Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
2001-04-06
2003-07-22
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
C424S195150
Reexamination Certificate
active
06596518
ABSTRACT:
This invention relates to a compound named Percyquinnin, its pharmaceutically acceptable salts and derivatives, and to methods for obtaining the compound. One method of obtaining the compound is by cultivation of the Basidiomycete
Stereum complicatum
, ST 001837 (DSM 13303). The present invention further relates to a process for the production of Percyquinnin, to the fungus ST 001837 (DSM 13303), to the use of Percyquinnin and its pharmaceutically acceptable salts and derivatives as pharmaceuticals, in particular to their use as lipase inhibitors, and to pharmaceutical compositions comprising Percyquinnin or a pharmaceutically acceptable salt or derivative thereof.
Lipid metabolism normally keeps a delicate balance between synthesis and degradation. When the balance is upset, hyperlipidemia may occur, which in turn can cause atherosclerosis, hypertension, diabetes etc. Modulators of lipid metabolism may be expected to be useful in controlling these and other disorders.
Inhibition of lipolysis in non-insulin-dependent diabetes mellitus (NIDDM) is supposed to reduce hyperglycemia. The initial event in the utilization of fat as an energy source is the hydrolysis of triacylglycerol by lipases, e.g., hormone sensitive lipase, and monoacylglycerol lipase. Hydrolyses of triacylglycerols may lead to increased levels of glycerol and fatty acids in the blood. Lipase inhibitors may be expected to reduce both plasma fatty acid levels and hyperglycemia with reduced side effects.
Obesity and hypercholesterolemia may be to a relevant degree related to high nutritional fat intake. An enzyme involved in dietary triglyceride absorption is pancreatic lipase. Inhibition of pancreatic lipase may therefore result in inhibition of fat absorption.
It has now been found that a novel compound named Percyquinnin inhibits lipase. The present invention thus relates to Percyquinnin, 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.
Percyquinnin has the molecular formula C
12
H
16
O
3
(208 Da) 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, provided in Tables 1 and 2.
Percyquinnin may be described as a new &bgr;-lactone with an annelated five membered ring carrying a hydroxymethyl moiety and a 2,3-isopentenyl sidechain at the &agr;-position of the lactone. Percyquinnin has a hitherto unreported new structure. A chemical abstract literature search established Percyquinnin to be a new compound.
A method for obtaining Percyquinnin is by cultivation of a microorganism referred to as Culture No. ST 001837 (henceforth referred to as ST 001837). This fungus, used for the production of Percyquinnin, was collected at Percy Quinn, Mississippi State Park in Pike County, USA. The fungus ST 001837 belongs to the order of Basidiomycetes species
Stereum complicatum
and was deposited according to the rules of the Budapest Convention on Feb. 4, 2000 with the German Collection of Microorganisms and Cell Cultures (DSMZ—Deutsche Sammlung von Mikroorganismen und Zelikulturen GmbH), Mascheroder Weg 1B, D-38124 Braunschweig, Germany and has been given the accession number DSM No. 13303.
Thus, the present invention further provides a process for the production of the novel compound named Percyquinnin from Basidiomycetes species ST 001837, and from its Basidiomycete mutants and variants, under aerobic conditions in a nutrient medium comprising at least one source of carbon atoms and at least one source of nitrogen atoms and optionally further comprising at least one component chosen from nutrient inorganic salts and trace elements, followed by isolation of said compound and purification in a customary manner.
The nutrient medium comprises at least one source of carbon atoms, at least one source of nitrogen atoms and optionally comprises at least one nutrient inorganic salt. The carbon atom sources may be chosen from a variety of sources, examples include starches, glucose, sucrose, dextrins, fructose, molasses, glycerol, lactose and galactose. In one embodiment, the carbon atom source is glucose. The nitrogen atom sources may be chosen from a variety of sources; examples include soyabean meals, peanut meals, yeast extracts, beef extracts, peptones, malt extracts, corn steep liquors, gelatins and casamion acids. In one embodiment, the nitrogen atom source is chosen from malt extracts and yeast extracts. The nutrient inorganic salts may be chosen from a variety of salts; examples include sodium hydrogen phosphate, potassium hydrogen phosphate, ammonium hydrogen phosphate, sodium chloride, calcium chloride, calcium carbonate, potassium nitrate, ammonium sulphate and magnesium sulphate. In one embodiment, the nutrient inorganic salt is ammonium hydrogen phosphate.
The cultivation of ST 001837 may be carried out at temperatures ranging from 20 to 35° C. and at a pH ranging from 3.0 to 8.0. In one embodiment, ST 001837 is cultivated at 25° C. (±1° C.) and at a pH ranging from 3 to 5.
In one embodiment, the cultivation of ST 001837 is carried out for a time period ranging from 96 to 300 hours. During this time period, a yield of the lipase inhibitor Percyquinnin of the invention may be obtained. In a further embodiment, the cultivation by fermentation is carried out for a time period ranging from 216 to 264 hours under submerged conditions. Examples of submerged conditions include being in containers chosen from shake flasks and laboratory fermenters. The progress of fermentation and formation of the Percyquinnin can be detected by High Pressure Liquid Chromatography (HPLC) and by measuring the bioactivity of the culture broth. In the resulting culture broth, Percyquinnin is present in the culture filtrate and in mycelium. In one embodiment, the Percyquinnin is isolated from the mycelium. Percyquinnin can be isolated using known separation techniques. For example, one method for recovering Percyquinnin from the culture filtrate is by extraction with a water immiscible solvent, examples include ethyl acetate, dichloromethane, chloroform and butanol, at a pH ranging from 5 to 8.
Another method for recovering Percyquinnin, for example, is by hydrophobic interaction chromatography using polymeric resins; examples include “Diaion HP-20®”, “MCl® Gel CHP-20P” (Mitsubishi Chemical Industries Limited, Japan), and “Amberlite XAD®” (Rohm and Hass Industries U.S.A.), and activated charcoal. Another separation method may be ion exchange chromatography at a pH ranging from 5 to 8. In one embodiment, the Percyquinnin is recovered using chromatography on MCl® Gel CHP-20P. The active material can also be recovered from mycelium by extraction with a water miscible solvent, examples include methanol, acetone, acetonitrile, n-propanol and iso-propanol, or a water immiscible solvent, examples include ethyl acetate, dichloromethane, chloroform and butanol, at a pH ranging from 5 to 8. In one embodiment, the Percyquinnin is extracted from mycelium using methanol as a solvent. Extracts may be further concentrated and lyophilized to give the active crude material.
The inhibitor Percyquinnin of the present invention may, for example, be recovered from the crude material by fractionation using any of the following techniques:
normal phase chromatography (using alumina or silica gel as stationary phase and 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 dimethyloctadecylsilyl-silica gel, also called RP-18 or dimethyloctylsilyl silica gel also called RP-8 as stationary phase and eluents such as water, buffers viz. phosphate, acetate, citrate (pH 2-8) and organic solvents such as methanol, acetonitrile, acetone, tetrahydrofuran or combinations of these solvents),
gel permeation chromatogr
Hopmann Cordula
Kurz Michael
Mueller Guenter
Toti Luigi
Aventis Pharma Deutschland GmbH
Finnegan Henderson Farabow Garrett & Dunner LLP
Srivastava Kailash C.
Tate Christopher R.
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