GB2259450A - Compositions with endothelin antagonist activity - Google Patents

Abstract

Pharmaceutical compositions comprise as the active ingredient, WF12880A (also known as TAN 1415A or asterric acid) of formula: <IMAGE> and a carrier. WF12880A can be obtained by fermentation of a strain, such as Penicillium citreonigrum F-12880, in a nutrient medium. Composition has activity as a vasodilator for hypertension, heart disease, Raynaud's disease, cerebral stroke, asthma or renal failure.

Description

A PHARMACEUTICAL COMPOSITION COMPRISING WF12880A SUBSTANCE HAVING ENDOTHELIN ANTAGONISTIC ACTIVITY This invention relates to a pharmaceutical composition comprising WF12880A substance having endothelin antagonistic activity.

WF12880A substance of this invention can be produced by fermentation of a WF12880A substance-producing strain such as Penicillium citreonigrum F-12880 in a nutrient medium.

The fermentation process is explained in detail in the following.

( Microorganism: Particulars of the microorganism used for producing WF12880A substance are explained in the following.

The microorganism which can be used for the production of WF12880A substance is WF12880A substance-producing strain belonging to the genus Penicillium, among which Penicillium citreonigrum F-12880 was isolated from a soil sample collected at Niigata-ken, Japan.

A lyophilized sample of the newly isolated Penicillium citreonigrum F-12880 was deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology (1-3, Higashi 1 chome, Tsukuba-shi, Ibaraki-ken, 305 Japan) under the accession number of FEDI P-11360 (deposited date: 16 March, 1990).

Characteristics of Penicillium citreonigrum F-12880: The fungus strain F-12880 grew rapidly on various culture media, and formed greyish green colonies. The strain F-12880 formed anamorph, consisting of Penicillate conidiophores and conidial chains on various agar media.

The conidiogenesis was phialidic (enteroblastic). The strain did not produce teleomorph structures. On the basis of its morphological characteristics, the strain appears to belong to the hyphomycete genus Penicillium Link 1809. Its mycological characteristics were as follows.

Cultural characteristics on various agar media are summarized in Table 1. Culture on malt extract agar grew rather restrictedly, attaining 3.0-3.5 cm in diameter after two weeks at 250C. This colony surface was plane, felty to cottony, dull green, and produced yellow soluble pigment.

The reverse was olive brown. Conidial structures formed abundantly. Colonies on Czapek's solution agar grew more rapidly, attaining 4.5-5.0 cm in diameter under the same condition. The surface was radially sulcate, felty to powdery, white to dull green and produced yellow soluble pigment. The reverse was yellowish brown. Conidial structures were observed.

The morphological characteristics were determined on basis of the cultures on Czapek yeast extract agar1). The conidiophores were born from aerial vegetative hyphae, hyaline, septate, semi-macronematous, mononematous, and formed in penicllate-fashion. The stipes were 90-120 pm long and 2.0-2.5 pm thick, smooth, monoverticillate. The phialides were in verticils of 6-10, hyaline, smooth, ampulliform, 8.0-10.0 pm x 2.0-3.5 pm, with short collula.

They produced hyaline conidia, which were short disordered chains up to 80 pm. The conidia were aseptate, spherical, 1.5-2.5 pm in diameter, smooth to very finely roughtened.

The vegetative hyphae were septate, hyaline, smooth and branched. The hyphal cells were sylindrical and 1.0-2.0 pm in diameter.

The strain F-12880 was able to grew at the temperature range from 70C to 340C with the growth optimum at 270C to 290C. These temperature data were determined on potato dextrose agar (made by NISSUI).

According to the taxonomic criteria of the genus Penicillium, the strain F-12880 resembled Penicillium citreonigrum Dierckx 1901. And above characteristics corresponded with this species descriptions by Pitt1)' 2), without colony size on Czapek yeast extract agar. Then we named the producing strain Penicillium citreonigrum F-12880.

Table 1 Cultural characteristics of the strain F-12880 Medium Cultural characteristics malt extract agar G: rather restrictedly, 3.0-3.5 cm (Blaskeslee 1915) S: circular to irregular, plane, felty to cottony, dull green (29E3), abundantly formed conidial structures, producing yellow soluble pigment R: olive brown (4F6) potato dextrose agar G: rather rapidly, 3.5-4.0 cm (Difco 0013) S: irregular, radially sulcate, felty to powdery, dull green (29E3), abundantly formed conidial structures, producing yellow soluble pigment R: yellowish brown (5F5) Czapek's solution agar G: rapidly, 4.5-5.0 cm (Raper and Thom 1949) S: circular, radially sulcate, felty to powdery, white to dull green (28E3), formed conidial structures, producing yellow soluble pigment R: yellowish brown (5F4) Medium Cultural characteristics Sabouraud dextrose G: spreading broadly, 5.5-6.0 cm agar (Difco 0190) S: circular to irregular, wrinkly, felty, white to pale grey (lib1), formed no conidial structures, producing brownish orange soluble pigment R: yellowish brown (SF8) oatmeal agar G: spreading broadly, 6.5-7.0 cm (Difco 1552) S: circular, radially sulcate, felty to powdery, white to dull green (29E3), formed conidial structures, producing yellow soluble pigment R: olive (2E4) Emerson Yp Ss agar G: spreading broadly, 5.5-6.0 cm (Difco 0739) S: circular to irregular, plane, felty to powdery, greenish grey (28C2) to dull green (28E4), abundantly formed conidial structures, producing yellow soluble pigment R: yellowish brown (5E4) corn meal agar G: spreading broadly, 5.5-6.0 cm (Difco 0386) S: circular, plane, thin to powdery, pastel yellow (3A4) to greenish grey (29D2), formed conidial structures R: yellowish white (2A2) Abbreviation: G: growth, measuring colony size in diameter S: colony surface R: reverse These characteristics were observed after 14 days of incubation at 250C. The color descriptions were based on the Methuen Handbook of Colour3) 1) Pitt, J. I.: A Laboratory Guide to Common Penicillium Species (2nd ed.), 187 P., Commonwealth Scientific and Industrial Research Organization Division of Food Processing, North Ryde, 1988.

2) Pitt, J. I.: The Genus Penicillium and its Teleomorphic States Eupenicillium and Talaromyces, 634 P., Academic Press, London, 1979.

3) Kornerup, A. and J. H. Wanscher: Methuen Handbook of Colour (3rd ed.), 525 P., Methuen, London, 1988 (2) Physico-chemical properties of WF12880A substance: WF12880A substance as obtained according to the fermentation process as mentioned below has the following physico-chemical properties.

Appearance: Colorless needles Nature: acidic substance Melting point: 213 - 2150C Molecular formula: 17 16 8 Elemental Analysis: Calcd. for C17Hl608.1/2H2O: C, 57.14; H, 4.80 Found: C, 57.87; H, 4.88 Molecular weight: 348 FAB-MS m/z 371 (M + Nazis HRFAB-MS m/z 349.0920 (C17H1608 + H requires 349.0923) Solubility: soluble: acetone, dimethylsufoxide insoluble: n-hexane, water Color reaction: positive: cerium sulfate-sulfuric acid reaction, ferric chloride reaction, iodine vapor reaction negative: ninhydrin reaction Thin layer chromatography (TLC): Stationary phase Developing solvent Rf value silica gel plate chloroform:methanol: (10:1) 0.3 ** ODS 50% methanol:0.1% trifluoroacetic acid (x:x) 0.2 * Kiesel Gel 60 F254 (made by E.Merck) ** silica gel plate for reverse phase TLC, RP-18 F254S (made by E. Merck) Ultraviolet absorption spectrum: methanol nm (E) 250(14,000), 314 (9700) max Xmethanol+HCl nm 250, 314 max methanol+NaOH nm 230 (sh), 305 max Infrared absorption spectrum: KBr vmax : 3400, 3260, 3000, 1710, 1690, 1660, 1630, 1600, 1580, 1480, 1460, 1440, 1350, 1250, 1200, 1060, 1000, 900, 850, 820, 800 cm 1 1H Nuclear magnetic resonance spectrum: (400 MHz, acetone-d6) d: 7.08 (1H, d, J=3Hz), 6.94 (1H, d, J=3Hz), 6.50 (1H, s), 5.92 (1H, s), 3.83 (3H, s), 3.76 (3H, s), 2.18 (3H, s) From the above-mentioned physico-chemical properties, WF12880A substance is assumed to have a chemical structure described below:

which is identical with the chemical structure of asterric acid reported by Frank et al4) 4) Frank R. Stermitz, H. A. Schroeder and J. Geigert: Phytochemistry, 12, 1173 (1973) Pharmaceutically acceptable salts of WF12880A substance can be prepared by a conventional method, e. g. by treating them with a base.

ThQ suitable base may be an alkali metal (e. g. sodium, potassium, etc.,), an alkaline earth metal (e. g. magnesium, calcium, etc.), the hydroxide or carbonate thereof, alkali metal alkoxide (e. g. sodium methoxide, sodium ethoxide, potassium tert-butoxide, etc-.) and the like.

Pharmaceutically acceptable salts of WF12880A substance are the salts with said base. The salts of WF12880A substance are also included within the scope of this invention.

(3) Production of WF12880A substance: WF12880A substance of this invention is produced when a strain which belongs to the genus Penicillium (e. g.

Penicillium citreonigrum F-12880), and which is capable of producing WF12880A substance is grown in a nutrient medium containing sources of assimilable carbon and nitrogen under aerobic conditions (e. g. shaking culture, submerged culture, etc.).

The preferred sources of carbon in the nutrient medium are carbohydrates such as glucose, starch, sucrose, fructose, glycerin, or the like.

The preferred sources of nitrogen are yeast extract, peptone, gluten meal, cotton seed flour, soybean meal, corn steep liquor, dried yeast, wheat germ, potato protein, or the like as well as inorganic and organic nitrogen compounds such as ammonium salts (e. g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea, amino acid, or the like.

The carbon and nitrogen sources, though advantageously employed in combination, need not to be used in their pure form because less pure materials, which contain traces of growth factors and considerable quantities of mineral nutrients, are also suitable for use.

When desired, there may be added to the medium mineral salts such as sodium or calcium carbonate, sodium or potassium phosphate, sodium or potassium hydrogenphosphate, sodium or potassium dihydrogenphosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, zinc salts, cobalt salts, or the like.

If necessary, especially when the culture medium foams seriously a defoaming agent, such as liquid paraffin, fatty oil, plant oil, mineral oil or silicone may be added.

As in the case of the preferred methods used for the production of other biologically active substances in massive amounts, submerged aerobic cultural conditions are preferred for the production of WF12880A substance in massive amounts.

For the production in small amounts, a shaking culture in a flask is employed.

Further, when the growth is carried out in large tanks, it is preferable to use the vegetative cells of the microorganism for inoculation in the production tanks in order to avoid growth lag in the process of production of WF12880A substance. Accordingly, it is desirable first to produce vegetative cells of the microorganism by inoculating a relatively small quantity of culture medium with cells of the microorganism and culturing said inoculated medium, and then to transfer the cultured vegetative cells to large tanks. The medium, in which the vegetative cells is produced, is substantially the same as or different from the medium utilized for the production of WF12880A substance.

Agitation and aeration of the culture mixture may be accomplished in a variety of ways. Agitation may be provided by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermentor, by various pumping equipment or by the passage of sterile air through the medium. Aeration may be effected by passing sterile air through the fermentation mixture.

The fermentation is usually conducted at a temperature between about 100C and 400C, preferably 200C to 300C, for a period of about 50 hours to 200 hours, which may be varied according to fermentation conditions and scales.

When the fermentation is completed, the culture broth is then subjected for the recovery of WF12880A substance to various processes conventionally used for recovery and purification of biological active substances, for instance, solvent extraction with an appropriate solvent or a mixture of some solvents, chromatography or recrystallization from an appropriate solvent or a mixture of some solvents.

According to this invention, in general, WFl2880A substance is found in the filtered broth as well as in the mycelia. Accordingly, it is preferable that the whole cultured broth is subjected to the isolation process of WF12880A substance, for example, by means of extraction using an appropriate solvent such as acetone, ethyl acetate or the like, a mixture of these solvents, or the like.

The extract is treated by a conventional manner to provide WF12880A substance, for example, the extract is concentrated by evaporation or distillation to a smaller amount and the resulting residue containing active material, i. e. WF12880A substance is purified by conventional purification processes, for example, chromatography or recrystallization from an appropriate solvent or a mixture of some solvents.

(4) Biological properties of WF12880A substance: For showing endothelin antagonistic activity of WFl2880A substance, some test data are explained in the following.

Test 1 Radioligand receptor binding assay in vitro: (a) Crude receptor membrane preparation: Porcine aorta was purchased form Pel-Freez Biologicals (U. S. A.) and stored at -800C until use.

Porcine aorta (50 g) was thawed and dissected free from fatty tissue, minced with scissors and then homogenized with a polytron (Brinkmann PT-20, maximal speed for 3 x 10 sec) in 100 ml buffer (0.25 M sucrose, 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5).

The homogenate was centrifuged at 10,000g for 20 minutes at 40C.

The supernatant, containing the plasma membrane fraction, was centrifuged at 100,000g for 60 minutes at 40C, and then resultant pellets were referred to as crude membrane fractions.

The pellets were resuspended in 25 ml of binding assay buffer (50 mM Tris-HCl, 100 mM Nail, 5 mM MgCl2, 1.5 Vg/ml phenylmethylsulfonyl fluoride (PMSF), 120 Vg/ml bacitracin, 12 pg/ml leupepcin, 6 pg/ml chymostain, 0.1% bovine serum albumin (BSA), pH 7.5) The aorta membrane fractions were stored at -800C until use.

(b) 125I-endothelin binding assay: 1251-Endothelin (1.67 x 10'11 M) (Amersham Japan, specific activity: 2000 Ci/m mol) was incubated with 50 pl of aorta membrane preparation in binding assay buffer at room temperature (20-220C) for 60 minutes in a final volume of 250 pl.

After incubation, the incubation mixture were filtered through Glass-fiber GF/C filter (pretreated with 0.1% polyethylene imine for 3 hours prior to use) using cell harvester (Brandel M-245). The filters were then washed ten times with a total of 3 ml of the washing buffer (50 mM Tris-HCl, pH 7.5) at OOC. The filters were counted in a gamma counter (Packard Auto Gamma Model 5650).

WF12880A substance competes with 125I-endothelin for binding to porcine aorta preparations.

The results are shown in Table 2.

Table 2 Radioligand receptor binding assay in vitro: Test compound IC50 value WF12880A substance 5.0 x 10 M Test 2 Effect of WF12880A substance on rabbit thoracic aorta of contraction response of endothelin: Thoracic aorta were isolated from freshly killed male albino rabbits (11 weeks old) and cut into 25 mm strips with the intima denuded. After removing fatty tissues, these arterial segments (2 mm width and 25 mm length) were suspended in 25 ml organ chambers filled with Krebs-Ringer solution (113 mM NaCl , 4.8 mM KCl, 2.2 mM CaCl2, 1.2 mM MgC12, 25 mM NaHCO3, 1.2 mM KH2PO4, 5.5 mM glucose) maintained at 370C and gassed with 95% 02/5% CO2.

A preload of I g was applied after the aorta had been conditioned by application of increasing concentration of KC1. Contractions were measured as an increase in isometric tension.

WF12880A substance was tested against contraction response of endothelin (3.2 x 10 9 M). Synthetic endothelin was obtained form Peptide Institute Inc. (Osaka, Japan).

WF12880A substance was added after the full contraction response induced endothelin.

The activity of WF12880A substance is expressed as the percentage inhibition of maximum contraction response induced by endothelin and shown in Table 3.

Table 3 Effect of WF12880A substance on rabbit thoracic aorta of contraction response of endothelin: Concentration of Inhibition against contraction WFl2880A substance (M) response of endothelin (%) 1.0 x 10 5 14.7 1.0 x 10 4 36.6 n=3 Test 3 Assay of in vitro pressor effect in conscious rats: Male Wister rats (10 weeks old) were used. The mean arterial blood pressure was recorded from femoral artery via polyethylene catheter connected to the pressure tranducer which was coupled to a Biophysiograph 180 system (Nihondenki-San-Ei Instrument Co., Ltd.).

WF12880A substance and endothelin were dissolved in saline and injected in bolus (0.2 ml) through polyethylene cateter inserted via femoral vein.

WF12880A substance was administered 30 minutes before the injection of endothelin. Inhibitory activity of WF12880 substance (100 mg/kg, orally) against endothelin (2.4 pg/kg, intravenously) induced pressor response was evaluated with a group of three rats and shown in Table 4.

Table 4 Assay of in vitro pressor effect in conscious rats Average blood pressure (mmHg) / Change rate (%) Number Group animals before after dosage (minutes) dosage 1 3 5 10 20 30 45 60 Reference 4 118 99 118 130 135 140 141 137 135 (saline) #4 #2 #2 #3 #2 #3 #2 #3 #3 0.0 -15.8 0.5 10.2 14.7 18.6 19.8 16.5 14.2 #0.0 #3.3 #3.6 #4.7 #3.6 #5.0 #3.3 #2.5 #2.6 WF12880A 4 121 93 120 129 134 135 130** 125* 121* substance #4 #2 #1 #2 #2 #3 #2 #2 #2 0.0 -2.29 -0.1 7.2 11.4 11.9 7.9* 3.9** 0.7** #0.0 #1.8 #3.3 #2.4 #2.8 #1.5 #3.0 #1.7 #1.3 * P < 0.05 ** P < 0.01 Test 4 Urinary recovery of WF12880A substance: Urinary recovery of WF12880A substance in ddY mice (male, 7 weeks old) given peroral dosing of 100 mg/kg was approximately 20%.

(5) Pharmaceutical Use of WF12880A substance: From the results of the above-mentioned biological test, WF12880A substance has endothelin antagonistic activity, therefore can be used as vasodilator for the treatment of hypertension such as peripheral circulatory failure, heart disease such as angina pectoris, cardiomyopathy, arteriosclerosis, myocardial infarction or the like, Raynaud's disease, cerebral stroke such as cerebral arterial spasm, cerebral ischemia, late phase cerebral spasm after subarachnoid hemorrhage or the like, asthma such as bronchoconstriction or the like, renal failure such as acute renal failure, or the like.

(6) Pharmaceutical compositions comprising WF12880A substance: The pharmaceutical composition of this invention can be used in the form of a pharmaceutical preparation, for example, in solid, semisolid or liquid form, which contains WF12880A substance, as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for external, oral or parenteral applications. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions; suspensions, and any other form suitable for use.And, if necessary, in addition, auxiliary, stabilizing, thickening and coloring agents and perfumes may be used. WFl2880A substance may be included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the process or condition of diseases.

For applying the composition to human, it is preferable to apply it by intravenous, intramuscular or oral administration. While the dosage of therapeutically effective amount of WF12880A substance varies from and also depends upon the age and condition of each individual patient to be treated, in the case of intravenous administration, a daily dose of 0.1 - 100 mg of WF12880A substance per kg weight of human being, in the case of intramuscular administration, a daily dose of 0.1 - 100 mg of WF12880A substance per kg weight of human being, in case of oral administration, a daily dose of 1 - 1000 mg of WF12880A substance per kg weight of human being is generally given as vasodilator for the treatment of above-mentioned diseases.

(6) Example of preparation of WF12880A substance: The following examples are given for the purpose of illustrating the present invention in more detail.

Preparation (i) Fermentation: A aqueous seed medium (160 ml) containing sucrose (4%), cotton seed flour (2%), dried yeast (1%), peptone (1%), potassium dihydrogenphosphate (0.2%), calcium carbonate (0.2%) and Tween 80 (0.1%) was poured into each of twenty 500 ml-Erlenmeyer flasks and sterilized at 1200C for 30 minutes.

A loopful of slant culture of Penicillium citreonigrum F-12880 (FERM P-11360)was inoculated to each of the media and cultured at 250C for 3 days on a rotary shaker (220 rpm, 5.1 cm throw). The resultant seed culture was transferred to a 20 liters of sterilized fermentation medium containing sucrose (2%), glycerine (0.5%), ground soy bean meal (1%), gluten meal (1%) and CuSO4 (O .' b018) in a 30-liter stainless steel jar-fermentor. The fermentation was carried out at 250C for 5 days under aeration of 20 liters/minutes and agitation of 300 rpm.

(ii) Isolation and purification: An equal volume of acetone was added to the culture broth (60 liters) with stirring. The mixture was allowed to stand at room temperature for one hour and then filtered.

The filtrate was concentrated in vacuo to a volume of 10 liters, and was adjusted to pH 2.0 with 1N hydrochloric acid, and then extracted with 10 liter of ethyl acetate.

The extract was concentrated to dryness under reduced pressure and applied to a column chromatography of silica gel (Silicar CC-4, made by Mallinckrodt, 2 liters). The column was washed with n-hexane (2 liters), n-hexane-ethyl acetate (1:1) (2 liters) and the active substance was eluted from the column with ethyl acetate (4 liters). The active fractions were evaporated in vacuo to give an oily material.

The oily material was dissolved in 20 ml of 50% aqueous methanol, and then applied to pre-packed column (LiChroprep RP-18, 40-63 pm, 25 mm inside diameter, 310 mm length, made by E. Merck) and eluted with 50% aqueous acetonitrile in 0.1% acetic acid solution. The active fractions (300 ml) to appear first contained WF12880A substance. The fraction was concentrated in vacuo to give a oily material. Thus obtained product was purified by crystalization from methanol to give colorless needles of WF12880A substance (16 mg).

(7) Examples of pharmaceutical compositions comprising WF12880A substance: Example 1 The ingredients in the following formula are blended and compressed into tablets in a conventional manner.

Formula for a tablet: WF12880A substance 10 mg methyl cellulose 5 mg corn starch 10 mg lactose 100 mg magnesium stearate 5 mg Thus obtained tablets are, when desired, coated with film coating or enteric coating.

Example 2 The ingredients in the following formula are blended and granulated into fine granules in a conventional manner.

Formula for fine granules: WF12880A substance 50 mg hydroxypropyl cellulose 50 mg lactose 900 mg Example 3 The ingredients in the following formula are blended and granulated into granules in a conventional manner.

Formula for granules: WF12880A substance 50 mg sucrose 950 mg

Claims (1)

1. What we claim is: 1. A pharmaceutical composition which has endothelin antagonistic activity, and which comprises, as an active ingredient WF12880A substance and a non-toxic, pharmaceutically acceptable carrier.