Title of Invention	A PROCESS FOR PREPARING A STORAGE - STABLE, ENTERIC RELEASE THERAPEUTIC COMPOSITION CONTAINING ONE OR MORE ANTIBIOTICS AND MICROORGANISM(S) AS THE ACTIVE INGREDIENTS .
Abstract	This invention relates to a process for preparing a storage - stable, enteric relea therapeutic composition containing one or more antibiotics and microorganism(s) a, the active ingredients. The process resides in sequentially carrying out the steps of (i) preparation of enteric matrix formulation, (ii) preparation of enteric - release granules of Lactic Acid Bacillus, (iii) preparation of acid release matrix formulation, (iv) preparation of acid - release granules containing antibiotic or mixture thereof and (v) preparation of encapsulated form of drug containing the active ingredients aforesaid.

Title of Invention

A PROCESS FOR PREPARING A STORAGE - STABLE, ENTERIC RELEASE THERAPEUTIC COMPOSITION CONTAINING ONE OR MORE ANTIBIOTICS AND MICROORGANISM(S) AS THE ACTIVE INGREDIENTS .

Abstract

This invention relates to a process for preparing a storage - stable, enteric relea therapeutic composition containing one or more antibiotics and microorganism(s) a, the active ingredients. The process resides in sequentially carrying out the steps of (i) preparation of enteric matrix formulation, (ii) preparation of enteric - release granules of Lactic Acid Bacillus, (iii) preparation of acid release matrix formulation, (iv) preparation of acid - release granules containing antibiotic or mixture thereof and (v) preparation of encapsulated form of drug containing the active ingredients aforesaid.

Full Text	The present invention relates to a process for preparing storage stable, therapeutic composition containing one or more antibiotics and microorganism(s) as the active ingredients. More particularly this invention is directed to a process for producting a therapentic composition in an enteric release system obtained by embedding the aforesaid active ingredients in an enteric - release matrix which not only serves as an effective barrier between the active ingredients but also ensures gradual release in the intestine, simultaneously protecting the drug(s) from degradation in the acidic pH environment of the stomach. The term "enteric coating" used in this specification refers to technique, usually known in the art, refers to providing a coat or layer on substrates like tablets, pellets, granules, flakes or the like to protedct the inner core, particularly if the core material is reactive, or prone to degradation with passage of time or in contact with other material(s). Normally human body harbours different types of micro-organisms. One class of microorganism inhabiting the human body, called 'pathogens , ma> be harmful to the host by causing infectious diseases. The other class of microbodies, called 'commensals', may be present in the human body for performing various useful functions, such as , for instance, providing essential vitamins to the body or preventing growth of harmful organisms within the human body. To treat and/or prevent infectious diseases caused by pathogens, anti-infective agents are used, which are, more often than not, non-specific for pathogen and may result in eliminating commensals zs well. Reduction in the number of commensals below optimum level may result in various adverse / Side effects. Diarrhoea is one such effect of treatment with antibiotics. Normal bacterial flora of the intestine provides Vitamin B complex to the human body and also prevents growth of other micro-organisms which are not congenial to the human system. Alteration of the normal flora on account of administration of antibiotics, either alone or in combination, may lead to transient dysfunction of normal colonic flora or superinfection of intestine by pathogens. The most common cause of antibiotic associated diarrhoea, e.g. megacolon and pseudomembranous colities,is the result of colonisation of the colon bya toxin-producing gram-positive anaerobic bacteria, Clostridium difficile.Some of the antibiotics normally used alone or in combination are, inter alia, ampicillin, amoxycillin, oliandomycin, Cepnalosporins marketed in various brand/trade names like Cephalexin, Cefixime, Certriaxone, Cefuroxine, Axetil, Proxetil, etc.), ampicillin + clanvanic acid, ampicillin+Sulbactum, fluoroquinolones, lincomycin, amoxycillin + cloxacillin, ampicilin + cloxacillin and the like.Early and specific anti-infective therapy should be given to patients suffering from diseases/disorders of the above nature. Treatment aimed at recolonisation of the gut with non-pathogenic microorganisms has included administration of lactic acid producing microorganisms such as Lactobacillus, Bifidobacterium. Streptococcus boulardii and Saccharomyces boulardii, Lactic acid producing organisms were introduced as a therapeutic agent by Metchnikoff with the idea of acidifying intestinal contents in order to prevent the growth of putrefactur organisms.The organism chosen by him for this purpose was Lactobacillus bulgaricus.These are stable and with beter therapeutic effect. Anion-exchange resins cholestyramine and cholestipol hydrochloride have been shown to bind the toxin of CI. difficile in vitro, and cholestyramine has been used to treat pseudomembranous colitis. Antibiotic associated colitis or colonic perturation has also been treated with drugs like vancomycin and metronidazole. To prevent diarrhoea or colitis, lactic acid producing microorganisms have been administered alone (e.g. Lactiflora, Lactobacil, Lactocap, Lactovit, Sporlac, etc.;, or in combination with antibiotic as two separate formulations. This necessitates consumption of two different drugs, namely, an anti-infective agent and a micro-organism, thereby decreasing patient compliance. A simple admixture of lactic acid bacillus (non-protected) with amoxycillin is not associated with any intrinsic type of resistance of lactic acid bacteria to amoxycillin. Hence, no particular safety concern is linked with such admixture. Attempts have also been made to enhance both compliance and efficacy. For this purpose, microorganisms and antibiotics have been made into a single formulation, some of which are commercially available. Anti-infective agents used in such formulations are, for instance, ampicillin, amoxycillin, which are made into simple formulation with susceptible micro-organisms. As pointed out earlier, "enteric coating" is application or formation of a layer on the tablets, granules or pellets which protects the inner core. In the event of enteric coating of tablets or granules, these are usually subjected to one or more subsequent mechanical operation, such as dry-coating, punching, capsuling and/or strip/blister packing. There is a possibility of partial rupture of the enteric coating-layer by abrasion while carrying out the subsequent mechanical operation(s). In the case of enteric-coated tablet, the live organisms are subjected to heavy compaction forces at which there is every possibility that the live organisms may lose their viability. Moreover, it has been observed that enteric-coated tablets of lactic acid bacillus fails to deliver the aggregated spores to their site of proliferation. The enteric-coated tablet dissolves in the duodenum and thus some organisms (Lactobacillus) are inactivated due to physical contact with anti-infective agent like amoxycillin in the course of its presence in duodenum till they are fully absorbed. It has also been reported that liquid formulations have been prepared in which the organisms are coated with barrier film and then mixed with other ingredients in the dry form including anti-infective agent. The product is to be reconstituted before use by addition of adequate amount of liquid, which is a rather cumbersome procedure. It has also been reported that the organisms are coated with barrier film and suspended in a liquid containing anti-infective agent(s) or vice versa. The barrier film is supposed to get disintegrated in body with the variation in pH. Such liquid formulations have not yet made their appearance in the market, and it seems unlikely that they will ever see the light of the day. When microorganisms are combined with anti-infective agents, namely, antibiotics, for convenience and ease of administration, the combination has been found to be unstable at room temperature, since microorganisms are sensitive and susceptible to the antibiotics and are destroyed by them. Analysis of commercially available formulations as well as similar formulations prepared at our end has revealed that organisms incorporated into formulation seldom perform any useful function for which they are intended. Neither the micro organisms nor their activity could be detected as early as 7 days after putting lactobacilli with various antibiotics like ampicillin, amoxycillin, amoxycillin -cloxacillin, amoxycillin + clavulanic acid, cephalosporin (sold under brand names as mentioned earlier), macrolides, fluoroquinolones, oliandomycin. lincomycin, etc. Although 60 million spores are put into formulation, none of them could be grown or demonstrated as viable on glucose yeast extract agar piate. It also failed to produce lactic acid as evaluated by consumption of Sodium Hydroxide (NaoH). The principal object of this invention is to overcome the difficulties and defects encountered in the conventional procedure followed hithertofore. A further object of this invention is to provide a process for preparing a storage - stable, enteric release therapeutic composition containing one or more antibiotics and microorganism(s) as the active ingredient. A still further object is to provide a process for preparing a therapeutic composition containing site-specific, enteric release granules in encapsulated form with an antibiotic like amoxycillin which help in avoiding the physical contact of the live organism(s) with amoxycillin throughout the gut length i.e. upto duodenum, where amoxycillin is present. Another object of this invention is to provide a process for preparing granular form of enteric release formulation wherein the granules are so designed as to be able to release their contents beyond duodenum. Yet another object of this invention is to provide a process for preparing enteric release granules capable of releasing their contents in intesting beyond duodenum in weakly acidic to alkaline pH of around 6.8 - 7.2, thus avoiding physical contact between antibiotic and lactic acid bacillus. The foregoing objects are achieved by the present invention according to which there is provided a process for preparing a storage - stable, enteric - release therapeutic composition containing one or more antibiotics and microorganism(s) as the active ingredients, which comprises in combination the following steps : (a) preparing enteric matrix formulation in a manner such as herein described ; (b) embedding micro organism(s) in an amount of around 80% overage by being mixed with the enteric matrix formulation to form granules ; (c) admixing enteric release granules from step (b) with gastric release granules of antibiotics in presence of pharmaceutically acceptable diluents, additives, fillers and/or adjuvants such as herein described so that the ratio of microorganism to antibiotic in the final composition varies between 1.2 and 1.75 and (d) finally filling the granules in hard gelatin capsule cells which are thereafter strip - sealed and packed as may be required or needed. As may be noted the foregoing discussions, this invention resides in a process of preparation of a stable, fixed dose combination of antibiotic with live microorganism as active, ingredients, various dosage forms for oral route like capsules, tablets and liquid formulations may be prepared according to the process of this invention. An appropriate barrier is provided for the microorganisms by means of a selected coating, which has been, found to be stable for 3-36 months under ambient conditions. The lactic acid bacillus is embedded on an enteric release matrix. The ratio of the microorganism to the antibiotic in the composition may be varied between 1.2 - 1.75 by weight, 1.5 being the optimal. The amount of coating depends on the type of coating technique, dosage form and desired shelf-life. For oral route formulation, organisms are coated by enteric coating, e.g. with cellulose acetate phthalate alone or in admixture with a resin polymer (Endragit). Antibiotic can be penicillin or ampicillin alone or in combination with resistant semi synthetic penicillin, e.g. amoxycillin alone or combined with penicillanase resistant penicillin. In the course of work, it was observed that it was difficult to produce a culture of this microbody in the intestine and preferred using L. acidophilus, which is a common commercial of human intestine. Preparations using various Lactobacillus spp. and other lactic acid producing organisms have been used in the treatment of gastrointestinal tract disorders. Metabolic acidosis has been observed following the use of products contaning L. acidophillus. These organisms have been found to be capable of eradicating or helpong in eradication of pathogens like CI. difficile by a number of mechasims which include, inter alia, production of hydrogen peroxide, or inhibition of adherence of pathogens to intestinal wa11s. In the enteric release matrix technique adopted in the present instance, the drug is embedded in the said matrix wherein the possibility of rupture of enteric coating layer due to abrasion or other mechanical reasons is absolutely remote. This, therefore, ensures better protection to the drug which is intended to be released in the intestine and be protected in the acidic pH environment of the stomach. The enteric - release technique used in the present instance achieves another desirable result. The admixture of site - specific enteric release granules of lactic acid bacillus in amoxycillin capsule helps to avoid physical contact between the living organisms with amoxycillin throughout the entire length of the gut, i.e. upto duodenum. These granules are so designed that they ensure release of the microorganisms beyond duodenum in intestine after around 4 hours of intake of the medication. The enteric - release granules dissolve in a slightly acidic to a low alkaline medium, between, say, a pH range of 6.8 and 7.2, somewhere in jejumum, thereby avoiding physical contact between the antibiotic (say. amoxycillin) and lactic acid baciPus. The invention will be further amplified by means of the following examples which are given by way of illustration and not by way of limitation. Preparation of encapsulated medication is carried out in a number of sequential steps given below. B. PROCEDURE i. Ingredients of Sl.No.l,2,3,4 & 5 are accurately weighed/ measured in separate, properly identified containers. ii. Ingredients of Sl.No.3,4,5 & 6 are added and mixed in a suitable polythene bucket with lid. Ingredient of Sl.No.l is added slowly and mixed with these ingredients under constant stirring using a suitable mechanical stirrer. Then Ingredient of SI.No.2 is added slowly & mixed with these ingredients under constant stirring by same way. iii. After 5 minutes 1000 ml. of Ingredient of SI.No.7 is slowly added and mixed by continuous strring until the solution becomes clear. iv. The solution of Statge iii is kept overnight and the next morning the volume is made upto 3.375 Its. by the addition of Acetone (item no.7) and necessary strring subsequently. v. The final solution is covered and set aside for later use. Stop 2 : Preparation of anfric - release qranules of Lactic Acid Bacillus A. FORMULATION SL. NO. INGRADIENTS LABEL CLAIM OVERAGE OTY. 1. Dibasic Calcium Phosphate - - 3.720 kg. 2. Titanium Dioxide - - 0.450 kg. 3. Tartrazine Supra - - 0.023 kg. 4. Brilliant Blue FCF - - 0.007 kg. 5. Purified Water - - 0.600 It. 6. Lactic Acid Bacillus 60 million spore 80% 2.700 kg. (6000 million spore/gm) (=10mg.) 7. Enteric matrix Solution - - 3.375 Its B. PROCEDURE: i. Item No. 1, 2, 3, 4 & 6 are accurately weighed & kept separately in properly identified polythene lined container. ii. Item No. 1 & 2 is passed through # 30 mesh sieve & mixed in a suitable plastic bowl which is degerminated by mopping with a clean fibre free cloth soaked in alcohol. iii. Item No.3 & 4 are dissolved in item No.5 with constant stirring till complete dissolution. iv. The mixture prepared in stage (ii) is kneaded in conventional manner with solution-of stage (iii) & passed through # 12 mesh sieve. The granules are dried in a tray drier with application of heat at 70°C. After complete drying the mass is passed through # 30 mesh sieve. v. Item No.6 is passed through # 30 mesh sieve & mixed in above mentioned suitable plastic degerminated be with the coloured granules prepared in stage (iv). This mixture is kneaded in the conventional manner by item No.7 to form granules & passed through # 12 mesh sieve. The granules are dried without application of heat. The operation of kneading with intermittent drying may be repeated twice / thrice as required to consume the full quantity of the enteric matrix solution. vi. The dried granules prepared in stage (v) are passed through # 16 mesh sieve to obtain the necessary final granules. vii. The enteric release granules of lactic acid bacillus are sent for intermediate testing for assay & confirmation of its enteric nature. Step 3. : Preparation of acid release matrix formulation - Batch Size : 15.000 Its B. PROCEDURE i. Ingredients of SI. No. 1 & 2 are weighed / measured accurately and kept separately in properly identified containers. ii. Item no.l is dissolved in item No.2 with continuous stirring until completely dissolved. iii. The solution is covered & set aside for use later. PROCEDURE: i. Item no. 1 is weighed accurately into a degerminaied plastic bowl with proper identification. ••i. Item No. I is kneaded in the conventional manner by item No.2 to form granules & passed throughy # 12 mesh sieve. The granules are dried in tray drier, initially without application of heat & subsequently at a low temperature (40°C - 45°C for four hours or till such time that the material is fully dried). The dried mass is passed initially through # 12 mesh size, & then through # 16 mesh sieve and necessary granules are obtained. Step 5 : Preparation of encapsulated form of drug containing enteric - release Lactic Acid Bacillus and Amoxycillin Trihydratc : A: FORMULATION SI. No. Ingredients Label Claim Overage Orv. used/cap Qty. in kg. used/batch 1. Enteric release L.A.B. 50.8 mg 7.620 Granules (6000 million spore/gm) 2. Acidic release granules of 306 mg. 45.900 Amoxycillin Trihydrate 3. Talcum 0.150 4. Magnesium Stearate 0.090 5. Empty hard gelatin capsule shell containing approved colours size ' 1'(printed green / CT) nos. 1.0% 1 no. 1,51,500 B, PROCEDURE: i. Item no.3 & 4 are weighed accurately & passed through ~ 100 r.iesh sieve & mixed together. ii. Item no. 1 & 2 are mixed together & bleaded in a suitable blender for 3 to 5 minutes. Then mixture prepared in stage (i) is added to it & blended in same manner for 2 minutes. i ii. The blended granules prepared in stage (ii) are sent to Quality Assurance department for intermediate approval regarding complete assay & confirmation of enteric release nature of the coated of L.A.B. granules & acidic release value of Amoxycillme Trihydratc. k. After approval of Q.A. Department, the granules are filled in item No.5. v. The filled capsules are tested for final approval by Q.A. Department. vi. On being duly approval by Q.A, the capsules are strip-sealed & packed as required. Quantitative Analytical results : The enteric - release therapeutic compositions of Examples 1 and 2 were subjected to quantitative analysis and the results are given below : From the ingredients marked above with asterisks (*), it may be noted that these are not present in the final composition in the encapsulated form. Materials other than Amoxycillin Trihydrate and Lactic Acid Bacillus may be regarded as falling within the categories of diluents / excipients / adjuvants/ fillers, etc. Other adjuvants like Starch, dextrin, Kaolin, colloidal Silica, etc., may be used without affecting the therapeutic activity of the final product. Cellulose acetate phthalate, ethyl cellulose and polyethylene glycol 6000 are some of the materials enhancing storage, stability and/or prolongation of time of release of medication. The final products of both Examples 1 and 2 were subjected to detailed analysis and assay in accordance with the procedcure laid down by the competent authority which is now in vogue and given below : ANALYTICAL PROTOCOL CATEGORY Antibiotic Dosage : Usual oral dose in adults is 250 mg. to 500 mg. of Amoxycillin thrice daily. Children under 20 kg. body - weight a dose of 20 to 40 mg. of Amoxycillin per kg. daily. USUAL STRENGTH : Amoxycillin Trihydrate LP. eq. to Amoxycillin 250 mg. Lactic Acid Bacillius 60 million spores. (As enteric released granules) STANDARDS The capsules contain not less than 95% and not more than 105% of the stated amount of Amoxycillin. It also contains not less than 95% of the stated amount of Lactic Acid Bacillus. APPEARANCE Size "1" or "00" capsules with opaque green cap. printed words in white and clear - transparent body, printed K logo in green. The inner contents of the capsule having white and green separate granules will be visible through the body oi the capsule UNIFORMITY OF WEIGHT : It should confirm the test of "Uniformity of weight" as per I.P. 96 monograph of "Capsules" (vide LP. '96, page - 134). DISINTEGRATION TIME : It should confirm the test of "Disintegration of Capsules" as per I.P. 96 monograph of'Capsules" (vide I.P.96, page -135). IDENTIFICATION Shake a quantity of the contents of the capsules equivalent to 0.5 g of amoxycillin with 5 ml. of water for 5 minutes, filter, wash the residue first with ethanol and then with ether and dry at a pressure not exceeding 0.7 kPa for 1 hour. The residue complies with the following test: The infra - red absorption spectrum, Appendix 5.4 (LP. 96) concordant with the reference spectrum of amoxycillin trihydrate or with the spectrum obtained from amoxycillin tnhydrate RS. DISSOLUTION DETERMINATION OF SPORE COUNT OF LACTIC ACID BACILLUS IN EACH CAPSULE. MEDIA USED FOR DISSOLUTION: 1. Acidic medium consists of 0.IN HCL. 2. Buffer medium consists of 6.8 buffer as per LP. PROCEDURE: 1. Place one capsule in the vessel of dissolution apparatus, which consists of 900 ml. of acidic medium. 2. Run the dissolution for 2 hours at 100 RPM with paddle attachment. 3. Collect the supernatant solution from the vessel. -. Taking one ml. from this solution, further dilutions are done from it to attain 60 spores / ml. 5. To the residue left over in the vessel, add 900 ml. of buffer solution and run the dissolution for 45 minutes. 6. After 45 minutes, take one ml. of the solution ando do further serial dilutions in order to attain spores / ml. 7. The contents of the last diluted test tube, which consists of app. 60 spore/ ml. is to be kept in water bath at 75° for 30 minutes. 8. The remaining procedure continues from "PLATING" of the assay procedure starting with "Pipette accurately —" and ending at"------60 million". PRECAUTIONS: J • All the glass ware, buffer solutions, acidic solutions should be autoclaved before use. 2. Pipetting with mouth should be avoided while collecting the sample from vessles, in order to avoid contamination. ASSAY A. CONTENT OF AMOXYCILLIN : Weigh accurately a quantity of the mixed contents of 20 capsules equivalent to 0.15 g of amoxycillin, add sufficient water to produce 500 m!., shake for 30 minutes and filter. To 10.0 ml of the filtrate add 10 ml. of alkaline borate buffer pH 9.0 followed by 1 ml. of acetic anhydride - dioxan solution, allow to stand for 5 minutes and add sufficient water to produce 100 ml. Transfer 2 ml. of the resulting solution into each of two stoppered tubes. To one tube add 10 ml. of imidazole - mercury reagent, mix, stopper the tube and immerse in a water-bath at 60° for exactly 25 minutes with occasional swirling. Remove the tube from the water-bath and cool rapidl) to 20° (solution A). To the second tube add 10 ml. of water and mix (solution B). Without delay measure i .e absorbances of solutions A and B at the maximum at about 325 nm, using as the blank a mixture of 2 ml of water and 10 ml. of imidazole - mercury reagent for solution A and water for solution B. Calculate the content of C16H19N3OsS from the difference between the absorbances of solution A and solution B, from the difference obtained by repeating tnc operation usmg 0. IT gsn. of amoxycillin trihydrate RS instead of the substance being examined and from the declared content of C,6H19N305S in amoxycillin trihydrateRS. B. DETERMINATION OF SPORE COUNT OF LACTIC ACID BACILLUS I EACH CAPSULE: PROCEDURE: Take contents of 5 capsules and transfer it into homogenizing Cup (Mixie). Add exactly 300 ml. of sterile normal saline solution and homogenize at about 12000 - 15000 R.P.M. for five minutes. This makes TEST SOLUTION of 1:300 dilution. Mix well the TEST SOLUTION (1:300 dilution) by hand shaking and dilute it further 'stepwise through a series of test tubes (size 25 mm x 150 mm), containing 9 ml. of sterile normal saline blank solution by an appropriate decimal dilution method. The .final dilution should be 300 x 104. Allow to stand the final diluted tube in a water bath at 75°C for 30 minutes (Heat -Shock), cooi immediately to about 45°C. Pipette accurately 1 ml. for Heat - Shock solution and add into each of five sterile petridishes. Add about 15 ml. of Glucose Yeast Extract Agar medium previously sterilized, melted and cooled to 45°C, to each of the five petri-dishes. Incubate the plates at 37°C for 72 hours and count the number of colonies in each plate. The average number of colonies, multiplied by the dilution factor and divide by five represents the SPORES COUNT per capsule. For example, if average count per plate is 100 colonies and final dilution factor is 300 x 10 4 then viable count : MEDIA AND REAGENTS: All chemicals to be used should be either A.R. or G.R. grade unless othei wise specified. Adjust (he pH of the medium to 6.3 using pH meter and sterilize the medium with steam at 1.2 Kg/Cm2 at 120°C for 15 minutes PROCEDURE: Weigh required quantity of salts and add in small quantity of Distilled Water and dissolve well. Raise the volume to 50 ml. in volumetric flask. The solution will attain faint pink colour. This solution can be kept in refrigerator for 2 months. Then prepare fresh solution. While the in\ ention has been described in detail with reference to specific embodii lents thereof , it will be apparent to one skilled in the art that various changes and modifications can be made therein without deviating or departing from the spirit and scope of this invention. Thus the disclosure contained herein includes within its ambit the obvious equivalents and substitutes as well. Havb-2 described the invention in detail with particular reference to the appended Examples, it sill now be more specifically defined by means of claims appended hereafter. Wo claim : 1. A process for preparing a storage-stable, enteric release therapeutic composition containing one or more antibiotics and microorganism(s) as the active ingredients, which comprises - (a) combining enteric release granules containing microorganism(s) with gastric release granules of antibiotics in presence of pharmaceutically acceptable diluents, fillers and/or adjuvants such as herein described and (b) filling the granules in hard gelatin capsules or compressing the granules into tablet form which in turn may be stripped, sealed and packed, characterized in that the said enteric release granules containing desired microorganism!s) are prepared by embedding the said microorganism(s) in an amount of around 80% overage by mixing with an enteric matrix formulation prepared from cellulose acetate phthalate, polyvinyl pyrrolidone, propylene glycol, isopropyl alcohol, ~Polysorbate 80" and acetone in amounts and manner such as herein described followed by conversion into granular form. 2. A process as claimed in Claim 1, wherein the ratio of microorganism to antibiotic in the final composition varies between 1.2 and 1.75, optimally 1.5. _j. A process as claimed in Claims 1 and 2, wherein the enteric release granules tend to give up their contents and disperse/ dissolve in the intestine after about 4 hours of intake with the rise in pH to around 6.8, thereby ensuring physical isolation of the active ingredients. 4. A process as claimed in Claims 1 to 3, wherein the composition is effective against toxin producing gram positive anaerobic bacteria Clostridium difficile. 5. A process as claimed in any of the preceding Claims, wherein there is added to the composition one or more chemicals selected from the group of cellulose acetate phthalate, ethyl cellulose and polyethylene glycol 6000 for enhancing storage stability and/or prolonging the time of release of medication in human system. 6. A process for preparing a storage—stable, enteric release therapeutic composition containing one or more antibiotics and microorganism(s) as the active ingredients, substantially as hereinbefore described with particular reference to the appended Examples. Dated this 16th day of May, 2002. This invention relates to a process for preparing a storage - stable, enteric relea therapeutic composition containing one or more antibiotics and microorganism(s) a, the active ingredients. The process resides in sequentially carrying out the steps of (i) preparation of enteric matrix formulation, (ii) preparation of enteric - release granules of Lactic Acid Bacillus, (iii) preparation of acid release matrix formulation, (iv) preparation of acid - release granules containing antibiotic or mixture thereof and (v) preparation of encapsulated form of drug containing the active ingredients aforesaid.

Full Text

The present invention relates to a process for preparing storage stable, therapeutic composition containing one or more antibiotics and microorganism(s) as the active ingredients. More particularly this invention is directed to a process for producting a therapentic composition in an enteric release system obtained by embedding the aforesaid active ingredients in an enteric - release matrix which not only serves as an effective barrier between the active ingredients but also ensures gradual release in the intestine, simultaneously protecting the drug(s) from degradation in the acidic pH environment of the stomach.
The term "enteric coating" used in this specification refers to technique, usually known in the art, refers to providing a coat or layer on substrates like tablets, pellets, granules, flakes or the like to protedct the inner core, particularly if the core material is reactive, or prone to degradation with passage of time or in contact with other material(s).
Normally human body harbours different types of micro-organisms. One class of microorganism inhabiting the human body, called 'pathogens , ma> be harmful to the host by causing infectious diseases. The other class of microbodies, called 'commensals', may be present in the human body for performing various useful functions, such as , for instance, providing essential vitamins to the body or preventing growth of harmful organisms within the human body.
To treat and/or prevent infectious diseases caused by pathogens, anti-infective agents are used, which are, more often than not, non-specific for pathogen and may result in eliminating commensals zs well. Reduction in the number of commensals below optimum level may result in various adverse / Side effects. Diarrhoea is one such effect of treatment with antibiotics. Normal bacterial flora of the intestine provides Vitamin B complex to the human body and also prevents growth of other micro-organisms which are not congenial to the human system. Alteration of the normal flora on account of administration
of antibiotics, either alone or in combination, may lead to transient dysfunction of normal colonic flora or superinfection of intestine by pathogens. The most common cause of antibiotic associated diarrhoea, e.g. megacolon and pseudomembranous colities,is the result of colonisation of the colon bya toxin-producing gram-positive anaerobic bacteria, Clostridium difficile.Some of the antibiotics normally used alone or in combination are, inter alia, ampicillin, amoxycillin, oliandomycin, Cepnalosporins marketed in various brand/trade names like Cephalexin, Cefixime, Certriaxone, Cefuroxine, Axetil, Proxetil, etc.), ampicillin + clanvanic acid, ampicillin+Sulbactum, fluoroquinolones, lincomycin, amoxycillin + cloxacillin, ampicilin + cloxacillin and the like.Early and specific anti-infective therapy should be given to patients suffering from diseases/disorders of the above nature.
Treatment aimed at recolonisation of the gut with non-pathogenic microorganisms has included administration of lactic acid producing microorganisms such as Lactobacillus, Bifidobacterium. Streptococcus boulardii and Saccharomyces boulardii, Lactic acid producing organisms were introduced as a therapeutic agent by Metchnikoff with the idea of acidifying intestinal contents in order to prevent the growth of putrefactur organisms.The organism chosen by him for this purpose was Lactobacillus bulgaricus.These are stable and with beter therapeutic effect.
Anion-exchange resins cholestyramine and cholestipol hydrochloride have been shown to bind the toxin of CI. difficile in vitro, and cholestyramine has been used to treat pseudomembranous colitis. Antibiotic associated colitis or colonic perturation has also been treated with drugs like vancomycin and metronidazole.
To prevent diarrhoea or colitis, lactic acid producing microorganisms have been administered alone (e.g. Lactiflora, Lactobacil, Lactocap, Lactovit, Sporlac, etc.;, or in combination with antibiotic as two separate formulations. This necessitates consumption of two different drugs, namely, an anti-infective
agent and a micro-organism, thereby decreasing patient compliance.
A simple admixture of lactic acid bacillus (non-protected) with amoxycillin is not associated with any intrinsic type of resistance of lactic acid bacteria to amoxycillin. Hence, no particular safety concern is linked with such admixture.
Attempts have also been made to enhance both compliance and efficacy. For this purpose, microorganisms and antibiotics have been made into a single formulation, some of which are commercially available. Anti-infective agents used in such formulations are, for instance, ampicillin, amoxycillin, which are made into simple formulation with susceptible micro-organisms.
As pointed out earlier, "enteric coating" is application or formation of a layer on the tablets, granules or pellets which protects the inner core. In the event of enteric coating of tablets or granules, these are usually subjected to one or more subsequent mechanical operation, such as dry-coating, punching, capsuling and/or strip/blister packing. There is a possibility of partial rupture of the enteric coating-layer by abrasion while carrying out the subsequent mechanical operation(s).
In the case of enteric-coated tablet, the live organisms are subjected to heavy compaction forces at which there is every possibility that the live organisms may lose their viability. Moreover, it has been observed that enteric-coated tablets of lactic acid bacillus fails to deliver the aggregated spores to their site of proliferation. The enteric-coated tablet dissolves in the duodenum and thus some organisms (Lactobacillus) are inactivated due to physical contact with anti-infective agent like amoxycillin in the course of its presence in duodenum till they are fully absorbed.
It has also been reported that liquid formulations have been prepared in which the organisms are coated with barrier film and then mixed with other ingredients in the dry form including anti-infective agent. The product is to be
reconstituted before use by addition of adequate amount of liquid, which is a rather cumbersome procedure. It has also been reported that the organisms are coated with barrier film and suspended in a liquid containing anti-infective agent(s) or vice versa. The barrier film is supposed to get disintegrated in body with the variation in pH. Such liquid formulations have not yet made their appearance in the market, and it seems unlikely that they will ever see the light of the day.
When microorganisms are combined with anti-infective agents, namely, antibiotics, for convenience and ease of administration, the combination has been found to be unstable at room temperature, since microorganisms are sensitive and susceptible to the antibiotics and are destroyed by them. Analysis of commercially available formulations as well as similar formulations prepared at our end has revealed that organisms incorporated into formulation seldom perform any useful function for which they are intended. Neither the micro organisms nor their activity could be detected as early as 7 days after putting lactobacilli with various antibiotics like ampicillin, amoxycillin, amoxycillin -cloxacillin, amoxycillin + clavulanic acid, cephalosporin (sold under brand names as mentioned earlier), macrolides, fluoroquinolones, oliandomycin. lincomycin, etc. Although 60 million spores are put into formulation, none of them could be grown or demonstrated as viable on glucose yeast extract agar piate. It also failed to produce lactic acid as evaluated by consumption of Sodium Hydroxide (NaoH).
The principal object of this invention is to overcome the difficulties and defects encountered in the conventional procedure followed hithertofore.
A further object of this invention is to provide a process for preparing a storage - stable, enteric release therapeutic composition containing one or more antibiotics and microorganism(s) as the active ingredient.
A still further object is to provide a process for preparing a therapeutic composition containing site-specific, enteric release granules in encapsulated form with an antibiotic like amoxycillin which help in avoiding the physical contact of the live organism(s) with amoxycillin throughout the gut length i.e. upto duodenum, where amoxycillin is present.
Another object of this invention is to provide a process for preparing granular form of enteric release formulation wherein the granules are so designed as to be able to release their contents beyond duodenum.
Yet another object of this invention is to provide a process for preparing enteric release granules capable of releasing their contents in intesting beyond duodenum in weakly acidic to alkaline pH of around 6.8 - 7.2, thus avoiding physical contact between antibiotic and lactic acid bacillus.
The foregoing objects are achieved by the present invention according to which there is provided a process for preparing a storage - stable, enteric - release therapeutic composition containing one or more antibiotics and microorganism(s) as the active ingredients, which comprises in combination the following steps :
(a) preparing enteric matrix formulation in a manner such as herein described ;
(b) embedding micro organism(s) in an amount of around 80% overage by being mixed with the enteric matrix formulation to form granules ;
(c) admixing enteric release granules from step (b) with gastric release granules of antibiotics in presence of pharmaceutically acceptable diluents, additives, fillers and/or adjuvants such as herein described so that the ratio of microorganism to antibiotic in the final composition varies between 1.2 and 1.75 and
(d) finally filling the granules in hard gelatin capsule cells which are thereafter strip - sealed and packed as may be required or needed.
As may be noted the foregoing discussions, this invention resides in a process of preparation of a stable, fixed dose combination of antibiotic with live microorganism as active, ingredients, various dosage forms for oral route like capsules, tablets and liquid formulations may be prepared according to the process of this invention. An appropriate barrier is provided for the microorganisms by means of a selected coating, which has been, found to be stable for 3-36 months under ambient conditions. The lactic acid bacillus is embedded on an enteric release matrix. The ratio of the microorganism to the antibiotic in the composition may be varied between 1.2 - 1.75 by weight, 1.5 being the optimal. The amount of coating depends on the type of coating technique, dosage form and desired shelf-life.
For oral route formulation, organisms are coated by enteric coating, e.g. with cellulose acetate phthalate alone or in admixture with a resin polymer (Endragit). Antibiotic can be penicillin or ampicillin alone or in combination with resistant semi synthetic penicillin, e.g. amoxycillin alone or combined with penicillanase resistant penicillin. In the course of work, it was observed that it was difficult to produce a culture of this microbody in the intestine and preferred using L. acidophilus, which is a common commercial of human intestine. Preparations using various Lactobacillus spp. and other lactic acid producing organisms have been used in the treatment of gastrointestinal tract disorders. Metabolic acidosis has been observed following the use of products contaning L. acidophillus. These organisms have been found to be capable of eradicating or helpong in eradication of pathogens like CI. difficile by a number of mechasims which include, inter alia, production of hydrogen peroxide, or inhibition of adherence of pathogens to intestinal wa11s.
In the enteric release matrix technique adopted in the present instance, the drug is embedded in the said matrix wherein the possibility of rupture of enteric coating layer due to abrasion or other mechanical reasons is absolutely remote. This, therefore, ensures better protection to the drug which is intended to be released in the intestine and be protected in the acidic pH environment of the stomach.
The enteric - release technique used in the present instance achieves another desirable result. The admixture of site - specific enteric release granules of lactic acid bacillus in amoxycillin capsule helps to avoid physical contact between the living organisms with amoxycillin throughout the entire length of the gut, i.e. upto duodenum. These granules are so designed that they ensure release of the microorganisms beyond duodenum in intestine after around 4 hours of intake of the medication. The enteric - release granules dissolve in a slightly acidic to a low alkaline medium, between, say, a pH range of 6.8 and 7.2, somewhere in jejumum, thereby avoiding physical contact between the antibiotic (say. amoxycillin) and lactic acid baciPus.
The invention will be further amplified by means of the following examples which are given by way of illustration and not by way of limitation.
Preparation of encapsulated medication is carried out in a number of sequential steps given below.

B. PROCEDURE
i. Ingredients of Sl.No.l,2,3,4 & 5 are accurately weighed/ measured in separate, properly identified containers.
ii. Ingredients of Sl.No.3,4,5 & 6 are added and mixed in a suitable polythene bucket with lid. Ingredient of Sl.No.l is added slowly and mixed with these ingredients under constant stirring using a suitable mechanical stirrer. Then Ingredient of SI.No.2 is added slowly & mixed with these ingredients under constant stirring by same way.
iii. After 5 minutes 1000 ml. of Ingredient of SI.No.7 is slowly added and mixed by continuous strring until the solution becomes clear.
iv. The solution of Statge iii is kept overnight and the next morning the volume is made upto 3.375 Its. by the addition of Acetone (item no.7) and necessary strring subsequently.
v. The final solution is covered and set aside for later use.
Stop 2 : Preparation of anfric - release qranules of Lactic Acid Bacillus
A. FORMULATION
SL. NO. INGRADIENTS LABEL CLAIM OVERAGE OTY.
1. Dibasic Calcium Phosphate - - 3.720 kg.
2. Titanium Dioxide - - 0.450 kg.
3. Tartrazine Supra - - 0.023 kg.
4. Brilliant Blue FCF - - 0.007 kg.
5. Purified Water - - 0.600 It.
6. Lactic Acid Bacillus 60 million spore 80% 2.700 kg. (6000 million spore/gm) (=10mg.)
7. Enteric matrix Solution - - 3.375 Its
B. PROCEDURE:
i. Item No. 1, 2, 3, 4 & 6 are accurately weighed & kept separately in properly identified polythene lined container.
ii. Item No. 1 & 2 is passed through # 30 mesh sieve & mixed in a suitable plastic bowl which is degerminated by mopping with a clean fibre free cloth soaked in alcohol.
iii. Item No.3 & 4 are dissolved in item No.5 with constant stirring till complete dissolution.
iv. The mixture prepared in stage (ii) is kneaded in conventional manner with solution-of stage (iii) & passed through # 12 mesh sieve. The granules are dried in a tray drier with application of heat at 70°C. After complete drying the mass is passed through # 30 mesh sieve.
v. Item No.6 is passed through # 30 mesh sieve & mixed in above mentioned suitable plastic degerminated be with the coloured granules prepared in stage (iv). This mixture is kneaded in the conventional manner by item No.7 to form granules & passed through # 12 mesh sieve. The granules are dried without application of heat. The operation of kneading with intermittent drying may be repeated twice / thrice as required to consume the full quantity of the enteric matrix solution.
vi. The dried granules prepared in stage (v) are passed through # 16 mesh sieve to obtain the necessary final granules.
vii. The enteric release granules of lactic acid bacillus are sent for intermediate testing for assay & confirmation of its enteric nature.
Step 3. : Preparation of acid release matrix formulation - Batch Size : 15.000 Its

B. PROCEDURE
i. Ingredients of SI. No. 1 & 2 are weighed / measured accurately and kept separately in properly identified containers.
ii. Item no.l is dissolved in item No.2 with continuous stirring until completely dissolved.
iii. The solution is covered & set aside for use later.

PROCEDURE:
i. Item no. 1 is weighed accurately into a degerminaied plastic bowl with proper identification.
••i. Item No. I is kneaded in the conventional manner by item No.2 to form granules & passed throughy # 12 mesh sieve. The granules are dried in tray drier, initially without application of heat & subsequently at a low temperature (40°C - 45°C for four hours or till such time that the material is fully dried). The dried mass is passed initially through # 12 mesh size, & then through # 16 mesh sieve and necessary granules are obtained.
Step 5 : Preparation of encapsulated form of drug containing enteric - release Lactic Acid Bacillus and Amoxycillin Trihydratc :
A: FORMULATION
SI. No. Ingredients Label Claim Overage Orv. used/cap Qty. in kg.
used/batch
1. Enteric release L.A.B. 50.8 mg 7.620 Granules (6000 million spore/gm)
2. Acidic release granules of 306 mg. 45.900 Amoxycillin Trihydrate
3. Talcum 0.150
4. Magnesium Stearate 0.090
5. Empty hard gelatin capsule shell containing approved colours
size ' 1'(printed green / CT) nos. 1.0% 1 no. 1,51,500
B, PROCEDURE:
i. Item no.3 & 4 are weighed accurately & passed through ~ 100 r.iesh sieve & mixed together.
ii. Item no. 1 & 2 are mixed together & bleaded in a suitable blender for 3 to 5 minutes. Then mixture prepared in stage (i) is added to it & blended in same manner for 2 minutes.
i ii. The blended granules prepared in stage (ii) are sent to Quality Assurance department for intermediate approval regarding complete assay & confirmation of enteric release nature of the coated of L.A.B. granules & acidic release value of Amoxycillme Trihydratc.
k. After approval of Q.A. Department, the granules are filled in item No.5.
v. The filled capsules are tested for final approval by Q.A. Department.
vi. On being duly approval by Q.A, the capsules are strip-sealed & packed as required.

Quantitative Analytical results :
The enteric - release therapeutic compositions of Examples 1 and 2 were subjected to quantitative analysis and the results are given below :

From the ingredients marked above with asterisks (*), it may be noted that these are not present in the final composition in the encapsulated form.
Materials other than Amoxycillin Trihydrate and Lactic Acid Bacillus may be regarded as falling within the categories of diluents / excipients / adjuvants/ fillers, etc. Other adjuvants like Starch, dextrin, Kaolin, colloidal Silica, etc., may be used without affecting the therapeutic activity of the final product. Cellulose acetate phthalate, ethyl cellulose and polyethylene glycol 6000 are some of the materials enhancing storage, stability and/or prolongation of time of release of medication.
The final products of both Examples 1 and 2 were subjected to detailed analysis and assay in accordance with the procedcure laid down by the competent authority which is now in vogue and given below :
ANALYTICAL PROTOCOL
CATEGORY Antibiotic
Dosage : Usual oral dose in adults is 250 mg. to 500 mg. of
Amoxycillin thrice daily. Children under 20 kg. body - weight a dose of 20 to 40 mg. of Amoxycillin per kg. daily.
USUAL STRENGTH : Amoxycillin Trihydrate LP. eq. to Amoxycillin 250 mg.
Lactic Acid Bacillius 60 million spores. (As enteric released granules)
STANDARDS The capsules contain not less than 95% and not more than
105% of the stated amount of Amoxycillin. It also contains not less than 95% of the stated amount of Lactic Acid Bacillus.
APPEARANCE Size "1" or "00" capsules with opaque green cap. printed
words in white and clear - transparent body, printed K logo in green. The inner contents of the capsule having white and green separate granules will be visible through the body oi the capsule
UNIFORMITY OF WEIGHT : It should confirm the test of "Uniformity of weight" as per
I.P. 96 monograph of "Capsules" (vide LP. '96, page - 134).
DISINTEGRATION TIME : It should confirm the test of "Disintegration of Capsules"
as per I.P. 96 monograph of'Capsules" (vide I.P.96, page -135).
IDENTIFICATION Shake a quantity of the contents of the capsules equivalent
to 0.5 g of amoxycillin with 5 ml. of water for 5 minutes, filter, wash the residue first with ethanol and then with ether and dry at a pressure not exceeding 0.7 kPa for 1 hour. The residue complies with the following test:
The infra - red absorption spectrum, Appendix 5.4 (LP. 96) concordant with the reference spectrum of amoxycillin trihydrate or with the spectrum obtained from amoxycillin tnhydrate RS.
DISSOLUTION DETERMINATION OF SPORE COUNT OF LACTIC ACID
BACILLUS IN EACH CAPSULE.
MEDIA USED FOR DISSOLUTION:
1. Acidic medium consists of 0.IN HCL.
2. Buffer medium consists of 6.8 buffer as per LP. PROCEDURE:
1. Place one capsule in the vessel of dissolution apparatus, which consists of 900 ml. of acidic medium.
2. Run the dissolution for 2 hours at 100 RPM with paddle attachment.
3. Collect the supernatant solution from the vessel.
-. Taking one ml. from this solution, further dilutions are done from it to attain 60 spores / ml.
5. To the residue left over in the vessel, add 900 ml. of buffer solution and run the dissolution for 45 minutes.
6. After 45 minutes, take one ml. of the solution ando do further serial dilutions in order to attain spores / ml.
7. The contents of the last diluted test tube, which consists of app. 60 spore/ ml. is to be kept in water bath at 75° for 30 minutes.
8. The remaining procedure continues from "PLATING" of the assay procedure starting with "Pipette accurately —" and ending at"------60 million".
PRECAUTIONS:
J • All the glass ware, buffer solutions, acidic solutions should be autoclaved before use.
2. Pipetting with mouth should be avoided while collecting the sample from vessles, in order to avoid contamination.
ASSAY
A. CONTENT OF AMOXYCILLIN :
Weigh accurately a quantity of the mixed contents of 20 capsules equivalent to 0.15 g of amoxycillin, add sufficient water to produce 500 m!., shake for 30 minutes and filter. To 10.0 ml of the filtrate add 10 ml. of alkaline borate buffer pH 9.0 followed by 1 ml. of acetic anhydride - dioxan solution, allow to stand for 5 minutes and add sufficient water to produce 100 ml. Transfer 2 ml. of the resulting solution into each of two stoppered tubes. To one tube add 10 ml. of imidazole - mercury reagent, mix, stopper the tube and immerse in a water-bath at 60° for exactly 25 minutes with occasional swirling. Remove the tube from the water-bath and cool rapidl) to 20° (solution A).
To the second tube add 10 ml. of water and mix (solution B). Without delay measure i .e absorbances of solutions A and B at the maximum at about 325 nm, using as the blank a mixture of 2 ml of water and 10 ml. of imidazole - mercury reagent for solution A and water for solution B. Calculate the content of C16H19N3OsS from the difference between the absorbances of solution A and solution B, from the difference obtained by repeating tnc operation usmg 0. IT gsn. of amoxycillin trihydrate RS instead of the substance being examined and from the declared content of C,6H19N305S in amoxycillin trihydrateRS.
B. DETERMINATION OF SPORE COUNT OF LACTIC ACID BACILLUS I EACH CAPSULE:
PROCEDURE:
Take contents of 5 capsules and transfer it into homogenizing Cup (Mixie). Add exactly 300 ml. of sterile normal saline solution and homogenize at about 12000 - 15000 R.P.M. for five minutes. This makes TEST SOLUTION of 1:300 dilution.
Mix well the TEST SOLUTION (1:300 dilution) by hand shaking and dilute it further
'stepwise through a series of test tubes (size 25 mm x 150 mm), containing 9 ml. of sterile normal saline blank solution by an appropriate decimal dilution method. The .final dilution should be 300 x 104.
Allow to stand the final diluted tube in a water bath at 75°C for 30 minutes (Heat -Shock), cooi immediately to about 45°C.
Pipette accurately 1 ml. for Heat - Shock solution and add into each of five sterile petridishes. Add about 15 ml. of Glucose Yeast Extract Agar medium previously sterilized, melted and cooled to 45°C, to each of the five petri-dishes. Incubate the plates at 37°C for 72 hours and count the number of colonies in each plate. The average number of colonies, multiplied by the dilution factor and divide by five represents the SPORES COUNT per capsule.
For example, if average count per plate is 100 colonies and final dilution factor is 300 x 10 4 then viable count :

MEDIA AND REAGENTS:
All chemicals to be used should be either A.R. or G.R. grade unless othei wise specified.

Adjust (he pH of the medium to 6.3 using pH meter and sterilize the medium with steam at 1.2 Kg/Cm2 at 120°C for 15 minutes

PROCEDURE:
Weigh required quantity of salts and add in small quantity of Distilled Water and dissolve well. Raise the volume to 50 ml. in volumetric flask. The solution will attain faint pink colour. This solution can be kept in refrigerator for 2 months. Then prepare fresh solution.
While the in\ ention has been described in detail with reference to specific embodii lents thereof , it will be apparent to one skilled in the art that various changes and modifications can be made therein without deviating or departing from the spirit and scope of this invention. Thus the disclosure contained herein includes within its ambit the obvious equivalents and substitutes as well.
Havb-2 described the invention in detail with particular reference to the appended Examples, it sill now be more specifically defined by means of claims appended hereafter.
Wo claim :
1. A process for preparing a storage-stable, enteric release therapeutic composition containing one or more antibiotics and microorganism(s) as the active ingredients, which comprises -
(a) combining enteric release granules containing microorganism(s) with gastric release granules of antibiotics in presence of pharmaceutically acceptable diluents, fillers and/or adjuvants such as herein described and
(b) filling the granules in hard gelatin capsules or compressing the granules into tablet form which in turn may be stripped, sealed and packed,
characterized in that the said enteric release granules containing desired microorganism!s) are prepared by embedding the said microorganism(s) in an amount of around 80% overage by mixing with an enteric matrix formulation prepared from cellulose acetate phthalate, polyvinyl pyrrolidone, propylene glycol, isopropyl alcohol, ~Polysorbate 80" and acetone in amounts and manner such as herein described followed by conversion into granular form.
2. A process as claimed in Claim 1, wherein the ratio of microorganism to antibiotic in the final composition varies between 1.2 and 1.75, optimally 1.5.
_j. A process as claimed in Claims 1 and 2, wherein the enteric release granules tend to give up their contents and disperse/ dissolve in the intestine after about 4 hours of intake with the rise in pH to around 6.8, thereby ensuring physical isolation of the active ingredients.
4. A process as claimed in Claims 1 to 3, wherein the composition is effective against toxin producing gram positive anaerobic bacteria Clostridium difficile.
5. A process as claimed in any of the preceding Claims, wherein there is added to the composition one or more chemicals selected from the group of cellulose acetate phthalate, ethyl cellulose and polyethylene glycol 6000 for enhancing storage stability and/or prolonging the time of release of medication in human system.
6. A process for preparing a storage—stable, enteric release therapeutic composition containing one or more antibiotics and microorganism(s) as the active ingredients, substantially as hereinbefore described with particular reference to the appended Examples.
Dated this 16th day of May, 2002.

This invention relates to a process for preparing a storage - stable, enteric relea therapeutic composition containing one or more antibiotics and microorganism(s) a, the active ingredients. The process resides in sequentially carrying out the steps of (i) preparation of enteric matrix formulation, (ii) preparation of enteric - release granules of Lactic Acid Bacillus, (iii) preparation of acid release matrix formulation, (iv) preparation of acid - release granules containing antibiotic or mixture thereof and (v) preparation of encapsulated form of drug containing the active ingredients aforesaid.

Documents:

311-cal-2002-granted-abstract.pdf

311-cal-2002-granted-assignment.pdf

311-cal-2002-granted-claims.pdf

311-cal-2002-granted-correspondence.pdf

311-cal-2002-granted-description (complete).pdf

311-cal-2002-granted-examination report.pdf

311-cal-2002-granted-form 1.pdf

311-cal-2002-granted-form 13.pdf

311-cal-2002-granted-form 18.pdf

311-cal-2002-granted-form 2.pdf

311-cal-2002-granted-form 3.pdf

311-cal-2002-granted-form 5.pdf

311-cal-2002-granted-pa.pdf

311-cal-2002-granted-reply to examination report.pdf

311-cal-2002-granted-specification.pdf

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Patent Number

228806

Indian Patent Application Number

311/CAL/2002

PG Journal Number

07/2009

Publication Date

13-Feb-2009

Grant Date

11-Feb-2009

Date of Filing

16-May-2002

Name of Patentee

KONTEST CHEMICALS LTD.

Applicant Address

80/1A, SARAT BOSE ROAD, KOLKATA

Inventors:

#	Inventor's Name	Inventor's Address
1	JYOTIRMOY DEY	80/1A, SARAT BOSE ROAD, KOLKATA 700 025
2	VICTOR EMANUEL AMBETT	80/1A, SARAT BOSE ROAD, KOLKATA 700 025

PCT International Classification Number

A61K 35/74

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA