FR-900216 SUBSTANCE OBTAINABLE BY CULTIVATING ATCC 20577 FOR USE AS AN ACTIVE THERAPEUTIC SUBSTANCE AND PHARMACEUTICAL ANTITUMOR COMPOSITION COMPRISING FR-900216 SUBSTANCE

Abstract: FR-900216 substance obtainable by cultivating ATCC 20577 in a nutrient medium containing sources of assimilable carbon and nitrogen under aerobic conditions and recovering it from the culture broth and its use as an active therapeutic substance especially for treatment of tumor. The invention covers also a pharmaceutical antitumor composition com­ prising FR-900216 substance in association with a phar­ maceutically acceptable, substantially non-toxic carrier or excipient.

Description: The present invention relates to a pharmaceutical composition, in particular to an anti-tumor composition, comprising FR-900 216 substance (Rhizoxin), a method for its preparation and its use in the treatment of tumor. The FR-900 216 substance, the active ingredient of the present invention, is the same compound as "Rhizoxin" which was published in the Symposium Papers of the 25th Symposium on the Chemistry of Natural Products held in Tokyo in 1982. Said FR-900 216 substance (Rhizoxin) has the following structural formula: and the following physical and chemical properties: 1) Molecular weight: 625 (Mass spectrum) 2) Molecular formula: C₃₅H₄₇NO₉ 3) Melting point: 130 - 135°C (dec.) 4) Specific rotation: [α] 20 / D = +140°C (c = 1, CHCl₃) 5) Ultraviolet absorption spectrum: 6) Infrared absorption spectrum: 7) ¹H Nuclear magnetic resonance spectrum (CDCl₃, inner standard: tetramethylsilane): It is shown in Figure 1 of the accompanying drawing. 8) ¹³C Nuclear magnetic resonance spectrum (CDCl₃, inner standard: tetramethylsilane): δ (ppm): 9.65, 11.41, 11.71, 13.77, 14.32, 16.93, 22.57, 29.24, 31.49, 31.85, 34.16, 35.86, 36.40, 37.98, 45.32, 54.18, 56.12, 63.77, 65.10, 76.81, 82.33, 89.25, 120.80, 123.77, 126.44, 129.29, 135.97, 136.21, 136.57, 137.67, 138.51, 139.54, 160.84, 168.01, 169.34 9) Solubility: Freely soluble: methanol, ethanol, acetone, ethyl acetate Soluble: chloroform, benzene Slightly soluble: hexane, diethyl ether Insoluble: water 10) Color reaction: Positive: Dragendorff's reaction, iodine reaction Negative: Ferric chloride reaction, Molisch's reaction, Ninhydrin reaction, Ehrlich's reaction 11) Nature: Weekly basic substance Subject-matter of the present invention is a pharmaceutical composition comprising FR-900 216 substance (Rhizoxin) or pharmaceutically acceptable salt thereof in association with a pharmaceutically acceptable, substantially non-toxic carrier or excipient. According to a preferred embodiment this pharmaceutical composition is an anti-tumor composition. A further subject-matter of the present invention is a method for producing a pharmaceutical composition, preferably an anti-tumor composition, which comprises mixing FR-900 216 substance (Rhizoxin) or pharmaceutically acceptable salt thereof as an active ingredient with an inert carrier. Furthermore, the present invention relates to FR-900 216 substance (Rhizoxin) or pharmaceutical acceptable salt thereof for use as a medicament, preferably for use in the treatment of tumor. In addition, the present invention relates to a method of making a pharmaceutical anti-tumor composition which is characterized in that FR-900 216 substance (Rhizoxin) or pharmaceutically acceptable salt thereof is put into a form for use recognizable by its composition or shape as being for use as a pharmaceutical anti-tumor composition. FR-900 216 substance (Rhizoxin) used according to the present invention can be produced by fermentation of FR-900 216 substance-producing strains belonging to the genus Rhizophus such as Rhizopus sp. No. F1360 (deposited with ATCC on November 21, 1979 under the Number of ATCC 20577) in a nutrient medium. Particulars of microorganism Rhizopus sp. No. F-1360 (ATCC No. 20577) which can be used for producing FR-900 216 substance (Rhizoxin) and production thereof will be explained in the following: The microorganism which can be used for the production of FR-9002l6 substance is a strain belonging to the genus Rhizopus, among which a strain of Rhizopus sp. No. F-1360 has been newly isolated from a soil sample collected at Uji-shi, Kyoto-fu, Japan. Taxonomic characteristics of Rhizopus sp. No. F-1360: (1) Morphological characteristics: (a) Macroscopic observation: (i) Cultures on malt extract agar: Colonies spread broadly, attaining over 8.0 cm in diameter after 3 to 5 days at 30°C, and finally the Petri dish is covered with the mycelia. The colony surface is plane and thick. While young cultures are white, they become gray or black with formation and maturation of sporangia and spores. The sporangia and spores are formed in abundance. The colony margin is cobweb-like, and aerial mycelia arise up to the walls of Petri dishes or slant tubes. The reverse is hyaline or pale yellow. Neither soluble pigments nor exudations are produced. The odor is sweet-smelling. It grows slowly at 25°C. (ii) Cultures on YpSs agar: Colonies are the same as on malt extract agar. (iii) Cultures on potato dextrose agar: They are the same as on malt extract agar. (iv) Cultures on Czapek's solution agar: They grow much more restrictedly. (b) Microscopic observation: The sporangia are covered with the thin-walled membrane. When they mature, they burst and discharge a number of enterogenous spores. They are globose and 30-200 µm in diameter. The sporangiospores are ellipsoidal, 7.5 x 15 µm in size, smooth to wrinkled. The substratal mycelia are non-septate, and they form the rhizoids. Each of the sporangiophores is connected by stolons and the columella is formed at the top of them. (2) Physiological characteristics: (a) Temperature range for growth: This strain can grow at temperature in the range from 10 to 48°C with the growth optimum at 33°C. And they can form sporangia from 14 to 39°C. These data were determined on potato dextrose agar. b) Growth in Pfeffer solution: This strain can grow at temperature range from 25 to 42°C; no growth at 10°C. c) pH range for growth: This strain can grow at pH 2.5 to 12.0 on malt extract agar and YpSs agar. d) Production of organic acids: This strain produce organic acids. From above-mentioned characteristics, the strain was considered to be one strain of the zygomycete genus Rhizopus Ehrenb. Consequently, the strain was named to Rhizopus sp. No. F-1360. A culture of Rhizopus sp. No. F-1360 has been deposited with ATCC (American Type Culture Collection, 12301 Parklawn Drive Rockville, MD 20852, U. S. A.) on November 21, 1979 under the number of ATCC 20577, and the deposit is now requested to be converted for Budapest Treaty purposes. This invention also includes the use of any mutants which are capable of producing FR-900216 substance, including natural mutants which are produced by natural mutation of the organism as well as artificial mutants which can be produced from the described organism by conventional means, such as X-rays, ultraviolet radiation, treatment with a mutating agent such as nitrogen mustard oils, azaserine, nitrous acid, 2-aminopurine, N,N'-nitro-N-nitrosoguanidine (NTG) or the like, treatment with phage, transformation, transduction, conjugation and the like. The FR-900216 substance of this invention is produced when a FR-900216 substance producing strain belonging to the genus Rhizopus (e.g. Rhizopus sp. No. F-l360 ATCC 20577) 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, sucrose, maltose, glycerin, starch, soluble starch, dextrin and the like. The preferred sources of nitrogen are meat extract, hydrolyzed casein, peptone, gluten meal, corn meal, cotton seed flour, soy bean meal, corn steep liquor, dried yeast, yeast extract, urea, ammonium phosphate, etc. 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 disodium or dipotassium hydrogen phosphate, magnesium chloride, magnesium sulfate, calcium carbonate and the like. If necessary, especially when the culture medium foams seriously a defoaming agent, such as plant oil (soy bean oil, linseed oil, etc.), liquid paraffin, fatty oil, higher alcohol (octadecanol, tetradecanol, heptanol, etc.), silicone may be added. Submerged aerobic cultural conditions are preferred for the production of FR-900216 substance in massive amounts. For the production in small amounts, a shaking or surface culture in a flask or bottle is employed. Furthermore, when the growth is carried out in large tanks, it is preferable to use the vegetative form of the organism for inoculation in the production tanks in order to avoid growth lag in the process of production of FR-900216 substance. Accordingly, it is desirable first to produce a vegetative inoculum of the organism by inoculating a relatively small quantity of culture medium with spores or mycelia of the organism and culturing said inoculated medium, and then to transfer the cultured vegetative inoculum aseptically to large tanks. The medium, in which the vegetative inoculum is produced, is substantially the same as or different from the medium utilized for the production of the FR-9002l6 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. Agitation may be effected by passing sterile air through the fermentation mixture. The fermentation is usually conducted at a temperature between about 20°C and 40°C, preferably 25-35°C, for a period of about 50 hours to 100 hours. Thus produced FR-9002l6 substance can be recovered from the cultured medium by conventional means which are commonly used for the recovery of other known biologically active substances. The FR-900216 substance may be included in both of filtered broth and cultured mycelia, but, in general, most of FR-9002l6 substance are found in the cultured mycelia. F-900216 substance can be extracted from the cultured mycelia with a solvent such as ethyl acetate and the like. Further, FR-900216 substance can be isolated or purified from thus obtained extract or filtered broth by a conventional manner such as concentration under reduced pressure, extraction with solvent, treatment with resin (e.g. anion or cation exchange resin, non-ionic adsorption resin), treatment with a conventional adsorbent (e.g. activated charcol, silicic acid, silica gel, alumina cellulose, etc.), crystallization, recrystallization, column chromatography using silica gel and the like, liquid column chromatography using silica gel and the like. Thus obtained FR-900216 substance can be converted to the corresponding pharmaceutically acceptable salt thereof by treating it with an acid by a conventional manner. Suitable pharmaceutically acceptable salt of FR-9002l6 substance may include inorganic or organic acid addition salts such as acetic acid salt, lactic acid salt, maleic acid salt, fumaric acid salt, oxalic acid salt, citric acid salt, methanesulfonic acid salt, hydrochloric acid salt, sulfuric acid salt, phosphoric acid salt and the like. 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 FR-9002l6 substance or its pharmaceutically acceptable salts, as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for external, enteral 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. FR-9002l6 substance is included in the pharmaceutical composition in an amount sufficient to produce the desired antitumor effect upon the process or condition of diseases. For applying this composition to human, it is preferably to apply it by intravenous, intramuscular or oral administration. While the dosage of therapeutically effective amount of FR-900216 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 0.01-1 mg/kg, preferably about 1 mg/kg, in the case of intramuscular administration, a daily dose of 0.1-10 mg/kg, preferably about 1 mg/kg, in case of oral administration, a daily dose of 0.5-50 mg/kg, preferably about 5 mg/kg of FR-900216 substance is generally given for treating tumor. The antitumor activity of FR-9002l6 substance is explained in the following. Test 1 Antitumor activity of FR-900216 substance against Lymphocytic Leukemia L1210 and P388: (1) Intraperitoneal administration: Method Lymphocytic Leukemia (L1210 or P388) cells were successively transferred to DBA/2 mice. On day 7, ascites were collected from the intraperitoneal cavity to isolate the tumor cells, from which the suspension of the tumor cells was prepared. The cell suspension (0.2 ml) containing 10⁵ cells (L1210) or 10⁶ cells(P388) was implanted intraperitoneally to female BDF₁ mice (7 weeks old and weighing 17.9 g to 21.7 g in the case of L1210, and 8 weeks old and weighing 18.4 to 22.5 g in the case of P388). The sample of FR-900216 substance used in the experiment was solubilized in methanol, concentrated in vacuo and then suspended in the sterilized water. After 24 hours of the implantation of the tumor cells to the mice, FR-900216 substance was administered intraperitoneally in the graded doses, respectively in each medicated group (water only in the control group) once a day for 4 days. Antitumor activity of FR-900216 substance was evaluated by the increase in life-span of the medicated group over the control groups (T/C x 100) in leukemias, wherein T is median survival time (MST) of the medicated group, and C is median survival time of the control group. Result The test result is shown in the following Table 1. (2) Oral administration: Method Lymphocytic Leukemia L1210 cells were successively transferred to DBA/2 mice. On day 7, ascites were collected from the intraperitoneal cavity to isolate the tumor cells, from which the suspension of the tumor cells was prepared. The cell suspension (0.2 ml) containing 10⁵ cells was implanted intraperitoneally to BDF₁ mice 7 weeks old and weighing 17.9 to 21.7 g. The sample of FR-900216 substance used in the experiment was solubilized in methanol, concentrated in vacuo and then suspended in the sterilized water. After 24 hours of the implantation of the tumor cells to the mice, FR-900216 substance was administered orally in the doses of 10,000 and 30,000 mcg/kg, respectively in each medicated group (water only in th control group) once a day for 4 days. Antitumor activity of FR-900216 substance was evaluated by the increase in life-span of the medicated group over the control group (T/C x 100) in leukemia, where T is median survival time (MST) of the medicated group, and C is the median survival time of the control group. Result The test result is shown in the following Table 2. Test 3 Antitumor activity of FR-900216 substance against Melanoma B16: Method On 14 days after subcutaneous implantation of Melanoma B16 cells to DBA/2 mouse, one gram of the tumor was aseptically separated from mouse and homogenized in 10 ml of Hank's solution. 0.5 ml of the homogenate was inoculated to BDF₁ mouse weighing 19.7 to 28.4 g of 12 weeks age. Twenty-four hours after the implantation of the tumor cells, graded doses of FR-900216 substance was administered to mice intraperitoneally once a day on day 1, 2, 3 and 4. FR-900216 substance was first dissolved into methanol, concentrated in vacuo by adding water and finally obtained in aqueous suspension. A tumor activity was evaluated by T/C (%) as described in Test 1. Result The test result is shown in the following Table 3 Test 3 Antitumor activity of FR-900216 substance for subrenal capsule human mammary carcinoma MX-1 xenograft: Method The antitumor activity of FR-900216 substance was determined in experimental tumor system in Swiss-nu/nu mice. Swiss-nu/nu mice were subcutaneously implanted with a 2 x 2 x 2 mm fragment of MX-1 cells. When donor tumor reached approximately 1 g (30 days after the implantation of tumor cells) , MX-1 cells were implanted with a 1 x 1 x 1 mm 3 fragment under the subrenal capsule, using a 16 gauge trocar after exposing the kidney with a 7 mm dorsal skin incision. The wound was closed with a 9 mm wound clip after closing the peritoneum with 1-4 silk sutures. One day after implantation of tumor cells, graded doses of FR-900216 substance were administered to mice intraperitoneally. The samples of FR-900216 substance used in the experiment were solubilized in methanol, concentrated in vacuo and then suspended in the sterilized water. Treatment is Q4D on days 1, 5 and 9. Control mice received intraperitoneal doses of physiological saline solution. The injection volume was 0.2 ml in all experiments. Six mice were used for each experimental group. At day 1 and day 11, tumor length and tumor width were measured. The antitumor activity was calculated according to the protocols for selected in vivo models in NCI, USA. Result The result is shown in Table 4. The following Preparations are given for the purpose of illustrating the preparation of FR-900216 substance. Preparation 1 Seed medium (80 ml) containing corn starch (1%), glucose (0.5%), gluten meal (1%), dried yeast (1%) and corn steep liquor (1%) (adjusted to pH 7.0 before sterilization) was poured into each of six 275 ml-Erlenmeyer flasks and sterilized at 120°C for 30 minutes. A loopful of slant culture of Rhizopus sp. No. F-1360 ATCC 20577 was inoculated to each of the media and cultured at 30°C for 48 hours at 250 rpm using a rotary shaker. The total volume of thus obtained seed culture was inoculated again to a seed medium (20 liters) containing a soluble starch (2%), glycerin (0.5%), glucose (0.5%), gluten meal (1%), dried yeast (1%), corn steep liquor (1%) (adjusted to pH 7.0 before sterilization) in a 30 liter stainless jar fermentor which had been sterilized at 120°C for 30 minutes and cultured at 30°C for 24 hours under aeration of 1 VVM and agitation of 200rpm. Further, total volume of thus obtained seed culture were inoculated to a production medium (150 liter) containing soluble starch (2%), glycerin (0.5%), dried yeast (1%), cottonseed flour (1%) soy been meal (0.5%) and calcium carbonate (%) (pH 7.0) in a 200-liter stainless jar fermentor which had been sterilized at 120°C for 30 minutes and cultured at 30°C for 72 hours under aeration of 0.7 VVM and agitation of 250 rpm. The cultured broth thus obtained was filtered with an aid of diatomaseous earth (5 kg). To the mycelia obtained were added 80 liters of ethyl acetate and extracted. This extraction procedure was carried out twice and the extracts were combined. The extract was concentrated in vacuo to a volume of 1 liter. The concentrate was washed with aqueous sodium bicarbonate solution (0.5%, 500 ml), dried over anhydrous sodium sulfate and further concentrated in vacuo to a volume of 300 ml. The concentrate thus obtained was applied to a column chromatography using silica gel (2500 ml). The column was washed with chloroform, and then eluted with a mixture of chloroform and methanol (100:1). Fractions containing FR-900216 substance were concentrated to a volume of 20 ml. To the concentrate hexane (1.5 liter) was added and filtered. Thus obtained precipitate was dried to give a crude powder (1.1 g). The crude powder was dissolved in methanol (100 ml) and then was applied to a reverse phase column chromatography using NS Gel (1500 ml, made by Nihon Seimitsukagaku Co., Ltd.). The column was eluted with methanol and fractions containing FR-900216 substance were collected (total 800 ml) and were concentrated to a volume of 10 ml. Hexane (1 liter) was added to the concentrate and filtered. Thus obtained precipitate was dried to give a power (630 mg). The powder was purified using thin-layer chromatography (developing solvent: chloroform : methanol = 20 : 1). Bands containing FR-900216 substance were collected and suspended in a mixture of chloroform and methanol (10 : 1). The extract was concentrated to a volume of 5 ml and added to hexane (500 ml) to give a colorless powder of FR-900216 substance (300 mg). Elemental Analysis: C: 66.30(%), H: 7.61 (%), N: 2.18(%) Preparation 2 Seed medium (80 ml) containing corn starch (1%), glucose (0.5%), gluten meal (I%), dried yeast (1%) and corn steep liquor (1%) (adjusted to pH 7.0 before sterilization) was poured into each of six 275 ml-Erlenmeyer flasks and sterilized at 120°C for 30 minutes. A loopful of slant culture of Rhizopus sp. No. F-1360 ATCC 20577 was inoculated to each of the media and cultured at 30°C for 48 hours at 250 rpm using a rotary shaker. The total volume of thus obtained seed culture was inoculated again to a seed medium (20 liters) containing a soluble starch (2%), glycerin (0.5%), glucose (0.5%), gluten meal (1%), dried yeast (1%), corn steep liquor (1%) (adjusted to pH 7.0 before sterilization) in a 30 liter stainless jar fermentor which had been sterilized at 120°C for 30 minutes and cultured at 30°C for 24 hours under aeration of 1 VVM and agitation of 200rpm. Further, total volume of thus obtained seed culture were inoculated to a production medium (150 liter) containing soluble starch (2%), glycerin (0.5%), dried yeast (1%), cottonseed flour (1%) soy been meal (0.5%) and calcium carbonate (pH 7.0) in a 200 liter stainless jar fermentor which had been sterilized at 120°C for 30 minutes and cultured at 30°C for 72 hours under aeration of 0.7 VVM and agitation of 250 rpm. The cultured broth thus obtained was filtered with an aid of diatomaseous earth (5 kg). To the mycelia obtained were added 80 liters of ethyl acetate and extracted. This extraction procedure was carried out twice and the extracts were combined. The extract was concentrated in vacuo to a volume of 1 liter. The concentrate was washed with aqueous sodium bicarbonate solution (0.5%, 500 ml), dried over anhydrous sodium sulfate and further concentrated in vacuo to a volume of 300 ml. The concentrate thus obtained was applied to a column chromatography using silica gel (2500 ml). The column was washed with chloroform, and then eluted with a mixture of chloroform and methanol (100:1). Fractions containing FR-900216 substance were concentrated to a volume of 50 ml. To the concentrate hexane (2 liter) was added and filtered. Thus obtained precipitate was dried to give a crude powder (2.5 g). The crude powder was dissolved in 10 ml of chloroform and subjected to high performance liquid chromatography (made by Nippon Water Limited Co., Ltd., column: Prepack 500 packed with silica gel) and eluted with a mixture of chloroform and methanol (100 : 15). Thus obtained fractions containing FR-900216 substance were collected, concentrated in vacuo to a volume of 10 ml and added to hexane (100 ml) to give a precipitate. Thus obtained precipitate was dried to give a power (1.2 g). This procedure was conducted once more to give a colorless powder of FR-900216 substance (500 mg). The following examples were given for the purpose of illustrating the present invention.

Claims: A pharmaceutical composition comprising FR-900216 substance (Rhizoxin) or pharmaceutically acceptable salt thereof in association with a pharmaceutically acceptable, substantially non-toxic carrier or excipient. A pharmaceutical composition as claimed in claim 1, characterised by the fact, that it is an anti-tumor composition. A method for producing a pharmaceutical composition which comprises mixing FR-900216 substance (Rhizoxin) or pharmaceutically acceptable salt thereof as an active ingredient with an inert carrier. A method as claimed in claim 3, characterised by the fact that an anti-tumor composition is produced. FR-900216 substance (Rhizoxin) or pharmaceutically acceptable salt thereof for use as a medicament. FR-900216 substance (Rhizoxin) or pharmaceutically acceptable salt thereof for use in the treatment of tumor. A method of making a pharmaceutical anti-tumor composition, characterised in that FR-900216 substance (Rhizoxin) or pharmaceutically acceptable salt thereof is put into a form for use recognisable by its composition or shape as being for use as a pharmaceutical anti-tumor composition.

Inventors: AOKI, HATSUO, IMANAKA, HIROSHI, KINO, EIKO, KIYOTO, SUMIO, KOHSAKA, MASANOBU, OKUHARA, MASAKUNI, TANAKA, HIROKAZU
Applicants: FUJISAWA PHARMACEUTICAL CO., LTD.
Application year: 1984
Main classification: C12P1
IPC codes: C07D493, C12P1