MXPA96003613A - Antibioti shelves - Google Patents

Abstract

The present invention relates to new antibiotics. In particular, this invention is related to the antibiotic stalobacins H and I, produced by Pseudomonas sp. PBJ-5360-STR-1-21. the microorganism that produces the antibiotics, and a process for the manufacture of the same ones

Description

ANTIBIOTIC STOOLS FIELD OF THE INVENTION The present invention relates to new antibiotics. In particular, this invention is related to the antibiotic stalobacins H and I, produced by Pseudomonas sp. PBJ-5360-STR-1-21, the microorganism that produces the antibiotics, and a process for their manufacture.

BACKGROUND OF THE INVENTION It is well known that the antibacterial activity of an antibiotic varies depending on the nature of the bacteria to be treated, and the effect of the antibiotic is often reduced due to the advent of resistant strains. The advent of multiple drug-resistant bacteria has recently become a major problem. Consequently, the development of novel and effective antibiotics that carry out effective treatments has been desired. First of all, Gram-positive bacteria, such as Staphylococcus, Streptococcus hemolytic or the like, can be resistant to antibiotics, and there is a continuing need for REF: 22892 the development of new antibiotics that have high potency for these Gram-positive bacteria.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides antibiotic stalobacines selected from the group consisting of antibiotic stalobacins H and I, produced by Pseudomonas sp. PBJ-5360-STR-1-21 previously mentioned. These antibiotics are peptide antibiotics that are produced by said Pseudomonas sp. PBJ-5360-STR-1-21. The H and I stalobacins (hereinafter sometimes referred to merely as "stalobacins") are obtained in the form of a mixture of closely related analogues, by cultivating said Pseudomonas. Their bacterial activities are much more powerful than those of the known antibiotics. The stalobacins H and I have physicochemical properties as shown in the following Table 1.

TABLE 1 Physicochemical Properties of Stalobacines H and I: Estalobacin H Estalobacin I p.f. (° C) (as salt 235 ° C (decomp. 240 ° C (decompo-de Na) puts) ne) proto-ned molecular ion LSI-MS (m / z) 1396 1325 IR (KBr) (cm_1) 3374, 1747, 3387, 1747, 1654, 1597, 1525 1651, 1596, 1527 UV (H "0) Final Absorption Final Absorption CD (S20) [?] 194-66980 [?] 212 + 9851 [?] 206 + 11530 [?] 232-31520 [?] 232-28660 [?] 257 + 4288 [?] 257 + 4749 Retention time ( min) in ero- 8.8 9.7 High Resolution liquid matography * Amino Acid Analysis (molar ratio) HyAsp1 HyAsp (1) HyAsp (1) Asp Asp (1) Asp (1) Ser Ser (1) Ser (1) Hylle 'Hylle (1) Hylle (1) Gly Gly (1) 'Gly (1) Ala Wing (1) * Column: Develosil 5C18, internal diameter 4.6 x 250 mm. Mobile phase: CH3CM / H3P04 2 mM (containing Na2S0,) 50 mm) = 43/57 Flow rate: 1 ml / min. 1) Hydroxiaspartic Acid 2) Hydroxyisoleucine DETAILED DESCRIPTION OF THE INVENTION The stalobaccine antibiotics of the present invention, characterized by the above properties, are. has found that they have excellent anti-bacterial activities in vitro and in vivo, showing potent effects especially on Gram-positive bacteria. Thus, the antibiotic estalobacins of the present invention exhibit excellent activities, and have higher activities especially against Gram-positive bacteria, as shown below in Table-2. No death was observed in the acute toxicity test. by intravenous administration of 300 mg / kg and 500 mg / kg of the antibiotics stalobacins, to mice. The stalobaccine antibiotics H and I of the present invention are produced by the cultivation of Pseudomonas sp. PBJ-5360-STR-1-21, a variant derived from Pseudomonas sp. PBJ-5360 (BIK0KEN reservoir Na 10578, FERM BP-4342), which produces a mixture of stalobacins, aerobically in a liquid medium containing assimilable carbon sources, nitrogen sources and mineral salts, in a conventional manner. This bacterium has been identified as the above-mentioned strain by culturing it according to the method as described below in Experiment 2, and comprehensively examining its morphology, cultivation properties, physiological and biochemical properties with reference to the description of Bergey's Manual of Systematic Bacterology, Vol. 1 (1984). This strain can undergo a spontaneous or artificial mutation, and it is obvious to one of skill in the art that such mutants should be included in the scope of the present invention, as long as they retain an ability to produce the stalobacins of the present invention. . In this way, the present invention also provides Pseudomonas sp. PBJ-5360-STR-1-21 which produce new antibiotic H or I stalobacins and mutants thereof, which * have an ability to produce said antibiotic stalobacines. The Pseudomonas sp. PBJ-5360-.STR-1-21 were deposited under accession number FERM P-14149 with the National Institute of Bioscience and Human Technology, Higashi 1-1-3, Tsu ubashi, Ibaraki Pref. Japan, on February 16 of 1994, and said original deposit was transferred to the International warehouse under the Budapest Treaty on April 28, 1994, and was given the Accession number FERM BP-4661. In addition, the present invention provides a process for the production of antibiotic stelobacins H and I by cultivating such a strain. Ordinary compositions of the medium and ordinary conditions used for conventional cultivation can be adopted to produce antibiotics. In principle, the medium includes carbon sources, nitrogen sources, mineral salts and the like. If necessary, vitamins, precursors or similar can also be added. Examples of carbon sources are glucose, starch, dextrin, glycerin, molasses, organic acids and the like, and these carbon sources can be used alone or in a mixture thereof. Examples of nitrogen sources are soybean powder, corn liquor, meat extract, yeast extract, cottonseed powder, peptone, wheat germ, ammonium sulfate, ammonium nitrate and the like, and these sources of nitrogen can be used alone or in a mixture thereof. Examples of mineral salts are calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate, cupric sulfate, manganese chloride, zinc sulfate, cobalt chloride, various phosphates and the like. These mineral salts can be added to a medium, when required. A sufficient quantity of the antibiotic stalobacins H and I is produced by the culture of Pseudomonas sp. PBJ-5360-STR-1-21 of the present invention, in an appropriate medium at temperatures from 20 to 35 ° C, preferably from 25 to 29 ° C, for about 1 to 7 days. The product is then purified and, if necessary, purified from the culture in a conventional manner. All such procedures are well known to one of skill in the art. The antibiotic estalobacins of the present invention are believed to be useful for the treatment of various infections, in particular, the treatment of infections caused by Gram-positive bacteria resistant to multiple drugs, since they show remarkable antibacterial activities in vivo and in vitro.

Figure 1 is a graph showing the infrared (IR) spectrum of stalobacin H.

Figure 2 is a graph showing the infrared spectrum of stalobacin I.

Figure 3 is a graph showing the nuclear magnetic resonance spectrum (NMR) of stalabakine H.

Figure 4 is a graph showing the NMR spectrum of stalabakine I.

The present invention will be explained in more detail below by means of the illustrative Examples and Experiments.

Example 1 (a) Fermentation Step: Eight hundred ml of a medium (adjusted to pH 7 with 2N NaOH) consisting of 1.0% glucose, 0.5% yeast extract (Difco) and tap water in a 2 liter Erlenmeyer flask, was inoculated with a stocking strain of Pseudomonas sp. PBJ-5360-STR-1-21 (maintained at -80 ° C in a 2 ml flask), and the resulting mixture was subjected to a shaking culture at 180 rpm with 70 mm shaking width at 28 ° C per 22 hours The culture (800 ml) was inoculated to 20 l of a medium (adjusted to pH 7 with 2N sodium hydroxide) containing 1.0% glucose, 0.4% yeast extract (Difco), 1.0% malt extract (Difco) ), 0.1% polypeptone (Nippon Seiyaku) and tap water, in a 30 lt fermenter, and the resulting mixture was cultivated with agitation at 200 rpm, with 14 l / min of aeration under 0.35 kg / cmJG of internal pressure, at 28 ° C for 21 hours. Subsequently, 8 lt of this culture was inoculated to 125 lt of a medium (adjusted to pH 7 with 2N sodium hydroxide) which consisted of 2.0% soluble starch, 2.0% yeast powder, 1.5% beta-cyclodextrin, 0.5 % olive oil, 0.3% magnesium chloride hexahydrate, 0.1% potassium diacid phosphate, 0.0008% antisugar agent ADECAN0L (Asahi Denka Kogyo) LG109 and tap water in a 250 lt tank, and the mixture will result it was cultivated with 65 lt / min of aeration, under 0.35 kg / cm2G of internal pressure, with agitation at 350 rpm at 28 ° G for 72 hours (b) Separation Step At 138 lt of the culture obtained in the previous stage, 1.4 lt of chloroform was added for sterilization *. Subsequently, 15 l of Amberlite XAD-7 (Organ) was added and the resulting mixture was mixed with stirring for 3 hours for a batch adsorption of the active substances on the resin. The resin was recovered using a stainless steel screen of N9 140 mesh. The resin was washed with water, placed on a glass column (internal diameter: 20 cm), washed with 40 l of water, with 40 l of methanol at 30% and then with 15 l of 50% methanol in 20 mM phosphate buffer (pH 7.5), and the active substances were eluted by 40 l of 60% methanol in 20 mM phosphate buffer (pH 7.5). The fractions (of 20 l) that show antibacterial activity for S. aureus JC-1 were collected, concentrated in vacuo to 3 lt. The concentrate was washed with 3 l of ethyl acetate to remove the lipophilic materials. The ethyl acetate contained in the aqueous layer was evaporated in vacuo. The active substances were absorbed on Amberlite XAD-7 (Organ) in a 1 liter column (internal diameter 6.5 cm) and the resin was washed with 2 l of water. Elution was carried out using 2 l of 30% aqueous methanol and 3 l of 50% aqueous methanol. The eluted fractions were subjected to high resolution liquid chromatography analysis, and the fractions containing the stalobacins were collected, adjusted to pH 7.0 with 2N hydrochloric acid, concentrated in vacuo and lyophilized to obtain 3890 mg of powder. (c) Purification Step: The First Step of Purification One thousand eight hundred twenty mg of the crude powder obtained in the previous step was dissolved in 60 ml of 20 mM phosphate buffer (pH 7.5). The solution was subjected to preparative high-speed liquid chromatography using the YMC 0DS column [S-15/30 μ, 5.0 x 50 cm, eluent: acetonitrile / 20 mM phosphate buffer (pH 7.5). Sodium sulphate 50 mM = 40/60, flow rate: 50 ml / min, UV detection; 210 nm] to obtain a fraction (2.4 lt) containing stalobacins H and I, as main ingredients. The acetonitrile in the reaction was distilled and the residue was passed through a column of Diaion HP-20 (Mitsubishi Kasei Corporation). The resin was washed with water, and the adsorbed components were eluted with 60% aqueous acetone. The acetone in the eluate was distilled under vacuum and the residue lyophilized to obtain 126 mg of pol.

The Second Step of Purification The powder obtained in the previous step was purified by preparative high-speed liquid chromatography under the following conditions to obtain stalobacins H and I. The powder (126 mg) was dissolved in 7 ml of 50 mM phosphate buffer (pH 7.0) 15 mg of the sample was loaded per procedure into an Asahipak 0DP-90 column (internal diameter 21.5 mm x 300 mm, eluent: acetonitrile solution of 20 mM AcOH / 20 mM AcOH aqueous solution = 40/60, flow rate 8 ml / min, UV detection: 220 nm), and stalobacin H was collected from the fractions of 144 ml to 184 ml, and stalobacin I was collected from the fractions of 216 ml to 288 ml. was repeated, and the collected fractions were neutralized to pH 7.0 with 1N aqueous sodium hydroxide.The acetonitrile was distilled in vacuo and sodium chloride was added to the residue to obtain a concentration of 5% sodium chloride. at pH 7.5 with hydr hydroxide 1N sodium and passed through MCI GEL CHP20P a (75 to 150 μ, Mitsubishi Kasei) column which had been equilibrated with aqueous solution of sodium chloride 5%. The column was washed with water and eluted with 70% aqueous methanol. The methanol and the eluate were distilled in vacuo and the residue lyophilized to give l- > 12 mg of pure stalobacin H, and 38 mg of pure stalobacin I. The physicochemical properties of the stalobacins H and I obtained in Example 1 are shown in Table 2. The IR spectra of the stalobacines H and I are shown in Figures 1 and 2 respectively, and the nuclear magnetic resonance spectra of the stalobacins H and I are shown in Figures 3 and 4, respectively.

Experiments 1 (Antibacterial activity in vitro and in vivo). 1) In vitro antibacterial activity The in vitro antibacterial activity of the antibiotic stalobacins H and I, obtained in Example 1, was tested by the agar dilution method. Results are shown in table 2.

TABLE 2 Stalobazine (μg / ml), 10 cfu / ml H I Bacteria Gram-positi va S. aureus FDA JC-1 0.1 0.05 S. faecalis SR1004 0.2 0.2 S. aureus 3626 (MRSA) 0.1 0.1 2) Antibacterial activity in vivo The in vivo antibacterial activity of stalobacin I was tested. Mice were challenged intraperitoneally with infectious bacteria. One hour after the challenge, the test compound was administered subcutaneously. The values of ED .. ^ were calculated based on the proportion of survival to the 7th. day after the challenge. The MIC was determined according to the agar dilution method. The results are shown in Table 3.

- - TABLE 3 Protective Effect of Stalobacin I in Systemically Infected Mice: ED 5-n0 (mg / kg) MIC (μg / ml) Estalobacin I Estalobacin I S. aureus SR3637 (H-MRSA) 0.12 0.012 S. pneumoniae Type I 0.046 0.006 E. faecalis SR1004 1.50 0.2 Experiment 2 Bacteriological properties of PBJ-5360 and PBJ-5360-STR-1-21: PBJ-5360-STR-1-21 of the present invention was obtained as a mutant of the aforementioned PBJ-5360 strain. PBJ-5360 was isolated from soil collected in Kyoto, Japan. Various bacteriological properties of PBJ-5360-STR-1-21 of the present invention are shown below. The culture was carried out at 28 ° C in principle.

A. Morphology: It is a Gram-negative bacillus. Its size is 1 0.3 - 0.5 (μm) x 0.8 - 1.3 (μm). It moves vigorously with one or more polar flagella.

B. Crop Characteristics. '5 1) Cultivation in the medium of meat infusion: The growth of the bacteria was hardly observed. Translucent white precipitates formed very lightly at the bottom of the test type. 2) Cultivation by meat infusion agar bite: Growth was observed in the form of yarn or in the form of a small tube along the sting line. Neither gas evolution nor pigment production was observed. A thin reddish bacterial plaque appeared on the surface, but this bacterial plaque became translucent and light brown with the passage of time, 20 and verrucous projections were observed at several sites. This is an aerobic bacterium. 3) Inclined culture in agar infusion of meat: The growth of the bacteria was not so rapid and started at 28 ° C after two days (observed with the naked eye). The bacterium developed in filamentous form, and its bacterial plaque was translucent and light yellow with flat swelling that had the appearance of spots. The periphery was complete peripheral. Subsequently, the bacterial plaque developed favorably in filamentous form or in warty form with luster. In this way, a translucent, slightly reddish, moist coffee bacterial plaque was obtained. The periphery was wavy or like long waves. Gas or pigment production was not observed. 4) Cultivation by gelatin bite of meat infusion: The culture was observed at room temperature (22-25 ° C). The gelatin liquefied slightly.

) Culture on flat medium of meat infusion agar: The growth of the bacteria was not so fast. The colony became visible at 28 ° C after two days. The colony was initially small, spotted, translucent and brown with a complete periphery. The colo- nia was too small to be observed with respect to His swelling. Subsequently, the colony developed in a dotted or circular shape and with complete periphery, and the swelling was flat or convex circular. The colony was translucent and coffee with brightness. No gas or soluble pigment was produced. 6) Characteristics in litmus milk culture No acid formation occurred, and pep-toning occurred but the reaction started after 14 days. In this way, the reaction was rather slow. There was no evolution of gas.

C. Physiological and Biochemical Properties 1) Catalase test: positive 2) Oxidase test: positive 3) OF test: negative (shown to be alkaline) 4) Haemolytic test: positive (weakly) 5) Feasibility at 5 ° C: negative 6) Production of H ^ S: negative 7) Reduction of nitrate: positive 8) Denitrification: negative (although nitrogen gas did not evolve, seemed to reduce N02 ~) 9) Citrate utilization: negative (Christensen medium and Simons medium) 10) Growth on NAC agar medium: negative (not viable) 11) Indole production: negative 12) Voges-Proskauer reaction (Voges test- Proskauer): negative 13) Methyl red test: negative 14) Arginine-dihydrolase test: weakly positive ) Lysine-decarboxylase test: positive 16) Ornithine-decarboxylase test: positive 17) Esculin hydrolysis: negative 18) DNA-loop test: negative 19) Starch hydrolysis: negative 20) ONPG test (grown at 37) ° C): negative 21) Test of acylamide: positive 22) Phosphatase test: negative 23) Chitin hydrolysis: negative 24) Productivity of sucrose from sucrose: positive 25) Acid and gas productivity from sugars : No acids or gases were produced from the following 13 sugars: glucose, fructose, galactose, mannose, xylose, arabinose, maltose, lactose, rhamnose, sucrose, cellobiose, trehalose and mannitol. 26) Accumulation of poly- (3-hydroxybutyrate in the cell: negative 27) Utilization of carbon sources: On the medium containing minerals, glucose and calcium 2-keto-gluconate can be used as a single source of carbon for the formation of cells. In this case, it seemed that specific vitamins for growth were not required. On the other hand, D- (+) - trehalose, DL-arginine, geraniol, β-alanine, L-valine and inositol were not used. 28)% mol of G + C (HPLC method): 60.4% (% mol of A + T = 39.6%) In view of the above test results, PBJ-5360-STR-1-21 is a Gram-negative aerobic bacillus and actively moves in a liquid medium using one or more polar flagella. This is catalase positive and contains oxidase. This was negative for the OF test (showing that it is alkaline). In view of these observations, it is apparent that the present bacterium belongs to the genus Pseudomonas in the Pseudomonadaceae family. When the inventors compared the above properties with those of the bacterial complexes that are unable to accumulate polyf-hydroxybutyrate (PHB) in their cells, whose complexes are described in Bergey's Systematic Bacterial Manual, Vol. 1 (1984) on the Pseudomonas Genus, the present inventors could not find any bacteria that had properties consistent or analogous to those properties described above. This bacterium looks like a considerably unusual strain of Pseudomonas, because it hydrolyzes arginine and the desire for boxinases from Usina and ornithine. The% mol value of G + C of 60.4% indicates that the strain belongs to a group that has a lower G + C value in Pseudomonas. Thus, in view of the various properties mentioned above, the present bacterium has been identified as Pseudomonas sp. PBJ-5360-STR-1-21. These properties were consistent with those of the parental strain PBJ-5360.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (3)

1. A starobacine antibiotic selected from the group consisting of H and I stalobacins, characterized because they have the physicochemical properties as shown below: Estalobacin H Estalobacin. I p.f. (° C) (as salt 235 ° C (decom240 ° C (decomposes Na) puts) ne) proto-ned molecular ion LSI-MS (m / z) 1396 1325 IR (KBr) (cm "1) 3374, 1747, 3387, 1747, 1654, 1597, 1525 1651, 1596, 1527 UV (H20) Final Absorption Final Absorption CD (s2o) [?] 194-66980 [?] 212 + 9851 [?] 206 + 11530 [?] 232-31520 [?] 232-28660 [?] 25? -4288 [?] 25? +4749 time of retention (min) in cro8.8 9.7 High Resolution liquid matography * Amino Acid Analysis (molar ratio) HyAsp1 HyAsp (1) HyAsp (1) Asp Asp (1) Asp (1) Ser (1) Ser (1) Hylle2 Hylle (1) Hylle (1) Gly Gly (1) Gly (1) Ala Ala (1) Column: Develosil 5C18, internal diameter 4.6 x 250 mm. Mobile phase: CH3C / H3P04 2 mM (containing Na2S04) 50 mm) - 43/57 Flow rate: 1 ml / min. 1) Hydroxiaspartic Acid 2) Hydroxyisoleucine

2. The Pseudomona sp. PBJ-5360-STR-1-21 characterized in that it produces antibiotic stalobacines as described in accordance with claim 1.

3. The process for the production of antibiotic estalobacines defined according to claim 1, characterized in the process because it comprises the cultivation of a microorganism belonging to the genus Pseudomonas and produces anti-biotic estalobacines, as described in accordance with claim 1, and antibiotic stalobacins are separated and recovered from the culture. ÍL fifteen twenty