GB2142026A - Desacetyl ravidomycin - Google Patents

Abstract

Desacetyl ravidomycin, a method for producing desacetyl ravidomycin from Streptomyces olivaceogriseus NRRL 15,357 and antibacterial and antitumor treatments for warm-blooded animals with desacetyl ravidomycin are described. The compound has the formula:- <IMAGE>

Description

SPECIFICATION Desacetyl ravidomycin BACKGROUND OF THE INVENTION The present invention concerns an antibacterial and antitumor compound, method of use and synthesis. More particularly, the present invention pertains to an antibiotic and antineoplastic agent isolated from a new species of Streptomyces, and its use in treatment of infections and inhibition of tumor growth.

Streptomyces, a family of the order Actinomycetales, are characterized by stable, branching filamentous growth and formation of aerial mycelia with spores. Streptomyces bacteria are ubiquitous in soil. In the soil, Streptomyces are widespread scavengers, breaking down proteins, cellulose and organic matter such as waxes, rubber and paraffin.

Until about 40 years ago, only a handful of soil microbiologists were concerned with Streptomyces. However, beginning with the isolation of actinomycin and then streptomycin, Streptomyces from soil samples from all over the world have been studied for production of antiobiotics.

Recently, a new antibiotic has been successfully isolated from a new species of Streptomyces, Stretomyces ravidus NRRL 11,300. Such antibiotic, known as ravidomycin, is described in U.S.

Patent No. 4,230,692, which issued on October 28, 1 980. Ravidomycin possesses not only antibacterial activity, but also antitumor activity.

DESCRIPTION OF THE INVENTION This invention relates to a new antibacterial and antitumor agent desacetyl ravidomycin, to its production by fermentation, to methods for its recovery and concentration from crude solutions, and to processes for its purification.

The molecular structure of desacetyl ravidomycin is shown in Fig. I:

Figure I Ravidomycin is described in U.S. Patent 4,230,692 which issued on October 28, 1980. The molecular structure of ravidomycin is shown in Fig. II:

Figure II Desacetyl ravidomycin is formed during cultivation, under controlled conditions, of a new species of Streptomyces, named Streptomyces olivaceo-griseus. It has been designated LL C23201+, and will be referred to hereinafter as desacetyl ravidomycin.

The new antibiotic-producing species Streptomyces olivaceo-griseus was isolated as an aerial contaminant at the Medical Research Division, American Cyanamid Company, Pearl River, New York and is maintained in the culture collection of the aforesaid Medical Research Division as culture number LL-C23201. A viable culture of this new microorganism was deposited on 5th April 1 983 with the Agricultural Research Culture Collection, Northern Regional Research Center, U.S. Department of Agriculture, Peoria, Illinois and has been added to its permanent collection. It has been assigned the strain designation NRRL 1 5357. It will be hereinafter referred to as NRRL 15357.

The culture NRRL 1 5357 was taxonomically characterized and identified as a new species of the gray-spored Streptomyces to be known as Streptomyces olivaceo-griseus. Observations were made of the cultural, physiological and morphological features of the culture in accordance with the methods detailed by Shilling, E.B. and Gottlieb, D., INTERNAT, J. SYST. BACTERIOL., 16: 313-340 (1966). Media used in this study were selected from those recommended by Pridham, T.G., et. al., ANTIBIOTICS ANNUAL, pp 947-953 (1956/57) and Gordon, R.E., et.

al., INTERNAT J. SYST. BACTERIOL., 24: 54-63 (1974) for the taxonomic study of Actinomycetes and soil bacteria, such as Streptomyces, respectively. Chemical composition of the cell walls of the culture was determined using the method of Lechevalier, et. al., ADV.

APPL. MICROBIOL., 14: 47-72 (1971). Details of such observations are recorded in Tables I-V and a general description of the culture is given below. Underscored descriptive colors are taken from Kelly, K.L. and Judd, D.B., NAT. BUR. STAND., SPEC. PUBL. 440(1976) and the accompanying Inter-Society Color-Council, National Bureau of Standards, Centroid Color Charts.

Isolate NRRL 1 5357 was compared to an appropriate reference strain, Streptomyces ravidus NRRL 11300, a culture which produces ravidomycin. See U.S. Patent No. 4,230,692. A comparison of 14-day growth of each of these cultures on Hickey-Tresner agar is shown below: Culture Spore Mass Color Soluble Pigments Reverse Color S. ravidus Light gray red grayish reddish NRRL 11300 brown S. olivaceo Light gray none blackish griseus green NRRL 15357 The gross colonial morphology of NRRL 1 5357 does not resemble S. ravidus and significant physiological differences were also observed. S. ravidus reduces nitrates and utilizes arabinose, but does not utilize mannitol or sucrose.A search of the current streptomycete literature failed to reveal any described species which resembled NRRL 1 5357. Accordingly, a new species was designated, to be known as Streptomyces olivaceo-griseus.

Micromorphology Spores are formed in coiled chains (Spira) on aerial sporophores of NRRL 15357. The spores are ovoid (about 1.5-1.8 micron by about 2.0-2.5 micron), and the surface of the mature spores is smooth when observed by scanning electron microscopy.

Cell Wall Composition Whole cell hydrolysates of cultured NRRL 1 5357 contain the LL-isomer of diaminopimelic acid, placing it in the Type I cell wall group of Lechevalier, et. al. (vide supra). This is typical of all Streptomyces species.

Amount of Growth of NRRL 15357 Good growth is observed on most media; moderate growth is observed on glycerol-asparagine agar; poor growth is observed on oatmeal agar; and no growth is observed on tomato pasteoatmeal agar.

Aerial Mycelium and Spore Color Aerial mycelium is white on most media but becomes tinged with pink on inorganic saltsstarch agar; spore masses are 264. light gray in color.

Soluble Pigments Absent on many media; brownish shades where produced.

Reverse Color Greenish black to olive black shades on all media.

Physiological Reactions Nitrates not reduced to nitrites in 14 days; no liquefaction of gelatin in 14 days; no black pigment (melanin) produced on either peptone-yeast extract-iron agar or tyrosine agar; strong peptonization of litmus milk in 14 days. Carbohydrate utilization as per the method of Pridham, T.G. and Gottlieb, D., J. BACTERIOL., 56, 107-144 (1948), good utilization of glucose; moderate utilization of fructose and inositol; poor utilization of galactose, mannitol, sucrose and xylose; no utilization of arabinose, raffinose, rhamose or salicin. Several organic acids were tested as sole carbon sources: citrate, malate and succinate were strongly utilized; lactate was weakly utilized; and benzoate, mucate and oxalate were not utilized. Adenine, hypoxanthine and tyrosine were hydrolysed in 14 days, while guanine and xanthine were not.

TABLE I Cultural Characteristics of Streptomyces olivaceo-griseus NRRL-15357 Incubation:14 days Temperature about 28 C

Amount of Aerial Mycelium and/or Spores Soluble Reverse Medium Growth Pigment Color Glycerol-Asparagine Moderate Relatively flat, waxy growth with Brown- Green Agar no aerial mycelia; growth 77. ish ish noderate yellowish brorn black Hickey-Tresner Good Raised waxy colonies with plicate none black Agar centers; vegetative growth 152. ish blcackish green; very little aerial green spores 264. light gray.

Inorganic Salts- Moderate Slightly raised waxy colonies; none Starch Agar to Good 110. grayish olive to 114. olive black, becoming powdery in sporulating areas; aerial mycelia white to 28. light grayish pink in areas; spores 264. light gray TABLE I (continued)

Nz- AMINE# Glucose Good Raised, ridged growth; vegetative Brown- Green Starch Agar mycelia 157. greenish black; heavy ish ish production of aerial mycelia and black spores; spores 264. light gray Oatmeal Agar Poor Flat, dull growth with no aerial none mycelia; vegetative mycelia 90.

grayish yellow to 112. light olive gray Yeast Extract Malt Good Raised, ridged colonies with Brown Olive Extract Agar heavy sporulation; vegetative black mycelia 114. olive black; spore mass 264. light gray TABLI II Micromorphoiogy of Streptomyces olivaceo-griseus NRRL-15357

Aerial Mycelium and/or Sporiferous Sproe Spore Spore Medium Structures Shape Size Surface about Inorganic Salts- Spore chains arise as coiled Ovoid 1.5-1.8 micron Smooth Starch Agar chains from aerial mycelia (Spira) about x 2.0-2.5 micron TABLE III Physiological Reaction of Streptomyces olivaceo-griseus NRRL 15357

Incubation Amount oE Physiological Medium period (Days) Growth Reaction Peptone-Iron 7 Good No blackening Agar 14 Good No blackening Tyrosine Agar 7 Good No blackening (ISP-7) 14 Good Greenish black pigment Litmus Milk 7 Good Slight proteolysis 14 Good Strong proteolysis Nutrien 7 Good No proteolysis Gelatin 14 Good No proteolysis Nitrate Broth 7 Good No reduction 14 Good No reduction Adenine Agar 7 Good Strong hydrolysis 14 Good Strong hydrolysis Guanine Agar 7 Good No hydrolysis 14 Good No hydrolysis Hypoxanthine 7 Good No hydrolysis Agar 14 Good Strong hydrolysis Tyrosine Agar 7 Good No hydrolysis 14 Good Weak hydrolysis Xanthine Agar 7 Good No hydrolysis 14 Good No hydrolysis TABLE IV Carbon Source Utilization of Streptomyces olivaceo-griseus NRRL 15357 Incubation: 14 Days Temperature: about 28"C Carbon Source Utilization I- Arabinose O Fructose 2 d- Ga lactose 1 d-Glucose 3 i- Inositol 2 d- Mannitol 1 d- Raffinose O I- Rhamnbse O Salicin O Sucrose 1 Xylose 1 Negative Control O .3 = Good utilization 2 = Fair utilization 1 = Poor utilization O = No utilization TABLE V Utilization of Organic Acids by Streptomyces olivaceo-griseus NRRL 15357 on Gordon's Modification of Koser's Basal Agar (Koser's Citrate Agar) Incubation: 14 Days Temperature: about 28'C Carbon Source Utilization Benzoate Citrate + Lactate + Malate + Mucic Acid Oxalate Succinate + -: no utilization +: strong utilization +: some utilization It is to be understood that for the production of this antibacterial and antitumor agent, the present invention is not limited to the particular Streptomyces organism designated NRRL 15357, or to organisms fully answering the above growth and microscopic characteristics, which are given for illustrative purposes only. It is intended to include the use, for production of desacetyl ravidomycin, of natural mutants of the organism designated NRRL 1 5357 and mutants produced from such by various means; e.g., exposure to x-radiation, ultraviolet radiation, nitrogen mustard, actinophages and the like.

The antibacterial and antitumor agent desacetyl ravidomycin is active in vitro against a variety of gram-positive bacteria and neoplasms.

The in-vitro antibacterial spectrum of desacetyl ravidomycin was determined by the agar plate dilution method with Mueller-Hinton agar and an inoculum of each test organism of approximately 104 colony-forming units delivered by a Steers replicating device. The minimal inhibitory concentration was defined as the lowest concentration of desacetyl ravidomycin that inhibited visible growth after about 18 hours incubation at about 35"C. The results appear in Table VI.

TABLE VI Antibacterial Spectrum of Desacetyl Ravidomycin

Minimal Inhibi tory Concentra Gram-positive Bacteria tion (mcg/ml) Staphylococcus aureus,. Smith < 0.25 I. LL No. 14 < 0.25 Ce LL No. 27 0.5 " LL No. 45 0.5 C. ATCC 25923 0.5 Staphylococcus epidermidis, CMC-83-56 1 CC ATCC 12228 0.5 Streptococcus faecalis, ATCC 29212 < 0.25 Streptococcus mutans, ATCC 27352 1 Streptococcus sanguis G9B(a) < 0.25 Streptococcus (enterococcus) sp OSU-75-1 < 0.25 SM-77-15 < 0.25 Micrococcus luteus, PCI 1001 < 0.25 Bacillus subtilis, ATCC 6633 < 0.25 Bacillus cereus, PCI 213 0.5 Certain in vivo testing systems and protocols have been developed by the National Cancer Institute for testing compounds to determine their suitability as antineoplastic agents. These have been reported in "Cancer Chemotherapy Reports", Part Ill, Vol. 3, No. 2 (1972), Deran, et. al. These protocols have established standardized screening tests which are generally followed in the field of testing for antitumor agents. Of these systems, lymphocytic leukemia P388 and melanotic melanoma B16 are particularly significant to the present invention. Such neoplasms are found in mice. Generally, good antitumor activity, as shown in these protocols by a percentage increase of mean survival times of the treated animals (T) over the control animals (C), is predictive of similar results in human leukemias. Id.

The compound desacetyl ravidomycin possesses the property of inhibiting the growth of transplanted mouse tumors as established by the following tests.

Lymphocytic Leukemia P388 Test The animals used were BDF1 mice, all of one sex, weighing a minimum of about 17 g and all within an about 3 g weight range. There were 5 or 6 mice per test group. The tumor transplant was by intraperitoneal injection of about 0.5 ml of dilute ascitic fluid containing about 106 cells of lymphocytic leukemia P388. The test compounds were administered intraperitoneally at a volume of about 0.5 ml in about 0.2% Klucel in normai saline on days 1,5 and 9 (relative to tumor inoculation) at the indicated doses. The mice were weighed and the survivors recorded on a regular basis for 30 days. The results of this test appear in Table VII.

TABLE VII Desacetyl Ravidomycin and Ravidomycin Activity Against P388 Leukemia in Mice

Dose Median T/Cx100 (mg/kg) Survival Compound (Days) Desacetyl Ravidomycin 25 20.5 174 (Test No. 1) 12 19.5 165 6 17.5 148 3 15.5 131 Control - 11.8 - 1.4-dihydroxy-5,8-bis[(2 (2-hydroxyethylamino)ethyli- 1.6 18.2 > 154 amino]anthraquinone, dihydrochloride (hereinafter called Positive Control) Desacetyl Ravidomycin 25 16.5 183 (Test No.2) 6 14 156 1.6 - 12.5 139 0.4 10.5 117 Control - 9 - Positive Control 0.4 14 156 Ravidomycin hydrochloride 50 21 178 25 20.5 174 12 17 144 6 12 102 3 12 102 Control - 11.8 Positive Control 1.6 18.2 > 154 Ravidomycin 400 16 178 100 17.5 194 25 14 156 6 10 111 1.6 10 111 Control - 9 Positive Control 0.4 14 156 Melanotic Melanoma B16 Test The animals used were BDF1 mice, all of the same sex, weighing a minimum of about 1 7 g and all within an about 3 g weight range. There are normally 6 animals per test group.An about one gram portion of melanotic melanoma B16 tumor was homogenized in about 10 ml of cold balanced salt solution and approximately 0.5 ml aliquot of the homogenate was implanted intraperitoneally into each of the test mice. The test compounds were administered intraperitoneally on days 1 through 9 (relative to tumor inoculation) at various doses. The mice were weighed and survivors recorded on a regular basis for 60 days. The median survival time and the ratio of survival time for treated (T)/control (C) mice were calculated. The positive control compound was the same as that used for the P388 test. The results appear in Table Vlil in comparison with ravidomycin.

TABLE VIII Desacetyl Ravidomycin and Ravidomycin Activity Against B16 Melanoma in Mice

Dose Median T/CxlO0 (mg/kg) Survival Compound (Days) Desacetyl Ravidomycin 12 38 184 3 -32 155 0.8 29.5 143 0.2 26 126 Control - 20.7 - Positive Control 0.8 > 39 > 190 Ravidomycin bydrochioride 12 39 188 3 32 155 0.8 -27.5 133 0.2 23.5 114 Control - 20.7 - Positive Control 0.8 > 39 > 190 General Fermentation Conditions Cultivation of Streptomyces olivaceo-griseus NRRL 1 5357 may be carried out in wide variety of liquid culture media. Media which are useful for the production of desacetyl ravidomycin include an assimilable source of carbon such as starch, sugar, molasses, glycerol, etc.; an assimilable source of nitrogen such as protein, protein hydrolysate, polypeptides, amino acids, corn steep liquor, etc.; and inorganic anions and cations such as potassium, sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, etc. Trace elements such as boron, molybdenum, copper, etc., are supplied as impurities of other constituents of the media.

Aeration in tanks and bottles is supplied by forcing sterile air through or onto the surface of the fermenting medium. Further agitation in tanks is provided by a mechanical impeller. An antifoam agent such as lard oil may be added as needed.

General Procedure for the Isolation of Desacetyl Ravidomycin Desacetyl ravidomycin is recovered from the fermentation mash by extraction with an organic solvent such as dichloromethane, concentrating the extract and chromatographing this extract on a column of silica gel and eluting with an appropriate solvent system.

The invention will be described in greater detail in conjunction with the following non-limiting specific examples.

Example 1 Inoculum Preparation An inoculum medium was prepared according to the following formulation.

Beef extract approx. 0.3% Tryptose approx. 0.5% Dextrose approx. 1.0% Yeast extract approx. 0.5% Water qs to 100% This medium was adjusted to about pH 6.8 and then sterilized. Approximately 50 ml of this medium were placed in a 250 ml flask, and were inoculated with mycelial scrapings from an agar slant of the culture Streptomyces olivaceo-griseus (NRRL 15357). The flask was incubated at about 32"C on a rotary shaker at approximately 180-200 rpm for about 48-72 hours.

Example 2 Fermentation A fermentation medium of the following formulation was prepared: Glucose approx. 1.5% Glycerol approx. 1.5% Soybean flour approx. 1.5% Calcium carbonate approx. 0.1% Sodium chloride approx. 0.3% Water qs to 100% The medium, adjusted to about pH 6.7, prior to sterilization, was distributed in about 100 ml aliquots in 500 ml Erlenmeyer flasks. The flasks were then sterilized and inoculated with about 5.0 ml of the inoculum prepared as described in Example 1. The fermentation was carried out at about 28"C for 6 days at which time the mash was harvested. This mash contained about 10 mcg/ml desacetyl ravidomycin.

Example 3 Fermentation With Defined Medium A fermentation medium of the following formulation was prepared.

Sodium nitrate approx. 0.1% Ferrous sulfate heptahydrate approx. 0.01% Magnesium sulfate heptahydrate approx. 0.02% Calcuim carbonate approx. 0.5% Dextrose approx. 1.5% Sodium acetate approx. 0.25% Water qs to 100% The medium, adjusted to about pH 7.2 prior to sterilization, was distributed in about 100 ml aliquots in 500 ml Erlenmyer flasks. The flasks were sterilized and then inoculated with about 50 ml of the inoculum described in Example 1. The fermentation was carried out at about 28"C for 6 days at which time the mash was harvested. This mash contained about 5 mcg/ml desacetyl ravidomycin.

Example 4 Isolation of Desacetyl Ravidomycin The one liter of harvest mash from Example 2 was extracted twice with about 500 ml portions of dichloromethane. The dichloromethane extracts were combined, centrifuged, dried over anhydrous sodium sulfate and then evaporated in vacuo at about 35"C. The residue was dissolved in about 2 ml of ethanol: dichloromethane (1:1) and applied to a 15 X 400mm column containing 40 g of silica gel packed with the same solvent mixture. The column was developed with the same solvent mixture at the rate of about 2-3 ml per minute, collecting fractions at approximately 1 2 minute intervals. Fractions 15-26 were combined and evaporated, giving about 8.5 mg of desacetyl ravidomycin.

Desacetyl ravidomycin may be administered or employed by itself or in combination with a pharmaceutically acceptable carrier. Of course, any material used as such carrier should be pure substantially non-toxic in amounts employed and not interfere with the biological activity of desacetyl ravidomycin.

Claims (6)

1. A compound having the following formula:

2. A method of inhibiting the growth of tumors in a warm-blooded animal which comprises administering to said animal an effective amount of a compound having the following formula:

3. A method for treating bacterial infections in a warm-blooded animal which comprises administering to said animal an effective amount of a compound having the following formula:

4. A process for producing desacetyl ravidomycin comprising cultivating Streptomyces olivaceo-griseus NRRL 1 5357 in a liquid medium containing assimilable sources of carbon, nitrogen and inorganic salts until substantial amounts of desacetyl ravidomycin are present in such medium;

5. The process according to Claim 4 wherein the cultivation is conducted at about 24-32'C for about 4-6 days.

6. A pharmaceutical composition comprising a compound having the following formula:

and a pharmaceutically acceptable carrier.