FI65085B - NOW FOERFARANDE FOER FRAMSTAELLNING AV SISOMICIN - Google Patents

Description

r, ADVERTISING PUBLICATION, n Q UTLÄCCNINGSSKRIFT 6 5 085 C (45)? r. ten tli '.2: 3 1724 ^ (51) K * .ikP / b * .ci.3 C 12 P 19 / 4-8 FINLAND — FINLAND (21) Patent application - PatantaraMcning 002138 (22) Hakemlspilvl — AnsSknlngsdai 03 · 07.80 (23) Alkuptlvi — Glklfhstsdag 03. 07.80 (41) Become Public - BllvK offentllj 0 ^. 01. 82

Patent and Registration Holders .... .........,. ,. , _. (44) Date of issue and date of publication. - is 11 Ao

Patent- och resisterstyrelsen '' AnsBkan uttajd och utUkrtftun publkurad -> u · x ± 'OJ> (32) (33) (31) claimed privilege —Beftrd prlorttet (71) Chinoin Pharmaceuticals and Chemicals GySra Rt., 1- 5 To utea, I0U5 Budapest, Hungary-Hungary (HU) (72) Miklos Järai, Budapest, Sändor Piukovich, Budapest,

Sandor Istvan, Szentendre, Istvän Gadö, Budapest,

Valeria Szell, Budapest, Istv & n Barta, Budapest,

Hungary-Hungary (HU) (7 **) Oy Kolster Ab (51 *) New method for the preparation of sisomicin -Nytt förfarande för framställning av sisomicin

The invention relates to a new process for the preparation of the antibiotic cisomycin and its pharmaceutically acceptable salts by fermentation.

Sisomycin, O-2,6-diamino-2,3,4,6-tetradeoxy-D-glycero-hexa-4-enopyranosyl 11 - [? 1 -> 47-O- (β-deoxy-4-C- methyl-3- (7-methylamino) - (3-L-arabinopyranosyl- (1-6) -2-deoxy-D-streptamine) is a broad spectrum antibiotic.

Several microbiological methods are known for the preparation of sisomicin. These are disclosed in U.S. Patent Nos. 3,832,286 and 3,907,771. According to these publications, the microorganism Micro-monospora inyoensis (NRRL 3292) produces it as a major component.

In addition to verdamisine (U.S. Pat. No. 3,951,746), Micromonospora grisea (NRRL 3800) produces Micromonospora zionensis (NRRL 5466) in addition to the antibiotic G-52 (U.S. Pat. No. 3,956,068) and Micromonospora purpurea var. nigrescens (MNG 00122) in addition to the gentamicin complex (Hungarian Patent 168,778) sisomycin.

Micromonospora rosea (MNG 00182) also produces cisomycin (FI patent application 800 360).

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When studying soil samples from Danubian sludge, a new microorganism was produced that produced cisomycin. This new microorganism was named JMS-1. This strain, which turned out to be different from all microorganisms known so far, was named Micromonospora danubiensis species nova. From this microorganism, a strain labeled with JMS-3 is obtained by various strain purification methods. Fermentation of this strain, deposited with the National Collection of Micro-organisms, Budapest on 19 June 1978 under the accession code MNG 00171, gave a very high content of cisomycin.

The invention relates to a new process for the preparation of the antibiotic cisomycin and its pharmaceutically acceptable salts by aerobically culturing a microorganism belonging to the genus Micromonospora in a nutrient medium containing assimilable carbon and nitrogen sources and mineral salts, and if desired the resulting cisomycin is is characterized in that a microorganism belonging to the species Micromonospora danubiens is cultured, preferably strain MNG-00171.

The microorganism used according to the invention has the following microscopic, macroscopic and biochemical properties:

Morphology

Macroscopic observations: A 10-day-old culture incubated at 37 ° C on Czapek agar has a fairly moderate growth, no aerial mycelium, colonies appear regularly round in shape, reddish brown in color. In addition to colonies, no secretion of soluble pigments is observed.

Microscopic observations: Long, branched filaments without a septum, filament diameter 0.5 μm, spores are simply held by sporophones 1-1.5 μm in diameter. The spores are ovoid spherical in shape.

Biochemical and physiological properties: Micromonospora danubiensis strain MNG-00171 grows well at temperatures between 28-37 ° C, but no growth is observed at and above 44 ° C.

The use of a carbon source was monitored with the following nutrient medium: yeast extract 0.5%, carbon source 1.0%, CaCO 3 0.1%, agar 1.5% in distilled water. The results are shown in Table 1: 1 3 m 1 yy 1 6 5 0 8 5

table 1

Carbon source Micromonospora danubiensis _________ _MNG 00171 arabinose weak (-) ribose moderate (+) xylose weak (-) rhamnose weak (-) fructose good (+++) galactose moderate (+) glucose good (+++) lactose low (-) sucrose good (+++) raffinose weak (-) dulcitol weak (-) mannitol good (+++) inositol weak (-) starch good (+++)

The use of a nitrogen source was studied with the following nutrient medium: glucose 1%, agar 1.5% in distilled water. The results are shown in Table 2:

Table 2

Nitrogen utilization

Nitrogen source Micromonospora danubiensis _______MNG 00171_ 0.5% yeast extract good (+++) 1% NZ-amine, type A good (+++) 1% asparagine moderate-weak (-) 1% glutamic acid weak (-) 1% KN03 weak (-)

The growing colony of Micromonospora danubiensis hydrolyzes gelatin, milk, and starch and reduces nitrate to nitrite. The microorganism tolerates up to 2% sodium chloride in the growth medium.

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Cultural Characteristics

Table 3 shows the culture characteristics of Micromonospora danubiensis JMS-3. (The following literature references have been used to describe color formation observations: Baumenns Farbtonkarte Atla II, Paul Baumann, Aue I-SA 87350 LAu 302, GFR.)

Table 3

Culture characteristics Medium Micromonospora danubiensis MNG 00717

Bennet agar weak growth, bright orange color,

Oc 97-98

Czapek agar good growth, reddish brown color, 0 142-145

Emerson 1 in agar does not grow

Glucose-asparagine agar does not grow

Glucose-yeast extract -agar good growth, bright orange color,

Oc 99

Nutritional agar good growth, burgundy in some points yellow-brown color Ro 193 respectively Or 165-166

Peptone-iron agar no growth or only traces, small colonies, light orange color Oc 97 and Co 69, no diffusible pigment

Traces of tyrosine agar growth, color cannot be determined, no diffusible pigment Slice of potato without CaCO3 no growth

Potato slice with CaCO 2 moderate growth, reddish brown color, O 136 and O 145-147

Based on the above facts, it was found that the Micromonospora strain labeled with JMS-3 differs from Micromonospora species known from the literature. When comparing the listed markers to Luedemann's Micromonospora system, strain JMS-3 is classified as a new species, which was named Micromonospora danubiensis species nova according to its isolation site.

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The new Micromonospora danubiensis strain MNG-00171 was compared with known Micromonospora species, which are characterized by, inter alia, M. purpurea being arabinose-positive and mannitol-negative, while M. danubiensis is arabinose-negative and mannitol-positive. M. danubiensis also differs from M. echinospora, which, in contrast to M. danubiensis, is arabinose- and rhamnose-positive and mannitol-negative. There are also differences between members of related groups of M. danubiensis and M. carbonacea strains that are arabinose- and melibiose-positive and mannitol-negative. In contrast to M. chalcea, M. danubiensis is raffinose-negative, mannitol-positive, melibiose-negative and arabinose-negative. M. danubiensis differs from M. coerulea in two carbon sources (raffinose and melibiosis) and M. purpureochromogenesis in three (melibiosis, mannitol, and raffinose). Considering data from Lundeman (1971), the NaCl tolerance range of M. danubiensis is worse than that of M. narashinoensis, M. megalomica, M. chalcea, and M. halophytica. Among others, M. danubiensis can be easily distinguished from cisomycin-producing M. rosea on the basis of two diagnostically important carbon sources (mannitol and raffinose) and is clearly different from other cisomycin-producing Micromonospora species. Comparison of M. danubiens with M. inyoensis, M. grisea and M. zionensis as described above gave the results shown in Table 4: 65085 TABLE 4

Carbon source M.danubiensis M.inyoensis M.grisea M.zionensis arabinose negative negative positive positive xylose negative (positive) positive positive rhamnose negative negative negative negative fructose positive negative positive (positive) galactose (positive) (positive?) Positive positive glucose positive positive lactose negative (positive) negative (positive) sucrose positive positive positive positive raffinose negative negative negative negative dulcitol negative negative negative negative mannitol positive negative negative negative inositol negative negative negative negative

From the above, it is apparent that M. danubiensis is a distinct species among the sisromycin-producing Micromonospora species. It differs from M. inyoensis in the use of xylose, fructose, lactose and mannitol as diagnostically important carbon sources. It also differs from M. grisea in the use of arabinose, xylose and mannitol and from M. zionensis in the use of arabinose, xylose, lactose and mannitol. It is thus evident that M. danubiensis is the "individual", "most special" species of Micromonospora that produces sisomycin. This conclusion is further supported by the comparison of diagnostic culture characteristics presented in Table 5. It is noted that M. danubiensis differs from M. inyoensis in that the former does not grow at all on Emerson's agar, while the latter forms reddish-brown colonies in the same substance. M. danubiensis colonies cannot be compared to M. grisea green colonies and are very different both morphologically and in color from M. zionensis colonies. It is noted that the M. danibuensis type strain differs sharply from all known and used cisomycin-producing strains. ΙΊ. the danubiensis type strain used in fermentation to produce cisomycin is not covered by the method protected by Hu Patents 160,408, 175,176 and 167,528.

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According to a preferred embodiment, the Micromonospora danubiensis strain is cultured as a submerged culture under aerobic conditions.

Several sources of carbon and nitrogen, which are assimilable by the microorganism Micromonospora danubiensis, inorganic salts, small amounts of trace elements, and antifoams, may be present in the nutrient medium to produce cisomycin. Starch, soluble starch, dextrin, glucose, sucrose, corn flour, soybean meal, soybean meal hydrolyzate, casein hydrolyzate, corn steep liquor, yeast extract, etc. can be used as carbon, nitrogen and energy sources, respectively.

Many ammonium, iron, zinc, manganese, magnesium, sodium, potassium and cobalt salts can be used as inorganic salts. To improve the buffer capacity of the nutrient medium, calcium carbonate and various vegetable oils (e.g. palm oil, sunflower oil, soybean oil) can be advantageously used as antifoams.

The use of different nutrient media components makes it possible to prepare different nutrient media. The nutritional requirements for making an inoculum differ from the requirements for main fermentation. For the preparation of sisomicin, the most preferred nutrient medium comprises soy flour, corn steep liquor, corn flour, starch, sucrose, calcium carbonate, cobalt chloride and palm oil.

The fermentation temperature ranges from 25 to 37 ° C. Preferably, the fermentation is performed at a temperature between 28-33 ° C.

The stirring speed varies between 100-600 rpm depending on the geometry of the fermenter. The preferred aeration is between about 1/1 v / v / min.

The activity of the antibiotics produced during fermentation (cisomycin and "minor" components) is determined by the test organism Staphylococcus epidermidis, by the agar diffusion method, compared to a standard cisomycin product.

During 96-120 hours of fermentation, the sum of the activity in the fermentation broth reaches a value of 400-450 U / ml (1 U = 1 ug of basic cisomycin activity). The cisomycin activity is then about 85% of the total antibiotic activity of the fermentation broth.

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Isolation of cisomycin can be performed by known methods. If desired, the cisomycin base can be converted to a pharmaceutically acceptable salt.

The amount of cisomycin produced by the microorganism Micromonospora danubiensis is many times higher than the amount produced by hitherto known methods. Under laboratory conditions it exceeds 450 U / ml and in experimental plant fermentation 350-400 U / ml. The fermentation broth contains only 10-15% other antibiotics in addition to cisomycin.

The method according to the invention is illustrated by the following examples. Examples 1) Inoculate under sterile conditions with 1 ml of subcooled

Micromonospora danubiensis strain JMS-3 (MNG 00171) with mycelium vegative spores 10-10 / ml) 100 ml of sterile nutrient medium in a 500 ml Erlenmeyer flask containing the following composition: starch 25.0 g maize steep liquor 7 .0 g (NH4) 2SO4 3.5 g

NaCl 5.0 g

CaCO 3 8.0 g tap water 1000 ml

The pH of the nutrient medium is adjusted to 7.5 before sterilization, so that the pH after sterilization is 7.0 to 7.2.

The culture is incubated for three days at 28 ° C with a plate shaker (220 rpm, deviation 7.5 cm). The culture thus obtained is inoculated with 6 l of sterile nutrient medium (in a 10 l glass fermenter) under sterile conditions. The composition of the nutrient medium is the same as that of the inoculum nutrient medium described above.

The culture in a glass fermentor is performed at 28 ° C, stirring at 400 rpm and aerating at 1/1 v / v / min. Palm oil is used as a defoamer as needed.

The inoculum developed in 40 hours is used to inoculate the fermentation medium. The volume of the inoculum is 600 ml, the amount of nutrient medium is 6 l, the pH is 7.0 to 7.2 and the volume of the fermenter is 10 1. The composition of the nutrient medium is as follows: 6 5 0 8 5 1 1 soy flour 50.0 g corn starch 10.0 g of sucrose 30.0 g

CaCO 3 5.0 g corn steep liquor 5.0 g

CoCl2-6H2O 4.5 mg tap water 1000 ml

Cultivation is performed at 31 ° C, rotary agitation 400 rpm, aeration 5 1 / min. Palm oil is used as an antifoam.

Antibiotic completion begins at 28-34. per hour of fermentation and the maximum value is reached after 110 hours of fermentation.

At this time, the total amount of antibiotic in the fermentation broth is determined to be 400 U / ml compared to standard sisomicin using Staphylococcus epidermid as test organism and agar diffusion method. (J.S. Simpson: Analytical Microbiology, Acad. Press,

New York. pp. 87-124, 1963.) 2) The following medium is inoculated with a culture culture produced with Micromonospora danubiensis strain JMS-3 (MNG 00171) with a level shaker according to Example 1: soy flour 10.0 g starch 10.0 g sucrose 10 .0 g

CaCO3 4.0 g tap water 1000 ml

The pH of the nutrient medium after sterilization is 7.0 to 7.2 and the amount of medium is 6 l (in a 10 l glass fermenter). Cultivation is performed at 31 ° C, rotation speed 400 rpm and aeration 6 l / min. Palm oil is used as antifoam as needed.

600 ml of inoculum developed in 36 hours is used for inoculation of fermentation medium with the following composition: soy flour 30.0 g corn flour 25.0 g

CaCO 3 5.0 g

CoCl 2 * 6H 2 O 10.0 mg tap water 1000 ml 12,65085

The pH of the nutrient medium after sterilization is 7.0 to 7.2. The culture temperature is 33 ° C, the mixing speed is 600 rpm and the aeration is 6 1 / min. Palm oil is used as an antifoam.

The formation of antibiotics starts at 26-30. fermentation at 1 hour and the maximum value is reached after 100 hours of fermentation. Currently, the total amount of antibiotic in the fermentation broth is 420 U / ml compared to the standard isisomine sample.

3) The following nutrient medium is inoculated with a growth culture produced with Micromonospora danubiensis strain JMS-3 (MNG 00171) with a level shaker according to Example 1: starch 25.0 g soy flour hydrolyzate 15.0 g (NH4) 2SO4 2.0 g

NaCl 3.0 g

ZnSO 4 0.5 g

CaCO3 8.0 g tap water 1000 ml

The pH of the nutrient medium after sterilization is 7.0 to 7.2, and the amount of medium is 6 L (in a 10 L glass fermenter). Hydrolysis of soybean meal was performed with NOVO bacterial protease at 60 ° C for 30 minutes at pH 7.5.

After inoculation, the culture was performed at 28 ° C, stirring at 400 rpm, with aeration at 6 l / min. Palm oil was used as the antifoam.

An inoculum of 600 ml per hour is used to inoculate the following fermentation broth: soy flour hydrolyzate 50.0 g starch 10.0 g dextrin 25.0 g

CaCO 3 5.0 g sucrose 10.0 g 4.5 mg tap water 1000 ml

The pH of the nutrient medium after sterilization is 7.0 to 7.2, and the amount of medium is 6 l (in a 10 l glass fermenter). Hydrolysis of soybean meal was performed with NOVO bacterial protease at 60 ° C for 30 minutes at pH 7.5.

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After inoculation, the culture was performed at 31 ° C, with stirring at 600 rpm and aeration at 6 l / min. Palm oil was used as an antifoam.

The formation of antibiotics starts at 26-30. per hour of fermentation, with the maximum value being reached after 100 hours of fermentation. At this time, the amount of antibiotic in the fermentation broth is 430 U / ml compared to the standard isisomycin sample.

4) The following nutrient medium, amounting to 200 l (stainless steel pre-fermenter with a volume of 300 l) is inoculated with a growth culture produced with Micromonospora danubiensis strain JMS-3 (MNG 00171) in a glass fermenter according to Example 2: soybean meal 10.0 g .0 g sucrose 10.0 g

CaCO3 4.0 g tap water 1000 ml

The pH of the nutrient medium after sterilization is 7.0 to 7.2. Palm oil is used as an antifoam.

The culture is carried out at 31 ° C, stirring at 180 rpm, aeration 200 l / min.

An inoculum developed in 36 hours is used to inoculate a fermentation broth having the following composition (medium volume 2,000 l): soy flour 30.0 g corn flour 25.0 g

CaCO3 5.0 g

CoCl 2 * 6H 2 O 10.0 mg tap water 1000 ml

The pH of the nutrient medium after sterilization is 7.0 to 7.2. The culture temperature is 33 ° C, stirring speed 220 rpm, aeration 2 m / min, a mixture of soybean oil and palm oil (50% to 50%) is used as antifoam.

The formation of antibiotics starts at 26-30. between the fermentation hour and the maximum value is reached after 100 hours of fermentation. At this time, the total amount of antibiotic in the fermentation broth is 380 U / ml compared to the standard isisomycin sample.

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At the end of the fermentation, 288 g of pure sisomicin base containing bound water are obtained by known methods.

Product features:

Melting point: 198-201 ° C

/ bc / Ό = + 188 ° (c = 0.3 water)

Elemental analysis for monohydrate:

Calculated: C 49.80 H 8.2 N 14.95% Found: C 49..15 H 8.37 N 15.15% IR spectrum (KBr) V: OH, NH 3170-3360, CH = COC 1690, COC 1060 cm -1.

NMR Spectrum (D 2 O): 1.20 (Me-4 ", s, 3H), 2.50 (Me-N-3", s, 3H), 2.56 (H-3 ", d, J 2" 3 "= 10Hz, 1H), 3.17 (H-6 ', bs, 2H), 3.30 (Hax-5H, d, Jgem = 12Hz1H), 3.80 (H-2", dd, J2 " Δ (= 10Hz, = 4Hz, 1H), 4.04 (He-5 ", d, Jgem = 12Hz, 1H), 4.88" (H-4 ', bt, 1H) 5.09 (H1 " , d, Jltl 2 (= 4Hz, 1H), 5.35 (H-1'f d, J ±, 2% = 2Hz, 1H) ppm.

Mass numbers of characteristic ions (m / e): 447, 332, 304, 160, 145, 127, 118, 110, 100.

Preparation of sisomic sulfate 15 g of pure sisomicin base are dissolved in 60 ml of deionized water, and 5N sulfuric acid solution is added dropwise to the solution thus obtained, so that the pH of the solution becomes 4.3. 1.5 g of activated carbon are then added to the solution and the mixture is stirred for 30 minutes and then filtered through a Seitz sheet. The filter is washed with 3 x 50 ml of deionized water and the combined filtrate is added to 1 l of methanol with constant stirring. The solution containing the separated precipitate is allowed to stand in a cooling state, after which the precipitate is filtered. The filtered cisomycin sulfate is washed with 3 x 50 mL of methanol and dried in vacuo at 50 ° C with P 2 O 3 to constant weight. 22 g of cisomycin sulphate are thus obtained.