Title of Invention

AN ENZYMATIC PROCESS FOR THE PRODUCTION OF RIBAVIRIN

Abstract

This invention relates to an enzymatic process for the production of Ribavirin. Brevibacterium acetylicum is treated with 10 to 100 mg/ml of nitrosoguanidine in the presence of a known buffer to isolate a higher yielding mutant strain therefrom. This high yielding strain is cultivated in a known culture medium to produce enzymatic cells 1,2,4'triazole-3-carboxamide and a known ribose donor are enzymatically reacted in the presence of a suspension of cells obtained from the high yielding mutant strain. The reaction is carried out under known non-proliferating conditions preferably about 60° C. The product is extracted and purified from the culture medium in a known manner. The mutagenically altered strain produces better yield, reduces contamination and affords continuity of the process.

Full Text

This invention relates to an enzymatic process for the production of Ribavirin. Chemical structure of Ribavirin is l-beta-D-Ribofuranosyl-1,2,4 triazole 3-carboxamide with a molecular weight of 244.21 Daltons. Ribavirin exhibits a broad spectrum of potent antiviral activity against DNA and RNA viruses and is used extensively to combat Heptatitis B and Hepatitis C viral infections. Ribavirin has also been found effective in the treatment of AIDS. Presently, repairing is produced by three different routes mêlée by synthetic route, by fermentative route and by enzymatic route. Each of these routes for producing ribavirin are found unsatisfactory due to various drawbacks of the processes. For instance, the synthesis of ribavirin requires blocking of the active groups of the starting material at high temperatures and the steps involved in relocking and subsequent admiration are cumbersome. Currently used fermentation methods are unsatisfactory as they are carried out under proliferating conditions, resulting in extensive contamination with by products. Further, the yield is very low and purification and isolation of pure ribavirin poses problems. Another negative aspect is the fermentation has to be effected for a considerable period to get satisfactory yields. This again results in accumulation of by-products, making subsequent purification difficult. Similarly the enzymatic method known in the art is found unsatisfactory as the ribose donor used is unstable and the preparation of pure enzyme from the microorganism is quite difficult. The object of this invention is to develop a process for preparing ribavirin which overcomes the problems faced with the state of the art production techniques. The first step of the invention is in the preparation of a high yielding mutant strain of Brevibacterium acetylicum. This organism is subjected to mutagenic treatment with nitrosoguanidine in the presence of a known buffer. Nitroguanidine concentration may range from 10 to 100 mg/ml and the buffer may be transform of malefic acid chemically known as limerick acid. The thus treated bacteria are allowed to segregate and visually large colonies are separated and tested for concentration of the enzyme. Selected mutants were then allowed to grow and were subsequently subjected to conventional steps of screening to assess enzyme concentration. The finally selected clone of the mutant bacterium is then inoculated into a culture medium consisting of bullion for 24 hours at about ambient temperature. Bullion consists of 0.5% of meat, 0.5% Di potassium hydrogen phosphate (K2HPO4) 1.5% of peptone, 0.5% sodium chloride in sterile water at pH 7. Cells are collected after cultivation by centrifugation, washed and suspended in sterile water. Highly productive strain of Brevobacterium acetylicum thus evolved by mutagenesis is then stabilized and used for the production of Ribavirin. A substrate solution is prepared by mixing Iodine, 1,2,4 Triazole-3-carboxamide, dipotassium hydrogen phosphate. A suspension of the mutagenically altered cells are added to the above substrate and the reaction is carried out at about 60°C to avoid proliferation conditions. The amount of Ribavirin formed is monitored by known methods and the product is isolated by known means. Advantages of the present process are higher yield, less reaction time, storability of the enzyme source for repeated use, easy purification, minimum by products and the like. An enzymatic process for the production of Ribavirin according to this invention comprises the steps of treating Brevibacterium acetylicum with 10 to 100 mg/ml of nitrosoguanidine in the presence of a buffer such as herein described, separating a high yielding mutant strain of said bacteria therefrom by known methods, cultivating said mutant strain in a known substrate extracting the enzyme produced from the culture medium enzymatically reacting 1,2,4-triazole-3-carboxamide and a known ribose donor in the presence of the enzyme cells obtained from said mutant strain under non-proliferating conditions such as herein described and recovering ribavirin therefrom in a known manner. In a specific embodiment of this invention Brevibacterium acetylicum is treated with nitro guanidine in the concentration of 10-100 mg/ml in the presence of malefic acid which acts as a buffer. Visually large colonies of the bacteria are collected and separated and the selected mutants were tested for enzyme concentration by screening in a known manner. The finally selected high yielding mutagenically altered clones of the bacterium is used for preparing the enzyme cells. The culture medium used for bacterial cultivation consists of 2% aqueous bullion which is made of 0.5% meat extract, 1.5% peptone, 0.5% of sodium chloride 0.5% of dipotassium hydrogen phosphate dissolved in sterile water at a pH of 7. The cultivation is preferably carried out at about 28®C for about 24 hours. The cells produced as collected by centrifugation and are then washed and suspended in sterile water. The cells prepared from the thus mutagenically altered bacterium manifests higher conversion rate of the reaction medium. In a specific embodiment 750ml of substrate solution containing 66.7 mM iodine 66.7mM of 1,2,4 triazyl-3-carboxamide and lOOmM of K2HPO4 is prepared and 250 ml of said cell suspension in sterile water is added. The reaction is carried out at 60°C for 24 hours. The solution obtained after completion of the reaction is passed through cation exchange (H+) resin. The resultant solution and washings are passed through a column of activated charcoal and Ribavirin is eluted therefrom by ethyl alcohol. Ammoniated ethyl alcohol eluent from the charcoal column is then passed through anion exchange resin to further purify. The resultant liquid from the anion exchange column is concentrated under reduced pressure and cooled to precipitate Ribavirin which is dried. It is observed that 750 ml of the substrate yielded 9.2 gm of Ribavirin. Other known ribose donors like adenosine, guanine, xanthosine uridine and cytosine may also be used instead of Inosin in the above reaction. As already stated, carrying out the reaction at 60°C or above minimises proliferation of the cells and resultant contamination of the reaction mixture with undesirable organisms. Further, the reaction time is much shortened and any ribose donor readily available can be used. Though this invention has been described hereinabove with a specific embodiment, alteration and modification known to person skilled in the art are within its scope and that of the appended claims. t WE CLAIM: 1. An enzymatic process for the production of ribavirin comprising the steps of treating Brevibacterium acetylicum with 10 to 100 mg/ml of nitrosoguanidine in the presence of a buffer such as herein described, separating a high yielding mutant strain of said bacteria therefrom by known methods, cultivating said mutant strain in a known substrate, extracting the enzyme produced from the culture medium; enzymatically reacting l,2,4-triazoie-3-carboxamide and a known ribose donor in the presence of the enzyme cells obtained from said mutant strain under non-proliferating conditions such as herein described and recovering Ribavirin therefrom in a known manner. 2. The process as claimed in claim 1, wherein said enzymatic reaction of 1,2,4-triazole-3-carboxamide with said ribose donor is carried at a temperature of about eO^C for 24 hours. 3. The process as claimed in claims 1 and 2, wherein a substrate solution containing l,2,4-triazole-3-carboxamide and said ribose donor is prepared and reacted in the presence of said enzyme to produce Ribavirin. 4. The process as claimed in claims 1-3 wherein said ribose donor is selected from Inosin, Adanosine, guanine, xanthosine, uridine and cystidine. 5. The process as claimed in claims 1 to 4, wherein ribavirin is separated and purified by first passing the solution through cationic resin and then eluting the resulting solution from an activated charcoal column with ethyl alcohol, ammoniating the eluant, passing the same through anion exchange resins, washing concentrating and drying crystalline Ribavirin obtained thereby. 6. The process as claimed in claims 1 to 5, wherein said altered high yielding strain of Brevibacterium acetylicum is cultivated in a medium consisting of meat extract, peptone, sodium chloride and di-potassium hydrogen phosphate in sterile water. 7. The process as claimed in claims 1 to 7, wherein Brevibacterium acetylicum is treated with 10 to 100 mg/ml of nitrosoguandine at a ten^erature of about 28°C to produce said mutant high yielding strain. 8. The process as claimed in claims 1 to 7, wherein said mutagenisis with nitroguandine is carried out in the presence of buffers such as trans form of maleic acid chemically known as fumaric acid. 9. The process as claimed in claims 1 to 8, wherein the mutagenic strains of said bacterium is cultivated in a medium comprising a 2% aqueous bullion solution made of 0.5% meat extract, 1.5% meat extract, 1.5% peptone, 0.5% sodium chloride, 0.5% of dipotassium hydrogen phosphate at a pH of 7 and is preferably carried out ambient temperatures for about 24 hours. 10. An enzymatic process for the production of Ribavirin substantially as herein described.

Documents:

0365-mas-2001 abstract.pdf

0365-mas-2001 claims.pdf

0365-mas-2001 correspondence-others.pdf

0365-mas-2001 correspondence-po.pdf

0365-mas-2001 description (complete).pdf

0365-mas-2001 form-1.pdf

0365-mas-2001 form-26.pdf

0365-mas-2001 form-3.pdf

0365-mas-2001 form-9.pdf