon, nitrogen and nutrient inorganic salts. The carbon sources are, for example, starch, glucose, sucrose, dextrin, fructose, molasses, glycerol, lactose, or galactose. A typical carbon source is starch. The sources of nitrogen are, for example, soybean meal, peanut meal, yeast extract, beef extract, peptone, malt extract, corn steep liquor, gelatin, casamion acids. Peptone and yeast extract are typical. The nutrient inorganic salts are, for example, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, calcium chloride, calcium carbonate, potassium nitrate, ammonium sulphate, and magnesium sulphate. Calcium carbonate, sodium chloride, and magnesium sulphate are typical.
- The cultivation of HIL-006734 may be carried out at temperatures between 25-35° C. and pH between 6.0 and 8.0. Typically, HIL-006734 is cultivated at 30° C. (±1° C.) and pH 7.0.
- A good yield of the antibiotic of the invention may be obtained by cultivating HIL-006734 for 60-96 hours. Typically, cultivation is carried out by fermentation for 68-96 hours under submerged conditions, for example in shake flasks, as well as in laboratory fermenters. The progress of fermentation and formation of the Vancoresmycin can be detected by High Pressure Liquid Chromatography (HPLC) and by measuring the bioactivity of the culture broth against Staphylococci and Enterococci species by the known microbial agar plate diffusion assay method. The preferred culture isStaphylococcus aureus 3066, which is a resistant strain to methicillin, a β-lactam antibiotic reported in the literature, and Entrococcus faecium (E. faecium VR-1), which is resistant to vancomycin. In the resulting culture broth, Vancoresmycin is present in the culture filtrate as well as in mycelium and can be isolated using known separation techniques. Thus, it can be recovered from the culture filtrate by extraction at pH 5-8 with a water immiscible solvent such as ethyl acetate, dichloromethane, chloroform, or butanol, or by hydrophobic interaction chromatography using polymeric resins such as “Diaion HP-20®” (Mitsubishi Chemical Industries Limited, Japan), “Amberlite XAD®” (Rohm and Hass Industries U.S.A.), activated charcoal, or by ion exchange chromatography at pH 5-8. Typically, the active material is extracted with ethyl acetate. The active material can also be recovered from mycelium by extraction at pH 5-8 with a water miscible solvent such as methanol, acetone, acetonitrile, n-propanol, or iso-propanol. Alternatively, it may be extracted at pH 5-8 with a water immiscible solvent such as ethyl acetate, dichloromethane, chloroform, or butanol. Typically, the active material is extracted at pH 5-8 with ethyl acetate. Concentration and lyophilization of the extracts gives the active crude material.
- The antibiotic Vancoresmycin of the present invention may, for example, be recovered from the crude material as follows:
- By fractionation using any of the following techniques: normal phase chromatography (using alumina or silica gel as stationary phase; eluents such as petroleum ether, ethyl acetate, methylene chloride, acetone, chloroform, methanol, or combinations thereof; and additions of amines such as NEt3); reverse phase chromatography (using reverse phase silica gel such as dimethyloctadecylsilylsilica gel, (RP-18) or dimethyloctylsilyl silica gel (RP-8) as stationary phase; and eluents such as water, buffers (for example, phosphate, acetate, citrate (pH 2-8)), and organic solvents (for example, methanol, acetonitrile, acetone, tetrahydrofuran, or combinations of these solvents)); gel permeation chromatography (using resins such as ®Sephadex LH-20 (Pharmacia Chemical Industries, Sweden), TSKgel ®Toyopearl HW (TosoHaas, Tosoh Corporation, Japan) in solvents such as methanol, chloroform, acetone, ethyl acetate, or their combinations, or ®Sephadex G-10 and G-25 in water); or by counter-current chromatography (using a biphasic eluent system made up of two or more solvents such as water, methanol, ethanol, iso-propanol, n-propanol, tetrahydrofuran, acetone, acetonitrile, methylene chloride, chloroform, ethyl acetate, petroleum ether, benzene, and toluene). These techniques may be used repeatedly, alone or in combination. A typical method is chromatography over reverse phase silica gel (RP-18).
- The compound Vancoresmycin may be converted into pharmaceutically acceptable salts and derivatives, like esters and ethers, and other obvious chemical equivalents, which are all covered by the present invention. The salts and derivatives can be prepared by standard procedures known to one skilled in the art. Salts like sodium and potassium salts, for example, may be prepared by treating Vancoresmycin with suitable sodium or potassium bases.
- Esters and ethers may be prepared by the methods given in the literature, for example, in Advanced Organic Synthesis, 4th Edition, J. March, John Wiley & Sons., 1992.
- The amino group of the sugar moiety can be alkylated or acetylated, e.g. with acid chlorides by standard procedures known to one skilled in the art.
- Chemical equivalents may be stable complexes with metal ions, e.g. transition metals like La3+, Sm3+, Eu3+, Gd 3+, which are typical for tetramic acid derivatives and may be prepared by the methods given in the literature (K. Tanaka et. al., Chem. Pharm. Bull. 1979, 27, 1901; K. Matsuo, Chem. Pharm. Bull. 1980, 28, 2494).
- The double bonds of the alkyl side chain may be reduced by the methods given in the literature, for example in P. N. Rylander, “Hydrogenation Methods”, Academic Press, New York (1985), Chpt. 2, or may be hydrohalogenated by methods described by H. O. House in “Modern Synthetic Reactions”, W. A. Benjymin, Inc., New York (1972), pp 446-452. Hydroxylated derivatives may be produced by reaction of the double bonds with reagents such as OsO4 as described in the literature, e.g. in Chem. Rev. 1980, 80, 187.
- Derivatives may also be formed by conversion of the double bonds into epoxides by oxidation, e.g. with MCPBA, as described in Advanced Organic Synthesis, 4th Edition, J. March, John Wiley & Sons., 1992.
- Vancoresmycin has antibacterial activity. Minimum inhibitory concentrations of Vancoresmycin against a wide range of bacterial are given in Table 3 below. Vancoresmycin and its pharmaceutically acceptable salts and derivatives can be administered to animals, such as mammals, including humans, as pharmaceuticals on their own, in mixtures with one another, and in the form of pharmaceutical compositions that permit parenteral administration. Accordingly, the present invention also relates to Vancoresmycin and its pharmaceutically acceptable salts and derivatives for use as pharmaceuticals and to the use of Vancoresmycin and its pharmaceutically acceptable salts and derivatives for the production of medicaments having antibacterial activity. The present invention further relates to pharmaceutical compositions which contain an effective amount of Vancoresmycin and/or one or more pharmaceutically acceptable salts and/or derivatives thereof, together with a pharmaceutically acceptable carrier.
- Vancoresmycin can be administered orally, intramuscularly, intravenously, or by other modes of administration. Pharmaceutical compositions which contain Vancoresmycin or a pharmaceutically acceptable salt or derivative thereof with other pharmaceutically active substances can be prepared by mixing the active compounds with one or more pharmacologically tolerated auxiliaries and/or excipients such as, for example, fillers, emulsifiers, lubricants, masking flavors, colorants, or buffer substances, and converting the mixture into a suitable pharmaceutical form such as, for example, tablets, coated tablets, capsules, granules, powders, emulsions, suspensions, or solutions suitable for parenteral administration.
- Examples of auxiliaries and/or excipients which may be mentioned are tragacanth, lactose, talc, agar-agar, polyglycols, ethanol, and water. Suitable and preferred for parenteral administration are suspensions or solutions in water. It is also possible to administer the active substances as such, without vehicles or diluents, in a suitable form, for example, in capsules.
- As is customary, the galenic formulation and the method of administration as well as the dosage range which are suitable in a specific case depend on the species to be treated and on the state of the respective condition or disease, and can be optimized using methods known in the art. On average, the daily dose of active compound in a patient of about 75 kg weight is at least 0.001 mg to at most 10 mg, typically at most 1.0 mg.
- The following are provided as illustrative examples of the present invention and do not limit the scope thereof:
- a) Composition of Nutrient Isolation Medium
Corn starch 10.0 g Casein 1.0 g Peptone 1.0 g Beef extract 1.0 g K2HPO4 0.5 g Agar powder 13.0 g Demineralized water 1.0 liter pH 7.5 - b) Soil Plating and Isolation
- 10 g of soil collected from National Park, Borivli, Mumbai, India were added to 90 ml of sterilized water in a 250 ml Erlenmeyer flask which was shaken for 2 hours on a rotary shaker (220 rpm). The above soil suspension was serially diluted in steps of 10 up to 10−5. From the last dilution, 1 ml of suspension was placed at the center of a sterile glass petri plate (15 cm diameter) to which was poured approximately 50 ml of the above isolation medium supplemented with 25 μg/ml of amphotericin B as antifungal agent and cooled to 45° C. and the plate swirled thoroughly. The mixture of soil suspension and medium was allowed to settle and incubated at 28° C. (±1° C.) for 7 days. The petri plate was periodically observed and HIL-006734 (culture No. Y-9439786) was isolated from amongst the growing microorganisms.
- Composition of Maintenance Medium
- HIL-006734 was maintained on the following medium:
Malt extract 10.0 g Glucose 4.0 g Yeast extract 4.0 g Actidiol 0.05 g Agar powder 13.0 g Demineralized water 1 liter pH 7.0-7.5 - After dissolving the ingredients thoroughly by heating, the resultant solution was distributed in test tubes and sterilized at 121° C. for 20 min. The test tubes were cooled and allowed to solidify in a slanting position. The agar slants were streaked with the growth of HIL-006734 by a wire loop and incubated at 28° C. (±1° C.) until a good growth was observed. The well-grown cultures were stored in the refrigerator at +8° C.
- Preparation of Glycerol Working Seed
- Composition of Medium
Yeast extract 4.0 g Soluble starch 15.0 g K2HPO4 1.0 g MgSO4 × 7 H2O 0.5 g Demineralized water 1 liter PH 7.0 - The above medium was distributed in 100 ml amounts in 300 ml Erlenmeyer flasks and autoclaved at 121° C. for 20 minutes. The flasks were cooled to room temperature and inoculated with the abovementioned agar slant. The incubation was carried out for five days on a rotary shaker at 180 rpm and 28° C. 1.5 ml of this culture was mixed with 1.5 ml glycerol (99%) and stored at −20° C.
- Composition of Seed Medium
Glucose 15.0 g Soybean meal 15.0 g Corn steep liquor 5.0 g CaCO3 2.0 g NaCl 5.0 g Demineralized water 1 liter pH 6.8-7.0 - The medium could be used with or without corn steep liquor.
- The above medium was distributed in 100 ml amounts in 500 ml Erlenmeyer flasks and autoclaved for 20 mins. The flasks were cooled to room temperature and each flask was inoculated with a loopful of the above mentioned well-grown culture of Example 2 and shaken on a rotary shaker for 72 hours at 240 rpm at 27° C. (±1° C.) to give seed culture.
- Composition of Production Medium
Glucose 20.0 g Soybean meal 10.0 g CaCO3 0.2 g CoCl2.6H2O 0.001 g Demineralized water 1 liter pH 6.8 or Starch 10.0 g Glucose 10.0 g Glycerol 99% 10.0 g Corn steep liquor 2.5 g Peptone 5.0 g Yeast extract 2.0 g NaCl 1.0 g CaCO3 3.0 g Demineralized water 1 liter pH 7.2 (before sterilization) - The production medium was distributed in 100 ml amounts in 500 ml Erlenmeyer flasks and autoclaved at 121° C. for 20 min. The flasks were cooled to room temperature and inoculated with the abovementioned seed culture (2% v/v). The fermentation was carried out on a rotary shaker at 240 rpm and 27° C. (±1° C.) for 48 hours. The production of the antibiotic was determined by testing the bioactivity againstS. aureus 3066 and Ent. faecium VR-1 using the well diffusion method in a known manner.
- Preparation of Seed Culture in Shake Flasks
- The seed medium of Example 3 was distributed in 150 ml amounts in 1000 ml Erlenmeyer flasks and autoclaved at 121° C. for 20 mins. The seed culture was grown in these flasks as described in Example 3.
- Large Scale Fermentation
- Composition of production medium:
Glucose 20.0 g Soybean meal 10.0 g CaCO3 0.2 g CoCl2.6H2O 0.001 g Demineralized water 1 liter pH 7.0 or Starch 10.0 g Glucose 10.0 g Glycerol 99% 10.0 g Corn steep liquor 2.5 g Peptone 5.0 g Yeast extract 2.0 g NaCl 1.0 g CaCO3 3.0 g Demineralized water 1 liter pH 7.2 (before sterilization) - 20 liters of the production medium in 22 liter fermenter (in two fermenters) and 9 liters of the production medium in 12 liter fermenter (in two fermenters) along with 1 ml(/10 liter fermenter) of ®Desmophen as antifoaming agent was sterilized in situ for 40 mins. at 121° C., cooled to 27° C. (±1° C.) and seeded with 1.5 liter(/22 liter fermenter) or 0.75 liter (/12 liter fermenter) of the seed culture mentioned above.
- The fermentation was run with the following parameters:
Temperature 27° C. (±1° C.) Agitation 200 rpm Aeration 15 lpm/22 liter fermenter 10 lpm/12 liter fermenter Harvest time 64 hours - The production of the antibiotic was determined by testing the bioactivity againstS. aureus 3066 and Ent. faecium VR-1 and HPLC analysis. The final pH of the culture broth was 7.0-7.5. The culture broth was harvested and centrifuged and the antibiotic was isolated and purified from the culture filtrate and the mycelium by the method described in the Example 5 or 6.
- The culture broth (60 liters) was harvested and centifuged to separate the mycelium (2.5 kg) and culture filtrate (50 liters, pH 7.3). The mycelium was extracted with methanol (2×20 liters) and the active extracts were pooled and concentrated under reduced pressure to get 70 g of crude material. The culture filtrate (50 liters, pH 7.3) was adjusted to pH 5.5 with 2N hydrochloric acid and passed through a column of ®Diaion HP-20 (2.5 liters). The column was washed with water (10 liters) followed by 15 liters of methanol:water (1:1). The active compound was found to be present in 15 liters of methanol:water (3:1) and 30 liters methanol eluates. The monitoring of the purification was done by bioassay againstS. aureus 3066 and Ent. faecium VR-1. The active eluates were pooled and concentrated to get 15 g of crude material. The combined crude was chromatographed on ®Sephadex LH-20 column (2.5 cm×90 cm) in methanol repeatedly and the active fractions were pooled and concentrated. This was further chromatographed on ®Toyopearl TSK HW 40F column (6 cm×35 cm) in methanol. The active fractions were pooled and concentrated under reduced pressure to get 3 g of semipure material. The final purification was done by preparative HPLC using the following conditions:
- The semi-pure material was finally purified by preparative HPLC on a 25 mm×250 mm Hibar -Lichrospher RP-18 (10 μ using 67.5:32.5 Methanol:Phosphate buffer (0.01 M, pH 6.5) as the eluant at a flow rate of 23 ml/min and detection at 220 nm).
- The active eluates were pooled and concentrated under reduced pressure to remove the solvent and then desalted on ®Diaion HP-20 (50 ml) column, eluted with acetonitrile:water (80:20) and concentrated under reduced pressure and lyophilized to get 70 mg of pure compound.
- The culture broth (200 liters) was harvested and centrifuged to separate the mycelium and the culture filtrate. The mycelium was exhaustively extracted with methanol (30-40 L) and the extract concentrated 1:10 to obtain a colorless precipitation which was filtered off to obtain 30-50 g of crude material. This material was further purified by HPLC:
- 1.) Column: Fractogel TSK-HW 40 (4 L, 500×100 mm)
- Eluent: MeOH
- Flow: 20 ml/min
- Detection: 204 and 236 nm
- The active fractions were eluted after 125 min. The pooled fractions were concentrated under reduced pressure and freeze dried.
- 2.) Column: Silica Gel 60 (Merck); Erimatech (300×20 mm, 100 ml)
- Eluent: A) CH2Cl2:MeOH 9:1 B) CH2Cl2:MeOH 3:1+1% NEt3 C) MeOH
Gradient: min % A % B % C 0 100 0 0 23 100 0 0 24 0 100 0 51 0 100 0 52 0 0 100 93 0 0 100 - The vancoresmycin-containing fractions eluted after 32 min. The pooled fractions were concentrated under reduced pressure and freeze dried. The yield of the two purification columns was 50%.
- The physico-chemical and spectral properties of Vancoresmycin are given in Tables 1 and 2. The minimum inhibitory concentrations (MIC) against various bacteria are listed in Table 3.
TABLE 1 Appearance White solid Solubility Methanol, DMSO Melting point 141-143° C. [α]D −13°(c 0.2, Methanol) TLC (Thin Layer Rf: 0.6 Chromatography) [Silica gel plate (Article No. 5554, E. Merck); n-BuOH:MeOH:water (4:1:2)] HPLC (High Pressure Retention time: 14.70 min Liquid Chromatography) [Column: LiChrocart (250 mm × 4 mm) RP Select B (5 μ; Eluant: Gradient of 0.1% aqueous orthophospharic acid (pH 2.5) to CH3CN in 20 min; Flow rate: 1 ml/min; Detection: 220 nm] Fig. 1 of the accompanying drawings or: Column: Purospher Star RP.18e (Merck), 55 × 4 mm, 3 μm Eluent: CH3CN/0,01% H3PO4 (85%) Gradient: time % CH3CN 0.00 5.0 3.00 95.0 5.00 95.0 6.00 5.0 10.00 5.0 Flow: 2 ml/min Temp.: 40° C. Detection: 210 nm, 254, 280, 320, 380 tR: 2.19 min ESI-MS (Electrospray 1342 (M − H) lonisation Mass) HR-FAB-MS (High 1343.89142 (M + H)+ resolution Fast Atom [Calculated for C71H127N2O21: Bombardment Mass) 1343.889915 (M + H)+] Mol. formula: C71H126N2O21 UV/VIS: MeOH, λmax (logε) = 234 nm (4.39), 280 (4.29) (Figure 2) IR: KBr, ν = 3384 cm−1 (br), 2934 (m), 1674 (m), 1616 (s), 1456 (s), 1381 (m), 1064 (Figure 3) 1H NMR: see Table 2 13C NMR: see Table 2 -
TABLE 2 1H and 13C NMR Spectroscopic Data of Vancoresmycin in MeOD at 300 K. 1H 13C 1 0.85 14.76 2 0.97 20.83 3 1.86 31.20 4 3.40 82.19 5 1.69 39.54 6 0.77 13.64 7 3.53 80.19 8 1.65 36.40 9 0.86 12.75 10 1.43 31.20 11 1.43 36.52 12 3.74 72.03 13 1.60/1.54 44.90 14 3.75 72.03 15 1.57/1.38 36.61 16 1.49/1.16 30.18 17 1.94 38.60 18 0.92 15.04 19 3.86 81.67 20 4.59 103.48 21 3.83 72.03 22 2.48 57.61 23 — — 24 3.08 75.08 25 3.21 74.66 26 1.27 18.29 27 1.52/1.42 39.35 28 3.96 65.82 29 1.49 42.39 30 3.79 72.47 31 4.31 73.26 32 6.61 142.09 33 — 138.52 34 1.82 12.75 35 — 206.04 36 4.20 41.01 37 1.14 17.77 38 5.36 128.35 39 — 138.72 40 1.67 12.75 41 3.98 80.53 42 1.63 41.30 43 0.88 8.10 44 3.58 73.85 45 1.48 36.52 46 1.62/1.32 23.51 47 1.55/1.32 33.36 48 3.79 75.08 49 1.73 42.71 50 0.77 11.91 51 3.40 77.82 52 1.61 36.28 53 0.85 12.70 54 1.54/1.33 31.34 55 1.61/1.44 36.28 56 3.77 71.83 57 1.67/1.62 44.53 58 3.90 71.28 59 2.29 37.53 60 5.75 129.78 61 — 141.66 62 1.82 13.98 63 — 195.36 64 — 174.52 65 3.22 27.78 66 — 136.51 67 — 183.80 68 — 100.31 69 5.35 116.05 70 3.07 26.50 71 1.09 24.46 72 1.09 24.46 -
TABLE 3 Test organism MIC (μg/ml) Staphylococcus aureus 209P 0.05 S. aureus 20240 0.05 S. aureus E-710 0.10 S. aureus SG511 0.05 S. aureus 3066 0.10 S. warneri 6563 II (2) 0.10 S. aureus E-712 0.10 S. aureus 20424 0.10 S. aureus Chantot 31153 0.05 S. aureus Wein 11 0.05 S. aureus Wein 120.05 S. aureus 503 (Mers) 0.025 S. aureus Brussel 4115 0.05 S. aureus MLS-16 0.39 S. haemolyticus 809 0.05 S. epidermidis 825 0.10 S. epidermidis 823 0.20 S. epidermidis 607 0.10 S. epidermidis 5747 IW 0.10 Enterococcus faecalis ATCC 29212 0.39 Ent. faecalis D 21777 0.39 Ent. faecalis D 23241 0.39 Ent. faecalis D 756 0.39 Ent. faecalis D 26777 0.39 Ent. faecalis D 7F 0.39 Ent. faecium D-65 0.39 Ent. faecium 5601 (H) 0.39 Ent. faecium P-1 0.39 Ent. faecium P-2 0.39 Ent. faecium VR-1 0.39 Ent. hirae 55 0.39 Ent. durans 4939H 0.39 Escherichia coli 9632 >100 E. coli super sen. 2231 >100 Pseudomonas aeruginosa M35 >100 - The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects as illustrative only and not restrictive.
Claims (19)
2. Vancoresmycin, a compound of the molecular formula C71H126N2O21, obtainable by cultivation of the microorganism Amycolatopsis species HIL-006734 (DSM 12216) or one of its variants or mutants under aerobic conditions in a nutrient medium containing sources of carbon and nitrogen, followed by isolation and purification, and its pharmaceutically acceptable salts, and derivatives, in all their stereoisomeric and tautomeric forms.
3. A process for the production of a compound as claimed in claim 1 comprising the steps of:
cultivating the microorganism Amycolatopsis species HIL-006734 (DSM 12216) or one of its variants or mutants under aerobic conditions in a nutrient medium containing sources of carbon and nitrogen to produce Vancoresmycin,
isolating the Vancoresmycin, and
purifying the Vancoresmycin.
4. A process for the production of a compound as claimed in claim 2 comprising the steps of:
cultivating the microorganism Amycolatopsis species HIL-006734 (DSM 12216) or one of its variants or mutants under aerobic conditions in a nutrient medium containing sources of carbon and nitrogen to produce Vancoresmycin,
isolating the Vancoresmycin, and
purifying the Vancoresmycin.
5. A process as claimed in claim 3 , further comprising the step of reacting the Vancoresmycin with a suitable agent to form a pharmaceutically acceptable salt or derivative.
6. A process as claimed in claim 4 , further comprising the step of reacting the Vancoresmycin with a suitable agent to form a pharmaceutically acceptable salt or derivative.
7. Amycolatopsis species HIL-006734 (DSM 12216) or one of its variants or mutants.
8. A pharmaceutical composition, comprising an effective amount of Vancoresmycin or a pharmaceutically acceptable salt or derivative thereof as claimed in claim 1 and a pharmaceutically acceptable carrier.
9. A pharmaceutical composition, comprising an effective amount of Vancoresmycin or a pharmaceutically acceptable salt or derivative thereof as claimed in claim 2 and a pharmaceutically acceptable carrier.
10. A method of treating an infection comprising administering to a patient in need thereof an effective amount of a compound as claimed in claim 1 .
11. A method of treating an infection comprising administering to a patient in need thereof an effective amount of a compound as claimed in claim 2 .
12. A method of treating a Staphylococcus aureas infection comprising administering to a patient in need thereof an effective amount of a compound as claimed in claim 1 .
13. A method of treating a Staphylococcus aureas infection comprising administering to a patient in need thereof an effective amount of a compound as claimed in claim 2 .
14. The method of claim 12 , wherein the Staphylococcus aureas is vancomycin-resistant and/or teicoplanin-resistant.
15. The method of claim 13 , wherein the Staphylococcus aureas is vancomycin-resistant and/or teicoplanin-resistant.
16. The method of claim 10 , wherein the infecting organism is a Staphylococcus.
17. The method of claim 11 , wherein the infecting organism is a Staphylococcus.
18. The method of claim 10 , wherein the infecting organism is an Enterococcus.
19. The method of claim 11 , wherein the infecting organism is an Enterococcus.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/103,880 US20020183267A1 (en) | 1998-11-09 | 2002-03-25 | Vancoresmycin, a process for its production and its use as a pharmaceutical |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP98121299.6 | 1998-11-09 | ||
EP98121299 | 1998-11-09 | ||
US09/436,718 US6387943B1 (en) | 1998-11-09 | 1999-11-09 | Vancoresmycin, a process for its production and its use as a pharmaceutical |
US10/103,880 US20020183267A1 (en) | 1998-11-09 | 2002-03-25 | Vancoresmycin, a process for its production and its use as a pharmaceutical |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/436,718 Division US6387943B1 (en) | 1998-11-09 | 1999-11-09 | Vancoresmycin, a process for its production and its use as a pharmaceutical |
Publications (1)
Publication Number | Publication Date |
---|---|
US20020183267A1 true US20020183267A1 (en) | 2002-12-05 |
Family
ID=8232941
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/436,718 Expired - Fee Related US6387943B1 (en) | 1998-11-09 | 1999-11-09 | Vancoresmycin, a process for its production and its use as a pharmaceutical |
US10/103,880 Abandoned US20020183267A1 (en) | 1998-11-09 | 2002-03-25 | Vancoresmycin, a process for its production and its use as a pharmaceutical |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/436,718 Expired - Fee Related US6387943B1 (en) | 1998-11-09 | 1999-11-09 | Vancoresmycin, a process for its production and its use as a pharmaceutical |
Country Status (26)
Country | Link |
---|---|
US (2) | US6387943B1 (en) |
EP (1) | EP1129208B1 (en) |
JP (1) | JP2002529477A (en) |
KR (1) | KR100623405B1 (en) |
CN (1) | CN1325656C (en) |
AR (1) | AR024226A1 (en) |
AT (1) | ATE225854T1 (en) |
AU (1) | AU761782B2 (en) |
BR (1) | BR9915161A (en) |
CA (1) | CA2351222A1 (en) |
CZ (1) | CZ20011593A3 (en) |
DE (1) | DE69903465T2 (en) |
DK (1) | DK1129208T3 (en) |
ES (1) | ES2181506T3 (en) |
HK (1) | HK1041026B (en) |
HU (1) | HU224610B1 (en) |
ID (1) | ID28381A (en) |
IL (1) | IL142848A0 (en) |
PL (1) | PL195995B1 (en) |
PT (1) | PT1129208E (en) |
RU (1) | RU2228337C2 (en) |
SI (1) | SI1129208T1 (en) |
TR (1) | TR200101279T2 (en) |
UA (1) | UA70974C2 (en) |
WO (1) | WO2000028064A1 (en) |
ZA (1) | ZA200103191B (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060149057A1 (en) * | 2004-12-22 | 2006-07-06 | Vilmos Keri | Method of purifying macrolides |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10060810A1 (en) | 2000-12-07 | 2002-06-20 | Aventis Pharma Gmbh | Coniosetin and derivatives thereof, processes for making and using the same |
WO2009035553A2 (en) * | 2007-09-11 | 2009-03-19 | University Of Tennessee Research Foundation | Analogs of tetramic acid |
JP6465299B2 (en) * | 2015-05-29 | 2019-02-06 | 株式会社山田養蜂場本社 | Honey fraction |
CN109608523B (en) * | 2018-11-23 | 2023-01-10 | 江苏九阳生物制药有限公司 | Production process of high-purity teicoplanin |
US20230106760A1 (en) * | 2020-03-17 | 2023-04-06 | The United States Of America,As Represented By The Secretary,Department Of Health And Human Services | Antibacterial and antifungal polyketides from environmental amycolatopsis spp |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH05163289A (en) * | 1991-08-27 | 1993-06-29 | Microbial Chem Res Found | New immunosuppressive substance, m1710-51f6 substance and its production |
JP3192723B2 (en) * | 1992-01-29 | 2001-07-30 | 明治製菓株式会社 | Novel macrolide antibiotics SF2748B, SF2748C1, SF2748D and SF2748E and their production |
JP3107455B2 (en) * | 1992-05-08 | 2000-11-06 | 財団法人微生物化学研究会 | New antibiotic MI481-42F4-A and method for producing the same |
JPH09157266A (en) * | 1995-12-04 | 1997-06-17 | Microbial Chem Res Found | New antibiotic epoxynomicin a and b and their production |
JPH1011477A (en) * | 1996-06-26 | 1998-01-16 | Dainippon Printing Co Ltd | Generating device for input file for simulation of integrated circuit |
EP0818539A1 (en) * | 1996-07-11 | 1998-01-14 | Hoechst Aktiengesellschaft | Methylsulfomycin I, a process for its production and its use |
-
1999
- 1999-11-04 CA CA002351222A patent/CA2351222A1/en not_active Abandoned
- 1999-11-04 JP JP2000581230A patent/JP2002529477A/en not_active Abandoned
- 1999-11-04 KR KR1020017005727A patent/KR100623405B1/en not_active IP Right Cessation
- 1999-11-04 IL IL14284899A patent/IL142848A0/en not_active IP Right Cessation
- 1999-11-04 CN CNB998131121A patent/CN1325656C/en not_active Expired - Fee Related
- 1999-11-04 BR BR9915161-8A patent/BR9915161A/en not_active Application Discontinuation
- 1999-11-04 CZ CZ20011593A patent/CZ20011593A3/en unknown
- 1999-11-04 PT PT99971862T patent/PT1129208E/en unknown
- 1999-11-04 RU RU2001116104/13A patent/RU2228337C2/en not_active IP Right Cessation
- 1999-11-04 ID IDW00200101016A patent/ID28381A/en unknown
- 1999-11-04 AT AT99971862T patent/ATE225854T1/en not_active IP Right Cessation
- 1999-11-04 AU AU13787/00A patent/AU761782B2/en not_active Ceased
- 1999-11-04 HU HU0104213A patent/HU224610B1/en not_active IP Right Cessation
- 1999-11-04 TR TR2001/01279T patent/TR200101279T2/en unknown
- 1999-11-04 UA UA2001063871A patent/UA70974C2/en unknown
- 1999-11-04 EP EP99971862A patent/EP1129208B1/en not_active Expired - Lifetime
- 1999-11-04 DE DE69903465T patent/DE69903465T2/en not_active Expired - Fee Related
- 1999-11-04 DK DK99971862T patent/DK1129208T3/en active
- 1999-11-04 ES ES99971862T patent/ES2181506T3/en not_active Expired - Lifetime
- 1999-11-04 PL PL99347658A patent/PL195995B1/en not_active IP Right Cessation
- 1999-11-04 SI SI9930169T patent/SI1129208T1/en unknown
- 1999-11-04 WO PCT/EP1999/008415 patent/WO2000028064A1/en active IP Right Grant
- 1999-11-05 AR ARP990105628A patent/AR024226A1/en unknown
- 1999-11-09 US US09/436,718 patent/US6387943B1/en not_active Expired - Fee Related
-
2001
- 2001-04-19 ZA ZA200103191A patent/ZA200103191B/en unknown
-
2002
- 2002-03-25 US US10/103,880 patent/US20020183267A1/en not_active Abandoned
- 2002-04-12 HK HK02102763.9A patent/HK1041026B/en not_active IP Right Cessation
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060149057A1 (en) * | 2004-12-22 | 2006-07-06 | Vilmos Keri | Method of purifying macrolides |
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6387943B1 (en) | Vancoresmycin, a process for its production and its use as a pharmaceutical | |
CA2047997C (en) | Antibiotic, balhimycin, a process for its production and its use as pharmaceutical | |
EP0818464B1 (en) | Methylsulfomycin l, a process for its production and its use | |
US5994543A (en) | Antibiotic bravomicins | |
US5571701A (en) | Antibiotic, balhimycin, a process for its production and its use as pharmaceutical | |
US6930130B2 (en) | Citrullimycines, a process for their production and their use as pharmaceuticals | |
US6642391B2 (en) | Amycomycin, a process for its production and its use as a pharmaceutical | |
EP0711785A1 (en) | Novel antitumor antibiotic compounds: hayumicins and analogs thereof | |
JPH06279481A (en) | Substance ws64826 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |