Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
1999-11-09
2002-05-14
Higel, Floyd D. (Department: 1626)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
C514S423000, C514S425000, C514S428000, C548S517000
Reexamination Certificate
active
06387943
ABSTRACT:
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.
FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
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. 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.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to Vancoresmycin, a compound of the formula:
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 C
71
H
126
N
2
O
21
(MW1343.80) and may be characterized by any one or more of its physico-chemical and spectral properties given below, such as its
1
H NMR spectroscopic data and its
13
C 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 Actinomycetalos, genus Amycolatopsis. It was deposited on Jun. 4, 1998 with the German Collection of Microorganisms and Cell Cultures (DSMZ—Deutsche Sammlung von Mikroorganismen und Zelikulturen 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-hydroxy4-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 is
Staphylococcus aureus
3066, which is a resistant strain to methicillin, a p&bgr;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 NEt
3
); 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, is acetonitrile, acetone, tetrahydrofuran, or co
Aszodi Jozsef
Bhat Ravi Gajanan
Hopmann Cordula
Kurz Michael
Le Beller Dominique
Aventis Pharma Deutschland GmbH
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Higel Floyd D.
Sackey Ebenezer
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