FR2550536A1 - DESACETYL-RAVIDOMYCIN, METHOD FOR PRODUCING THE SAME, AND THERAPEUTIC USE THEREOF - Google Patents

Abstract

THE INVENTION CONCERNS DESACETYL-RAVIDOMYCIN, WHICH IS A COMPOUND OF THE FORMULA: AND ITS PRODUCTION PROCESS FROM OLIVACEO-GRISEUS STREPTOMYCES. APPLICATION IN THERAPEUTICS.

Description

The present invention relates to an antibacterial and anti-tumor compound,

  In particular, the present invention relates to an antibiotic and antineoplastic agent isolated from a novel species of Streptomyces, and its application to treatment.

  infections and inhibition of tumor development.

  Streptomyces, a family of the order Actinomycetales, are characterized by stable branched filamentous growth and aerial mycelium formation with spores. Streptomyces-like bacteria are found throughout the soil In soil, Streptomyces are widely used cleaning agents which break down proteins, cellulose and organic materials such as waxes, rubber and paraffin. Until about 40 years ago, only a minority of microbiologists studying soil microbes

  However, starting with the isolation of actinomycin and then streptomycin, Streptomyces from soil samples from all parts of the globe have been studied for the production of antibiotics.

  Recently, a new antibiotic has been successfully isolated from a new species of Streptomyces, Streptomyces ravidus NRRL 11,300. This antibiotic, known as ravidomycin, is described in US Pat.

  United States of America No. 4,230,692 issued October 28, 1980 Ravidomycin possesses not only antibacterial activity, but also antitumor activity.

  The present invention relates to a new antibacterial and antitumor agent, deacetyl-ravidomycin,

  its fermentation production, processes for its recovery and concentration from crude solutions, and methods for its purification.

  The molecular structure of desacetyl-ravidomycin is indicated by Formula I: Formula I Ravidomycin is described in US Patent No. 4,230,692 issued October 28, 1980. Molecularity of Ravidomycin is represented by Formula II:

OH OCH 3 25

  HO, Deacetyl-ravidomycin is formed during the cultivation, under controlled conditions, of a new species of Streptomyces, called Streptomyces olivaceo-griseus. It has been designated LL-C 23201 Y and will be referred to herein as after deacetyl-ravidomycin. The novel Streptomyces olivaceo-griseus species producing an antibiotic is isolated as an aerial impurity at the Medical Research Division, American Cyanamid Company, Pearl River, New York, and is used in the culture collection of the Medical Research Division. above under the LL-C 23201 culture number A viable culture of this new microorganism was deposited on April 5, 1983 at the Agricultural Research Culture Collection, Northern Regional Research Center, US Department of Agriculture, Peoria, Illinois and was added to its permanent collection It has been assigned the designation

of strain NRRL 15357.

  The NRRL 15357 culture is taxonomically characterized and is identified as a new gray-spored Streptomyces species known as Streptomyces olivaceo-griseus. Observations have been made on the physiological and morphological characteristics of the culture according to the methods described in detail. by EB Shirling and D Gottlieb, INTERNAT J SYST BACTERIOL, 16: 313-340 (1966) The media used in this study were selected from those recommended by TG Pridham et al, ANTIBIOTICS ANNUAL, pp. 947-953 (1956/57). ) and RE Gordon et al, INTERNAT J SYST BACTERIOL, 24: 54-63 (1974) for the taxonomic study of Actinomycetes and soil bacteria such as Streptomyces, respectively.

  Cell wall chemistry of the culture is determined using the method of Lechevalier et al., ADV.

  APPLICATIONS MICROBIOL, 14: 47-72 (1971) Details of these observations are recorded in Tables IV and one of the general description of culture is given below. The descriptive colors underlined are according to KL Kelly and DB Judd, NAT BUR STAND, SPEC PUB Lt 440 (1976) and associated Inter-Society Color Council, National Bureau of

Standards, Centroid Color Charts.

  Isolated NRRL 15357 is compared to a suitable reference strain, Streptomyces ravidus NRRL 11300, a culture that produces ravidomycin. See U.S. Patent No. 4,230,692. A 14-day growth comparison of each of these crops on the

  Hickey-Tresner agar is indicated below.

  Cultivation Color of Soluble Pigments Color Spore mass by transparency S.ravidus Light gray Red Greyish-brown NRRL 11300 reddish S.olivaceo griseus Light gray None Blackish green

NRRL 15357

  The coarse morphology of the NRRL 15357 colonies does not resemble S ravidus and there are also significant physiological differences. S ravidus reduces nitrates and uses arabinose but does not use mannitol or sucrose. A review of current literature Streptomycetes do not reveal any species described which resemble NRRL 15357 Accordingly, a new species is designated, which must be known: as Streptomyces olivaceo-griseus 3 Micromorphology Spores are formed into coiled chains (Spiers) on Aerial sporophores of NRRL 15357 The spores are ovo Ydes (about 1.5-1.8 microns on about 2.0-2.5 microns), and the surface of the mature spores is

  smooth when viewed by scanning electron microscope.

  Cell wall composition Whole cell hydrolysates of NRRL

  15357 contain the LL isomer of diamino pimelic acid, which classifies it in the cell wall group.

  Lechevalier et al. Type I (see above).

  This is typical of all Streptomyces species.

  Growth Degree of NRRL 15357 Good growth is observed in most media; there is moderate growth in fast-growing glycerol and asparagine; poor growth on

  oatmeal agar; and no growth on agar with tomato purée and oatmeal.

  Aerial mycelium and spore color The aerial mycelium is white on most media but becomes pink-tinged on mineral salts and starch agar; the spore masses are colored

light gray 264.

Soluble pigments

  Absent in many environments: brownish hues possible.

  Transparency color Shades black greyish to olive black on all environments. Physiological Reactions Nitrates are not reduced to nitrite in 14 days; no liquefaction of gelatin in 14 days; no black pigment (melanin) produced on peptone yeast or tyrosine agar; strong uptake of sunflower milk in 14 days Use of carbohydrates as by the method of T G Pridham and D Gottlieb, J BACTERIOL, 56, 107-144 (1948), good use of glucose; moderate use of fructose and inositol; poor use of galactosol, mannitol, sucrose and xylose; no use of arabinose, raffinose, rhamnose or salicin Various organic acids are tested as the sole sources of carbon: citrate, malate and succinate are

  heavily used; lactate is weakly used; And benzoate, mucate and oxalate are not used.

  Adenine, hypoxanthine and tyrosine are hydrolyzed in 14 days, while guanine and xanthine are not.

  Culture characteristics Incubation: 14 days

TABLE 1

  of Streptomyces olivaceo-griseus NRRL-15357 Temperature: approximately 28 C I Medium Degree of I Aerial mycelium and / or spores Pigment Color by soluble growth transparency I Glycerol agar Waxy growth relatively Brownish Greenish black and asparagine Moderate flat without aerial mycelia;

  moderate yellowish brown growth 77.

  Agar of waxy colonies erected with Hickey-Tresner Good centers of plication; growth None Blackish green

  vegetative blackish green 152; very small aerial mycelia or spore production: light gray spores 264.

  Moderate salt agar with waxy colonies slightly mineral and well erect; greyish olive 110 to None of olive black starch 114, becoming powdery in the regions of sporulation; mycelium-like white to pale greyish-pink 28 in some areas; light gray spores 264, -J Ln U 1 u. cr> Lt 'C O% TABLE I (cont.) Coissaoe agar, ridiculous, mycelia NZ-AMINE -glucose Good vegetative greenish-black 157; strong starch production of mycelia Brownish greenish black

  aerial and spores; light gray spores 264.

  Flat dull growth agar without aerial mycelia o mycelium Oatmeal Good vemetato mycelia Nil

  vegetative Nonegreyish gray 90 to olive gray 112.

  Agar with erect wrinkled colonies with yeast and strong sporulation extract; mycelium malt Good vegetative olive black 114; Brown Black olive

mass of spores 9 lightning 264.

t 1 Ln A CD 0 Oi os

TABLE II

  Micronorphology of Streptomyces olivaceo-griseus NRRL-15357 Medium Aerial mycelium and / or structures Sporeform Spore surface Spore spore spores Salts agar Spore chains appearing About 1.5minerals and in the form of coiled chains Ovo Tdes 1.8 micron Smooth of starch from aerial mycelia over about (Spiers) 2.0-2.5 micron D Ma Ln Ln C> Ln os U 1 TABLE III Physiological reaction of Streptomyces olivaceo-griseus NRRL 15357

15 20 25

  Medium Incubation Period Degree of Physiological Reaction bation (days) growth Agar 7 Good No blackening peptone and 14 Good No blackening iron Agar 7 Good No blackening tyrosine (ISP-7) 14 Good Pigment greenish black Milk 7 Good Light Proteolysis Sunflower 14 Good Forte Proteolysis Gelatin 7 Light No Nutritional Proteolysis 14 Light No Proteolysis Broth 7 Good No Reduction Nitrate 14 Good No Reduction 7 Good Agar Strong Hydrolyses Adenine 14 Good Forte Hydrolysis Agar at 7 Good No hydrolysis guanine 14 Good No hydrolysis Agar at 7 Good No hydrolysis hypoxanthine 14 Good Z Strong hydrolysis Agar at 7 Good No hydrolysis tyrosine 14 Good Low hydrolysis Agar at 7 Good Pas d ' xanthine hydrolysis 14 Good No hydrolysis it

TABLE IV

  Use of carbon source by Streptomyces olivaceo-griseus NRRL 15357 Incubation: 14 Days Temperature: about 28 C

15 20

  Carbon source Use TM 1-Arabinose OI Fructose 2 d-Galactose 1 d-Glucose 3 i-Inositol 2 d-Mannitol 1 d-Raffinose O 1-Rhamnose O Salicin 0 Sucrose 1 Xylose 1 Negative control I * 3 = Good use 2 = Fairly good use 1 = Poor use 25 O = No use

TABLE V

  Use of organic acids by Streptomyces olivaceoqriseus NRRL 15357 on Gordon's modified Koser base agar (Koser citrate agar) Incubation: 14 days Temperature approx. 28 C Source of carbon Use Benzoate Citrate + Lactate Malate + Mucic acid Oxalate I uccinate + 15: no use +: strong use

+: some use.

  It is evident that for the production of this antibacterial and antitumor agent, the present invention is not limited to the particular Streptomyces-type microorganism referred to as NRRL 15357, or to microorganisms fully responsive to the growth and microscopic characteristics above, illustrative only. It is intended to encompass the use, for the production of deacetyl-ravidomycin, of natural mutants of the microorganism designated NRRL 15357, and the mutants produced therefrom by various means; for example, exposure to X-radiation, ultraviolet radiation, nitrogenous nitrogen, actinophages, etc. The antibacterial and antitumor agent deacetyl ravidomycin is active in vitro against various

  Gram-positive bacteria and various neoplasms.

  The in vitro antibacterial spectrum of desacetyl-ravidomycin is determined by the Mueller-Hinton agar plate dilution method and an inoculum of each test microorganism of approximately 104 colony forming units distributed by a Steers replication device The minimum inhibitory concentration is defined as the lowest concentration of desactetylravidomycin which inhibits visible growth after about 18 hours of incubation at about 35 ° C.

Table VI.

TABLE VI

  Antibacterial spectrum of desacetyl-ravidomycin Gram-positive bacteria

15 20

  Staphylococcus aureus, Smith "LL N 14" LL N 27 "LL N 45" ATCC 25923 Staphylococcus epidermidis, Minimum inhibitory concentration (micrograms / ml)

<0.25 <0.25

0.5 0.5 0.5

  Streptococcus Streptococcus Streptococcus Streptococcus Micrococcus Bacillus subti Bacillus cereu

CMC-83-56

ATCC 12228

  faecalis, ATCC 29212 mutans, ATCC 27352 bloodstream G 9 B (a) (enterococcus) sp OSU-75-1

"SM-77-15

lily, ATCC 6633 0.5

<0.25

  S 0.25 S 0.25 s 0.25 c 0.25 s 0.25 0.5 Some in vivo test systems and protocols have been developed by the National Cancer Institute to test compounds to determine their These antineoplastic agents have been shown in "Cancer Chemotherapy Reports", Part III, Vol 3, No. 2 (1972), Deran et al. These protocols have established standardized tests of selection which are generally followed in clinical trials. the domain of agent search tests

  Among these systems, P 388 lymphocytic leukemia and B 16 melanotic melanoma are particularly significant for the purposes of the present invention.

  These neoplasms are found in mice. In general, good antitumor activity as indicated in these protocols by a percentage increase in average survival times of treated animals (T) compared to

  control animals (C), suggests similar results in human leukemias Id.

  Deacetyl-ravidomycin has the property

  to inhibit the growth of transplanted tumors in mice as indicated by the following tests.

  Assay on P388 lymphocytic leukemia The animals used are BDF-1 mice, all of the same sex, weighing at least about 17 g and with a difference of plus or minus about 3 g Each group of es25 sai comprises 5 or 6 mice. Tumor transplantation is carried out by intraperitoneal injection of approximately 0.5 ml of diluted ascitic fluid containing approximately 10 6 cells of P-388 lymphocytic leukemia. The test compounds are administered intraperitoneally at a volume of 30%. 5 ml in approximately 0.2% Klucel in normal saline on days 1, 5 and 9 (relative to tumor inoculation) at the indicated doses The mice are weighed and the surviving animals are recorded

  on a regular basis for 30 days The results of this test are shown in Table VII.

TABLE VII

  Activity of deacetyl-ravidomycin and ravidomycin

15 20 25

  on P 388 leukemia in mice Compound Dose Mean time T / Cxl OO mg / kg) survival% (days) Desacetyl-ravidomycin 25 20.5 174 (Test N l) 12 19.5 165

6 17.5 148

3 15.5 131

  Control 11.8 1,4-dihydroxy dihydrochloride. 5,8-bis (2-hydroxyethylamino) -1.6 (18,2> 154 ethyl) -amino) anthraquinone, designated hereinafter as Positive Control) Deacetyl-Ravidomycin 25 16.5 1:83 (Test N 2) 6 14 156

1.6 12.5 139

0.4 10.5 117

  Control 9 Positive Control 0.4 14 156 Hydrochloride 50 21 178 Ravidomycin 25 20.5 174

12 17,144

6 12 102

3 12 102

  Control 11.8 Emery Positive 1.6 18.2> 154 TABLE VII (continued) Compound Dose Mean Time T / Cxl O O (mg / kg) Survival% _______ ___ ___ (days) Ravidomycin 400 16 178

17.5 194

14,156

6 10 111

1.6 10 111

  Control 9 Positive Control 0.4 14 156 Melanotic melanoma test B 16 The animals used are BDF 1 mice, all of the same sex, weighing about 17 g with a difference of plus or minus 3 g approximately. Each test group comprises About one gram of B 16 melanotic melanoma tumor is homogenized in about 10 ml of cold equilibrated saline solution and about an aliquot of about 0.5 ml of homogenate is implanted intraperitoneally into each of the mice. The test compounds are administered intraperitoneally on days 1 to 9 (relative to tumor inoculation) at various doses. The mice are weighed and the surviving animals are recorded on a regular basis for 60 days. average survival time and survival time ratio of treated (T) / control mice

  C are calculated The positive control compound is the same as that used for the P 388 test. The results are shown in Table VIII compared with ravidomycin.

15 20

TABLE VIII

  Activity of deacetyl-ravidomycin and ravidomycin against B16 melanoma in mice Compound Dose Mean time T / Cxl O O survival (%) (mg / kg) (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 Hydrochloride 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 The cultivation of Streptomyces olivaceo-griseus NRRL 15357 can be carried out in a wide variety of liquid culture media. The media which are used for the production of deacetylravidomycin include an assimilable source of carbon such as starch, sugar, molasses, glycerol, etc .; an assimilable source of nitrogen such as proteins, protein hydrolyzate, polypeptides, amino acids, ex-soluble trait of my Ts, 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 provided as impurities. other media constituents Aeration in tanks and bottles is obtained by repressing sterile air through or on the surface of the fermentation medium Additional agitation in the tanks is provided by a mechanical propeller An anti-foam agent such as lard can

to be added if necessary.

  General process for isolation of deacetyl-ravidomycin Deacetyl-ravidomycin is recovered from the fermentation slurry by extraction with an organic solvent such as dichloromethane, concentration

  extract and chromatography of this extract on a column of silica gel and elution with a suitable solvent system.

  The invention will be described in more detail with reference to the following non-limiting examples.

Example 1

  Inoculum Preparation An inoculum medium was prepared according to the following form: Beef Extract about 0.3% Tryptose about 0.5% Dextrose about 1.0% Envir yeast extract 0.5% Sn, Water This medium is adjusted to about pH 6.8 and then sterilized. About 50 ml of this medium are placed in a 250 ml flask and inoculated with mycelial smears from an inclined culture on Streptomyces olivaceous agar. griseus (NRRL 15357) The flask is incubated at about 32 ° C on a rotary shaker at approximately

  -200 rpm for about 48-72 hours.

Example 2

  Fermentation A fermentation medium having the following formulation is prepared: Glucose about 1.5% Glycerol about 1.5% Soy flour about 1.5% Calcium carbonate about 0.1% Sodium chloride about 0.3% Water, The medium, adjusted to about pH 6.7, before sterilization, is divided into about 1 ml aliquots in 500 ml Erlenmeyer flasks. The flasks are then sterilized and inoculated with about 5.0 ml. of the inoculum prepared as described in Example 1. The fermentation is carried out at about 28 ° C. for 6 days at the end of which the slurry is harvested.

  contains about 10 micrograms / ml of deacetyl-ravidomycin.

  E-xample 3 Fermentation with a defined medium A fermentation medium is prepared having the following formulation: Sodium nitrate about 0.1% Ferrous sulphate heptahydrate about 0.01% Magnesium sulphate heptahydrate about 0.02% Calcium carbonate about 0 5% Dextrose about 1.5% Sodium acetate about 0.25% Water, qs per 100% The medium, adjusted to about pH 7.2, before sterilization, is divided into 100 ml aliquots in vials. The flasks are sterilized and then inoculated with about 50 ml of the inoculum described in Example 1 Fermentation is carried out

  at approximately 28 ° C for 6 days after which the slurry is harvested This slurry contains about 5 micrograms / ml of deacetylravidomycin.

Example 4

  Isolation of deacetyl-ravidomycin One liter of the harvested porridge from

  Example 2 is extracted twice with portions of approximately 500 ml of dichloromethane. The dichloromethane extracts are combined, centrifuged, dried over anhydrous sodium sulphate and then evaporated under vacuum at approximately 35 ° C.

  The residue is dissolved in about 2 ml of a mixture (1: 1) ethanol: dichloromethane and applied to a column of x 400 mm containing 40 g of silica gel, filled with the same

  solvent mixture The column is developed with the same solvent mixture at a rate of about 2-3 ml per minute, collecting fractions at intervals of about 12 minutes. Fractions 15-26 are combined and evaporated to give about 8.5 mg of deacetylravidomycin.

  Deacetyl-ravidomycin can be administered alone or in combination with a carrier

  Of course, any material used as a carrier of this type must be pure and substantially non-toxic in the amounts used and not interfere with the biological activity of deacetyl-ravidomycin.

RE V E N D I C A T I O NS

  1 Compound corresponding to the formula: 10 OH OCH 3 CH. H 2 A process for producing deacetyl-ravido-20 mycine, characterized in that it consists in culturing Streptomyces olivaceo-griseus NRRL 15357 in a liquid medium containing assimilable sources of carbon, nitrogen and mineral salts up to what this

  medium contains significant amounts of deacetyl ravidomycin.

  Process according to Claim 2, characterized in that the cultivation is carried out at approximately 24-32 ° C

for about 4-6 days.

  Pharmaceutical composition, characterized in that it contains a compound having the following formula:

OH OCH 3

OCH 3 l O 15 H-i it O

  and a pharmaceutically acceptable carrier.