Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1997-03-05
2001-02-27
Wessendorf, T. (Department: 1627)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S009100, C514S014800, C514S015800, C514S002600, C530S318000, C530S317000, C530S359000, C435S071300, C435S071200, C435S071100, C435S252600, C435S252100, C435S827000
Reexamination Certificate
active
06194383
ABSTRACT:
The invention relates to lipopeptides with very homologous amino-acid sequences but different fatty acid residues (lipid portion) which are synthesized by Actinoplanes sp. during fermentation and are released into the culture medium, to a process for isolating the lipopeptides from the culture medium and purifying them, to the use of the lipopeptides as pharmacologically active substances, in particular against Gram-positive bacteria, and to Actinoplanes sp. DSM 7358 for producing the abovementioned lipopeptides.
Secondary metabolites from microorganisms are successfully employed for the treatment of infectious diseases. Secondary metabolites are low molecular weight compounds whose production takes place in “biosynthetic one-way streets” which branch off from the primary metabolism, and whose function for the particular producer is unclear. To date, about 8000 secondary metabolites isolated from cultures of various microorganisms (especially fungi and bacteria of the genus Streptomyces) are known.
The main area of use of these secondary metabolites is the therapy of infectious diseases. However, owing to the wide use, there is frequently development of resistance so that there is a continuous need for novel antibiotics and active substances with novel mechanisms of action (Neu H. C., Science 257, 1992, pages 1064-1073).
In addition, the area of indications for microbial active substances has also extended to diseases which are not included among infectious diseases (for example tumor therapy, immunomodulation or for the regulation of lipid metabolism) and to crop protection (herbicides and insecticides). However, the active substances which are employed are still frequently associated with deficiencies which are characterized by unsatisfactory effect levels, excessive toxicity and/or unwanted side effects.
There are descriptions in the literature of lipopeptides whose amino-acid content is identical in respect of sequence, or is the same or very similar in respect of amino-acid composition, to the lipopeptides according to the invention. However, these lipopeptides differ fundamentally from the lipopeptides according to the invention in the lipid portion.
Examples of lipopeptides as mentioned above are:
Amphomycin Antibiot. [J. Am. Chem. Soc 95, 2352 (1973)];
Glumamycin [Bull. Chem. Soc. Jap. 38, 517 (1965)];
Zaomycin [J. Antibiot. Ann., page 194 (1960)];
Aspartocin [Antibiot. Ann., 194 (1960)];
Tsuhimycin [J. Antibiotics, 21, page 439 (1968)];
Laspartomycin [J. Antibiotics 21, page 55 (1968)].
These lipopeptides, which are called amphomycin-type lipopeptides, are synthesized by microorganisms of the genus Streptomyces. They display their antibiotic activity against Gram-positive bacterias such as, for example, Strepto-, Staphylo- and Enterococci. Strains of the genera Staphylo- and Enterococcus in particular have proved in recent times to be increasingly problematic organisms. The skilled worker understands problematic organisms to be those microorganisms which it is now impossible to control efficiently because of resistance to conventional antibiotics (for example &bgr;-lactam antibiotics or glycopeptide antibiotics such as, for example, vancomycin or teikoplanin).
An example of one group of microorganism strains which have developed resistance comprises the methicillin-resistant
Staphylococcus aureus
strains, abbreviated to MRSA strains. It is now known that these MRSA strains have often developed resistance not only to methicillin but also to other antibiotics (for example vancomycin).
Apart from the abovementioned compounds from Streptomyces, there is known to be a compound from Actinoplanes nipponensis ATTCC 31145 which, by reason of its spectrum of action and the physicochemical properties described, has structural similarities to the lipopeptides according to the invention and is called compound 41.012 (U.S. Pat. No. 4,001,397). Fermentation of Actinoplanes nipponensis ATCC 31145 under various culture conditions always leads to relatively low yields of compound 41.012.
The object of the invention is to look for microbial natural substances with improved properties.
This object is achieved according to the invention by fermentation of Actinoplanes sp. in a nutrient solution with carbon source and nitrogen source as well as the customary in organic salts, until the lipopeptides, preferably the lipopeptides A 1437, accumulate in the culture medium, subsequent isolation of the lipopeptides from the culture medium and, where appropriate, separation of the mixture into its individual components. The lipopeptides have pharmacological activity and thus therapeutic efficacy and can be employed as antibiotics acting against Gram-positive bacteria, preferably against glycopeptide-resistant strains.
The invention thus relates to:
1. Lipopeptides wherein Actinoplanes sp. is fermented in a culture medium until one or more lipopeptides of the formula I
in which
R
1
is a singly or multiply unsaturated, saturated or, independently thereof, a branched or unbranched fatty acid or hydroxy fatty acid with a chain length of from 6 to 22, inclusive, carbon atoms
and
R
2
is Asp or Asn,
accumulate in the culture medium and, where appropriate, one or more lipopeptides of the formula I are purified from the culture medium, with the exception of the lipopeptide of the formula I in which
and
R
2
is Asp.
2. Lipopeptides wherein Actinoplanes sp. is fermented in a culture medium until one or more lipopeptides from the group:
a) iC
13
-fatty acid-Asp-Dab-Pip-MeAsp-Asp-Gly-Asp-Gly-Dab-Val-Pro,
b) iC
14
-fatty acid-Asp-Dab-Pip-MeAsp-Asp-Gly-Asp-Gly-Dab-Val-Pro,
c) ic
13
-fatty acid-Asn-Dab-Pip-MeAsp-Asp-Gly-Asp-Gly-Dab-Val-Pro,
d) iC
14
-fatty acid-Asn-Dab-Pip-MeAsp-Asp-Gly-Asp-Gly-Dab-Val-Pro,
e) aiC
13
-fatty acid-Asn-Dab-Pip-MeAsp-Asp-Gly-Asp-Gly-Dab-Val-Pro,
f) aiC
15
-fatty acid-Asp-Dab-Pip-MeAsp-Asp-Gly-Asp-Gly-Dab-Val-Pro,
g) aiC
15
-fatty acid-Asn-Dab-Pip-MeAsp-Asp-Gly-Asp-Gly-Dab-Val-Pro,
h) nC
12
-fatty acid-Asp-Dab-Pip-MeAsp-Asp-Gly-Asp-Gly-Dab-Val-Pro,
i) nC
13
-fatty acid-Asp-Dab-Pi-Me~sp-Asp-Gly-Asp-Gly-Dab-Val-Pro,
j) nC
14
-fatty acid-Asp-Dab-Pip-MeAsp-Asp-Gly-Asp-Gly-Dab-Val-Pro,
accumulate in the culture medium and, where appropriate, one or more of these lipopeptides are purified from the culture medium.
3. Lipopeptides of the formula II,
R
1
-R
2
-Dab-Pip-MeAsp-Asp-Gly-Asp-Gly-Dab-Val-Pro II
in which
and
R
2
=Asp in the three last-mentioned cases.
4. A process for the preparation of one or more lipopeptides of the formula I as defined in 1., of one or more lipopeptides as defined in 2., or of one or more lipopeptides of the formula II as defined in 3., which comprises fermenting Actinoplanes sp. in a culture medium until one or more lipopeptides accumulate in the culture medium and, where appropriate, purifying one or more lipopeptides from the culture medium.
5. A use of a lipopeptide of the formula I as pharmacologically active substance, in particular as antibiotic against Gram-positive bacteria, particularly preferably against glycopeptide-resistant bacteria.
6. The use of the lipopeptide of the formula III
R
1
-R
2
-Dab-Pip-MeAsp-Asp-Gly-Asp-Gly-Dab-Val-Pro III
in which
and
R
2
is Asp
as pharmacologically active substance, in particular as, antibiotic against Gram-positive bacteria, particularly preferably against glycopeptide-resistant bacteria.
7. Actinoplanes sp. DSM 7358.
The invention is described in detail hereinafter, especially in its preferred embodiments. The invention is furthermore defined by the contents of the patent claims.
Definition of terms:
Dab means 2,3-diaminobutyric acid;
Pip means pipecolic acid (synonym=homoproline);
MeAsp means &bgr;-methylaspartate;
Gly means glycine;
Asn means asparagine;
Asp means aspartic acid;
Val means valine;
Pro means proline;
n means normal/unbranched and
CID means collisional induced decay.
The abbreviation “i” stands for “iso”, while “ai” means “ante-iso”. These definitions are known to the skilled worker in the co
Hammann Peter
Markus Astrid
Meiwes Johannes
Seibert Gerhard
Vertesy Laszlo
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
Hoechst Aktiengesellschaft
Wessendorf T.
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