Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2000-12-07
2003-11-04
Gerstl, Robert (Department: 1626)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C435S121000, C514S423000
Reexamination Certificate
active
06642391
ABSTRACT:
This invention relates to a compound named Amycomycin, which is obtainable by cultivation of the microorganism Amycolatopsis species ST101170 (DSM 12216), and to its pharmaceutically-acceptable salts and derivatives. The present invention further relates to a process for the production of Amycomycin, to the use of Amycomycin and its pharmaceutically-acceptable salts and derivatives as pharmaceuticals, in particular to their use as antibiotics, and to pharmaceutical compositions comprising Amycomycin or a pharmaceutically-acceptable salt or derivative thereof.
Methicillin resistant
Staphylococcus aureus
(MRSA) infections are known to be predominant in infectious conditions such as wounds and burns. Vancomycin and teicoplanin, which belong to the glycopeptide class, are the only two antibiotics clinically used for the treatment of MRSA infections. However, due to the recent emergence of vancomycin- and teicoplanin-resistant strains, these infections are reported to have become life-threatening and fatal. An intensive search for a structurally-different class of compounds active against these vancomycin- and teicoplanin-resistant strains has, therefore, been initiated. For instance, methylsulfomycin I, a cyclic thiopeptide, has been described earlier (EP-A-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 Amycomycin has antibiotic activity. The present invention thus relates to a compound of the formula:
and to pharmaceutically-acceptable salts and derivatives thereof, such as esters, ethers and other obvious chemical equivalents, including all stereoisomeric forms and all tautomeric forms.
Amycomycin has a characteristic tetramic acid moiety with a highly oxygenated C45-side chain. It has the molecular formula C
65
H
115
NO
18
and is obtainable by cultivation of the microorganism Amycolatopsis species ST101170 (DSM 12216) under aerobic conditions in a nutrient medium containing sources of carbon and nitrogen, followed by isolation and purification in a customary manner. The microorganism ST101170 belongs to the order of Actinomycetales, genus Amycolatopsis, and was deposited on Jun. 4, 1998, with the German Collection of Microorganisms and Cell Cultures (DSMZ-Deutsche Sammlung von Mikroorganismen und Zelikulturen GmbH), Braunschweig, Deutschland and has been given the accession number DSM No. 12216.
The present invention further provides a process for the production of the compound named Amycomycin from Amycolatopsis species ST101170, its mutants and variants, 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, followed by isolation of the compound and purification in a customary manner.
Mutants and variants of the microorganism ST101170 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 with 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 that 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, in particular against vancomycin-resistant strains (see Table 2 below).
Preferred carbon sources suitable for aerobic fermentation are assimilable carbohydrate and sugar alcohols, such as glucose, lactose or D-mannitol as well as carbohydrate-containing natural products such as malt extract. Suitable sources of nitrogen are, for example, amino acids, peptides and proteins, including their degradation products such as peptones or tryptones, meat extract, ground seeds, for example, of maize, white beans, soya or the cotton plants, distillation residues from alcohol production, meat meal and yeast extracts, but also ammonium salts and nitrates. Suitable inorganic salts, which the nutrient solution may contain, are, for example, chlorides, carbonates, sulfates or phosphates of the alkaline metals or alkaline earth metals, ion, zinc, colbalt or manganese.
The formation of Amycomycin proceeds well, for example, in a nutrient medium comprising about 0.5 to 5% starch (soluble), preferably 1 to 2%, about 0.5 to 5% glucose, preferably 1 to 3%, about 0.5 to 5% glycerin, preferably 1 to 2%, about 0.1 to 0.5% corn steep liquor, preferably 0.2 to 0.3%, about 0.2 to 1% peptone, preferably 0.4 to 0.6%, about 0.1 to 0.5% yeast extract, preferably 0.2 to 0.4%, about 0.05 to 0.2% sodium chloride, preferably 0.1 to 0.2% and about 0.1 to 0.5% CaCO
3
, preferably 0.2 to 0.3%. These amounts are based on the weight of the whole nutrient medium.
The growth of ST101170 is carried out aerobically, for example, in liquid medium with shaking or stirring in shake flasks or laboratory fermenters, optionally with the introduction or air or oxygen. Growth by fermentation can be carried out, for example, in sterile wide-necked bottles or round-bottomed flasks of various volumes, in glass fermenters or V
2
A-steel tanks.
The growth of ST101170 may be carried out at temperatures between about 20° C. and about 35° C., preferably between about 25° C. and about 30° C. and pH between 4 and 10, preferably between 6 and 8. Under these conditions, the microorganism is grown in general over a period of 20 to 200 hours, preferably 24 to 150 hours.
Growth is advantageously carried out in several stages. Firstly, one or more pre-cultures may be prepared in a liquid nutrient medium. A main culture, the actual production medium, is then inoculated with the pre-culture in, for example, a volume ratio of 1:10. The pre-culture is obtained, for example, by inoculating a nutrient medium with sporified mycelium and allowing growth for about 20 to about 120 hours, preferably 24 to 90 hours. The sporified mycelium can be obtained, for example, by allowing the microorganism to grow for about 1 to about 40 days, preferably about 5 to about 12 days, on a solid or liquid nutrient medium such as yeast-malt agar or potato-dextrose agar.
The course of fermentation and the formation of Amycomycin can be monitored by methods known to the skilled person such as by measuring the biological activity of the culture broth in bioassays or by chromatographic methods such as thin-layer chromatography (TLC) or high-pressure liquid chromatography (HPLC).
The compound Amycomycin is present in the culture filtrate as well as in the mycelium. Usually, however, the highest amount is present in the mycelium. The compound can be isolated using known separation techniques. Thus, it can be recovered from the culture filtrate by, for example, filtration or centrifugation. The filtrate can be extracted with a water-immiscible solvent such as 1-butanol, ethyl acetate, chloroform, dichloromethane or the like, preferably 1-butanol or ethyl acetate.
The active material also can be recovered from mycelium by extraction with a water miscible solvent such as methanol, acetone, acetonitrile, n-propanol or iso-propanol, preferably methanol or acetone or a water immiscible solvent such as tert-butanol, ethyl acetate, chloroform, dichloromethanol or the like, preferably tert-butanol or ethyl acetate.
The extraction of the culture filtrate may be carried out within a wide pH range. It is preferable, however, for the extraction to be carried out in a neutral or weakly acidic medium, preferably at a pH of between 4 and 9. The organic extract may be concentrated in vacuum and dried to give the active crude material.
The isolation or purification of the compound Amycomycin may be carried out in a known manner taking into consideration the chemical, physical and biological characteristics of the natural compound.
One method of isolation of Amycomycin according to the invention
Aszodi Jozsef
Decker Heinrich
Hopmann Cordula
Kurz Michael
Le Beller Dominique
Aventis Pharma Deutschland GmbH
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
Gerstl Robert
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