FORM 2
THE PATENTS ACT, 1970 (39 of 1970) & THE PATENT RULES, 2003
PROVISIONAL SPECIFICATION
(See Section 10; rule 13)
"PROCESS FOR PRODUCTION OF POLYMYXIN B SULFATE"


RELIANCE LIFE SCIENCES PVT.LTD an Indian Company having its Registered Office at Dhirubhai Ambani Life Sciences Centre, R-282, TTC Area of MIDC, Thane Belapur Road, Rabale, NaviMumbai-400 701 Maharashtra India.

The following specification particularly describes and ascertains the nature of this invention and the manner in which it is performed:-



FIELD OF THE INVENTION:
The present invention relates to an improved process of production and purification of Polymyxin B sulphate. The present invention in particular relates to the fermentative production of Polymyxin B sulphate in high yields.
BACKGORUND OF THE INVENTTOiV
Polymyxin B sulphate is an antibiotic used in the treatment of infections caused by gram-negative bacteria, particularly the Pseudomonas aeruginosa and Escherichia coli. Polymyxins belong to a class of antibiotics that were discovered about 60 years ago. They are decapeptitfes with antimicrobial spectrum that includes gram-negative bacteria, such as Acinetobacter baumanii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Although there are five main polymyxins (A, B, D, E and M), only two-polymyxins, B and colistin (polymyxin E) have been used in clinical practice.
Although it has not been extensively used as penicillins, these antibiotics were widely used to treat patients with various gram-negative bacterial infections including meningial {Haemophilus influenzae), urinary tract infections (E. coli) during 1960s and 1970s. Besides, one of the major use of polymyxin B is treating a common but fatal disease called 'septic' shock caused by the endotoxin produced by gram negative bacteria (Schindler M, Osborn MJ. Interaction of divalent cations and polymyxin B with lipopolysaccharide. Biochemistry. 1979 Oct 2; 18(20): 4425-430). However, due to reports of toxic effects on the kidney and nervous system, polymyxins were abandoned in the 1980s.
Nevertheless, the pQlymyxins have been found to salvage antibiotics especially in treating multi-drug resistant gram-negative bacterial infections (ME.Falafas and A. Michalapolos, (2006) Polymyxins: old antibiotics are back. The Lancet, 367: 633-634) and have once again come into the foray of contemporary medicine.
This antibiotic is 4 complex mixture of closely related polypeptides obtained from cultures of various strains of Bacillus polymyxa and related species (J. Shoji, H. Hinoo, Y. Wakisaka, K. Koizumi, M. Mayama, S. Mitsuura. J. Antibiotics. 30 (1977) 1029-
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1034). Polymyxins contain characteristic constituents such as a,y-diaminobutyric acid, L-threonine and a fatty acid. They differ by the presence or absence of additional amino acid as well as the nature of the fatty acid.
Polymyxin B is a cyclic, branched decapeptide that binds to membrane phospholipids and thereby interferes with membrane function. Polymyxin B is subdivided into at least four components, polymyxin Bl, B2, B3 and B4. They differ from each other only in the fatty acyl moiety: Bl contains 6-methyloctanoic acid, B2 has 6-methylheptanoic cid, B3 has octanoic acid and B4 has heptanoic acid.
Literature on the biosynthesis or physiology of polymyxin production is sporadic. Effect of potassium ions, phosphorus, amino acids, and corn extract on polymyxin production has been done by some Russian groups (Effect of K+ ions on Bacillus polymyxa 153 growth and the biosynthesis of polymyxin B, Nefelova MV, Ermakova GN, Egorov NS, Antibiotiki. 1980 May;25(5):326 30; Effect of phosphorus on polymyxin B biosynthesis by B. polymyxa 1538 on media of varying makeup, Pavliuk IuV, Bogatskii MA, Orlova NV, Anakhova VA, Antibiotiki. 1979 Oct;24(10):723-7; Effect of amino acids on the growth of a B. polymyxa 1538 culture and the biosynthesis of polymyxin B, Pavliuk IuV, Bogatskii MA, Orlova NV, Anakhova VA., Antibiotiki. 1979 Nov;24(ll):815-20.; Importance of corn extract components for the biosynthesis of polymyxin B by B. polymyxa strain 1538, Pavliuk IuV, Bogatskii MA, Orlova NV, Anakhova VA, Antibiotiki. 1979 Aug;24(8):566-70.)
In M.J. Daniels article, the growth of the organism and polymyxin production was monitored (M.J. Daniels, (1968) Studies of the biosynthesis of polymyxin B. Biochimica et Biophysica Acta. 156: 119-127) and the antibiotic titre at the end of 45 h of fermentation was only 70 ug/ml. In another report (P.A. Stansly, M.E. Schlosser, N.H. Ananenko and M.H. Cook. (1948) Studies on polymyxin: the production of fermentation liquor. J. Bacteriol. 55: 573-578), changes in pH during fermentation has been reported wherein a successful fermentation, the pH of the broth decreases from 7 to 6 by 3rd day and then increases to a value close to neutrality by 5th day. More recently, a yield of 380
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mg/L was obtained in a medium having low P and low N content (A.E. Lantz, P. Jorgensenn, E. Poulsen, C. Lindermann and L. Olsson. (2006) Determination of cell mass and polymyxin using multi-wavelenght fluorescence. Journal of Biotechnology. 321: 544-554.)
UK patent GB991602 provides a purification process for Polymyxin which comprises treating an aqueous solution of the Polymyxin with a permanganate at pH below 8.0. The examples describe the treatment of solutions of Polymyxin B hydrochloride and of the sulphates of polymyxins A, B and E
Various processes of preparation of Polymyxin B sulphate is mentioned in patents US 2,759,868 , US 2,695,261, US 3,413,398, US 2,595,605, US 3,132,994, US 2,599,950 US 2,571,104, US 2,556,376, US 2,602,041, US 1899156, US 2599950, US 2565057, US 2602041, US 2511104, US 2595605, US 2556376, Us 3132994, US 3413398, GB 924652, GB 742589, GB 315263, JP11140189, RU2l 15721 herein incorporated as reference.
UK patent GB 645750 provides a process for sulphate or B-naphthalene sulphonate salts of Polymyxin B and E which is precipitated as the fr^e base by treating an aqueous solution of such a salt with an alkali such as aqueous ammonia at a temperature between 50 and 100°C, the precipitated polymyxin base is then quickly removed from the hot supernatant liquid and washed with hot water and dried Or converted to its hydrochloride or other desired compound. A theoretical yield of about 96.8% of Polymyxin E was obtained.
UK patent GB658766 provides a purification method by recrystallisation of the precipitated Polymyxin base with an alcohol containing 2-5 carbon atoms. The crystallization is repeated after charcoal treatment. The process employs many solvents and repeated recrystallisation.
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UK Patent GB 647925 provides the recovery of Polymyxin contained in a metabolism fluid by treating such fluid after removal of bacterial cells and any gummy impurities at a pH of 4.5 with one or more sulphated fatty alcohols or esters or their mixtures with salts. The resulting precipitate is separated by filtration and is dried. The salt of the Polymyxin is obtained by acid precipitation and then converted to the base again. The base is then by treatment with activated charcoal or fractional precipitation.
UK patent GB 65897 provides a fermentation process wherein Polymyxin is produced by inoculating a substantially neutral nutrient medium with Bacillus polymyxa, allowing fermentation to occur under aerobic conditions for 2-5 days at 20-30°C while aerating with 4-64 litres of air per hour per 8 litres of medium, removing bacteria and suspended material, and then use of other adsorbents, e.g. activated carbon or charcoal, for adsorbing the polymyxin from the culture medium, and contains additional examples of the preparation of derivatives of polymyxin, by treating the free base or hydrochloride of Polymyxin with formaldehyde, acetaldehyde or 4-nitrobenzaldehyde
Chinese patent CN 1800201 provides a method for preparing Polymyxin E, which employs foam separation method to extract and separate the Polymyxin E from fermentation liquor.
The PCT application WO/2007/145602 is the most recent literature on production of polymyxin B using Paenibacillus polymyxa. The patent describes production of polymyxin B with a yield of 1.8 to 2.8 g/L at 28oC, pressure of 40-70 kPa, aeration from 0.4 to 1 wm, minimum dissolve oxygen of 60 %, maintenance of pH from 5.2 to 5.6 and optimum concentration of glucose (maintained at 1.5-2 g/L) and ammonia ions (maintained at 0.3-0.5 g/L). The fermentation is carried out for 54 h using either corn steep liquid, wheat flour, wheat bran or soya flour as nitrogen source. The highest yield reported is 2.8 g/L under these conditions.
Many questions remain unanswered about polymyxins. Since most of the research was carried out before the 1980s, the methods for evaluation of the antibiotic were not as
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advanced as today. Therefore, a detailed investigation on the polymyxin production by Bacillus polymyxa is warranted.
Looking into the long felt need for an efficient process resulting in high yield of polymyxin, the present invention has focused on production of polymyxin by fermentation unlike the conventional production process using synthetic and semi synthetic approach. By doing so the inventors have been successful in producing high yield of polymyxin.
The present invention aims to provide an efficient method of producing polymyxin for large scale production, the present invention in.particular aims at providing optimal culture conditions that would result in high yield of polymyxin.
OBJECT OF THE INVENTION
It is the object of the present invention to provide an efficient process of polymyxin by fermentation.
It is the object of the present invention to provide an efficient process for producing high yield of Polymyxins.
It is the object of the present invention to provide a process, which is feasible on a large scale production.
The present invention in particular aims at providing optimal culture conditions that would result in high yield of polymyxin.
It is the object of the present invention to optimize the nutritional parameters of Polymyxin.
SUMMARY OF THE INVENTION
The present invention provides an improved process production of polymyxin by fermentation process resulting in high yield of polymyxin. The present invention has
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focused on fermentation process unlike the conventional production process using synthetic and semi synthetic approach. By doing so the inventors have been successful in producing high yield of polymyxin. In the fermentation process the inventors have has studied the effect of glucose and oat meal. This was done to maintain the carbon and nitrogen balance in the medium resulting in the production of high yield of polymyxin B sulphate
The present invention aims to provide an efficient method of producing polymyxin for large scale production. The present invention in particular aims at providing optimal culture conditions that would result in high yield of polyrnyxin.
In one embodiment the present invention provides a process wherein physiological parameters are studied which will result in better yields
In one embodiment the present invention provides a process wherein nutritional parameters are optimized. In one preferred embodiment the present invention has studied the effect of varying the concentrations of glucose an.d 0at meal. This was done to maintain the carbon and nitrogen balance in the medium for production of polymyxin B sulphate
In one embodiment the present invention has studied the effect of replacing yeast extract with bakers yeast autolysate.
In one embodiment the present invention provides the effect of the pH on the
fermentation medium. In one preferred embodiment the present invention provides the
effect of maintaining pH at ranging from _6.2_ to 6.8
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be
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better understood by reference to one or more of these drawings in combination with the
detailed description of specific embodiments presented herein.
Fig.l: shows Structure and composition of various polymyxins
Fig. 2. Effect of varying oatmeal concentration on production of polymyxin B
DETAILED DESCRIPTION OF THE INVENTION
Definitions:
The term "Polymyxin B" refers to the Polymyxin B sulphate
The present invention aims to provide a process for the preparation of Polymyxin, which results in high yields and can be produced on a large scale.
The present invention provides optimized conditions for fermentation The present invention has studied the effect of various physiological and nutrient requirements of the fermentation conditions. The present invention has focused on the nutritional parameters such as amount of glucose and oatmeal in the production medium. These parameters showed a significant effect on the production of Polymyxin B. Maximum yields were obtained with a medium having glucose at 1-2 % (w/v) and oat meal at 1-3 % (w/v).
The effect of replacing yeast extract with bakers yeast autolysate on the production of Polymyxin B was also studied. Bakers yeast autolysate was found to be more suitable for optimal production of Polymyxin B. By replacing the yeast extract with bakers yeast autolysate, the yield was increased by 2 fold.
Using 25% Liquid Ammonia for maintenance of pH at 6.5 was found to be crucial
for Polymyxin B production. There was no Polymyxin B production if pH was not
controlled
The following examples are included to demonstrate preferred embodiments of the
invention. It should be appreciated by those of skill in the art that the techniques
disclosed in the examples which follow represent techniques discovered by the inventor
to function well in the practice of the invention, and thus can be considered to constitute
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preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLE 1 : Bacteria and growth conditions:
Bacillus polymxya ATCC 10401 was maintained on nutrient agar. For production of polymyxin B sulphate, the seed culture was grown in nutrient broth. The culture was incubated at 30°C at 200 rpm for 24 h.
For bioreactor studies, the cells were grown in the production medium sterile production media containing 2% ammonium Sulphate, 0.2% dipotassium hydrogen phosphate, 0.05% magnesium Sulphate, 0.05% sodium chloride, 0.001% ferrous Sulphate, 0.5% bakers yeast autolysate, 1% glucose and 2% oatmeal.. The fermentation was carried out in 1.5 L bioreactor (B.Braun) at 30°C, 250 rpm agitation, 1.2 wm aeration for 72 h. The pH of the medium was maintained at 6.5 by addition of either 25% liquid ammonia or 5 NHC1.
EXAMPLE 2: Bioassay of polymyxin B sulphate
Polymyxin B sulphate produced during the fermentation was monitored by bioassay using Escherichia colt as an indicator strain by agar diffusion method.
EXAMPLE 3: Optimization studies
Physiological and nutritional parameters for maximum production of polymyxin B sulphate were optimized at shake-flask level in production medium. Nutritional parameters were optimized by varying the glucose, oatmeal and bakers yeast autolysate concentrations.
pH control for optimal production of PMB was revealed by the bioassay There was no production if pH was left uncontrolled. The maintenance of pH for production of PMB was found to be very stringent and bioassay was positive only if the pH was controlled.
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Nutritional parameters such as amount of glucose and oat meal in the production medium also had significant effect on the production of PMB. A medium having glucose at 2 % (w/v) and oat meal at 2-3 % (w/v) concentration gave maximum yield of PMB (Fig. 2). Nutritional parameters such as yeast extract also had a significant effect on PMB production. Yeast extract replaced with bakers yeast autolysate increased the PMB yield by two-fold.
EXAMPLE 4: Production of polymyxin B sulphate in high yields in optimized medium
Fermentation at 1.5 L scale in the optimized medium having 2 % glucose and 2 % oat meal at 30°C, 250 rpm, 1.25 vvm aeration and at pH 6.5 gave a yield of 2g/L in 72 h.
Example 5: Process for production of atleast 3 g/L of polymyxin B sulphate
Fermentation was carried out at 1.5 L scale in the optimized medium as in Example 4. The bakers yeast autolysate concentration was reduced from 0.5 % to 0.25 % and sodium carbonate at 0.025 % was used instead of sodium chloride in the medium. The fermentation conditions were 30°C, 250 rpm, 1.25-2 vvm aeration , pH 6.5 for 72 h. A yield of 3 g/L of polymyxin B sulphate was obtained at the end of 72 h.
Thus, while we have described fundamental novel features of the invention, it will be understood that various omissions and substitutions and changes in the form and details may be possible without departing from the spirit of the invention. For example, it is expressly intended that all combinations of those elements and/or method steps, which perform substantially the same function in substantially the same way to achieve the same results, be within the scope of the invention.
Dated this 24th day of November , 2008
For Reliance Life Sciences Pvt. Ltd