Title of Invention	A PROCESS FOR THE PREPARATION OF BETA-LACTAM ANTIBIOTIC-POLYSACCHRIDE COMPLEX
Abstract	ABSTRACT The present invention relates to a process for the preparation of Beta-lactam antibiotic-Polysaccharide complex having improved therapeutic efficacy as compared parent (known) Beta-lactam antibiotics such as Cephalosporin antibiotics. The present novel process results in complexes that facilitate optimisation in drug delivery with an objective to enhance the efficacy of the therapeutic moieties, with reduced toxicity.

Title of Invention

A PROCESS FOR THE PREPARATION OF BETA-LACTAM ANTIBIOTIC-POLYSACCHRIDE COMPLEX

Abstract

ABSTRACT The present invention relates to a process for the preparation of Beta-lactam antibiotic-Polysaccharide complex having improved therapeutic efficacy as compared parent (known) Beta-lactam antibiotics such as Cephalosporin antibiotics. The present novel process results in complexes that facilitate optimisation in drug delivery with an objective to enhance the efficacy of the therapeutic moieties, with reduced toxicity.

Full Text	FIELD OF INVENTION The present invention relates to a novel process for the preparation of Beta-lactum antibiotic-Polysaccharide complexes having improved therapeutic efficacy. The novel complex produced by the present process imparts an improvement in the therapeutic efficacy compared to uncomplexed Beta-lactum antibiotics, for example, Cephalosporin antibiotics. The present novel process results in complexes that facilitate optimisation in drug delivery with an objective to enhance the efficacy of the therapeutic moieties, with reduced toxicity. Preferably, the novel process is especially suitable to produce injectable Beta-lactum antibiotics complexes involving polysaccharides. BACKGROUND OF THE INVENTION Beta-lactum antibiotic compounds are derived from 6-APA (6 amino penicillanic acid), 7-ADCA (7 amino desacetoxy cephalosporanic acid) or 7-ACA (7 amino cephalosporanic acid). Beta-lactum antibiotics are widely used for treatment of infections caused by both gram-positive and gram-negative organisms. Beta-lactum antibiotic acts through inhibition of cell wall synthesis by the inhibition of the peptido-glycan synthesis and teichoic acid metabolism of the organism. It also inhibits the cross-linking of N-acetyl muramic acid with N-acetyl glucosamine, thereby preventing the integrity of the bacterial cell wall. Among the commonly known Beta-lactum antibiotics which are used for wide range of bacterial infections. Cephalosporin antibiotics have emerged as a preferred group of antibiotics. Cephalosporin antibiotics have broad spectrum activity covering both gram-positive and gram-negative organisms. These are administered in both parenteral and oral routes depending on their physico-chemical characteristics, pharmaco¬dynamic and pharmaco-kinetic profile. The choice of antibiotics depends on the type of infection and the dose and route of administration based on the severity of the infection. The injectable cephalosporins are employed for systemic use for prompt action and bio-availability of these dosage forms is found to be 100 percent. The pharmaco-kinetic studies show that injectable cephalosporins are plasma-bound by about 60 percent and remaining portion of cephalopsorins, being highly water soluble gets excreted through urinary route which does not elicit any therapeutic response at the target site. In case of other Beta-lactums, the plasma-protein binding may range upto about 85 percent depending on the molecule. Polysaccharides are carbohydrates derived from plant and microbial sources. Polysaccharides are used for many industrial applications. Beta-lactum antibiotics (including Cephalosporins) being life-saving antibiotics, any invention which enhances the therapeutic efficacy of the products while at the same time reducing the toxicity is a highly desirable objective. Usually the patients affected by infections tend to have altered and weakened physiological systems. Also, generally, patients tend to have multiple pathological conditions which cannot bear the risk of high toxicity burden. In this context, if the invention achieves an attendant lower dosage of active moiety, it reduces the risk of higher dose exposure which otherwise would be inevitable with onlv the conventional moiety. Moreover, these antibiotics are costly. If the novel complex utilises less amount of the active ingredient to achieve the same therapeutic efficacy, it would significantly reduce the cost of treatment. Conventionally, Beta-lactum antibiotics are synthesised from 6-APA, 7-ADCA and 7-ACA depending upon the molecule. More specifically, Cephalosporin antibiotics are synthesised from 7-ADCA or 7-ACA. The process of synthesis is such that the end product would be the active pharmaceutical ingredient of the required cephalosporin representing 100% of the active moiety. The applicant's invention encompasses the complexing of the active moiety, preferably injectable cephalosporins, with polysaccharide to obtain optimum usage of the therapeutic moiety. This process results in a novel lyophilised complex which provides efficacious, less toxic and more cost-effective drug delivery intended for freating infections in mammals, particularly humans. PRIOR ART There had been some earlier studies conducted for optimisation of anti-cancer drugs, namely Mitomycin and Methotrexate for the confrol of toxicity as the above drugs are highly toxic. This had been achieved through complexation involving polysaccharides. In the case of Mitomycin and Methotrexate the attachment of the polysaccharides to the molecule had been mediated through artificially synthesised spacer arm. Such synthesis is complex, time consuming and costly in nature since it requires the activation of the polysaccharide to link with this pre-synthesised spacer arm for the establishment of the fmal linkage with the active moiety viz.. Mitomycin and Methotrexate. However, complexation of Beta-lactum antibiotics with polysaccharides has so far not been reported. Beta-lactum antibiotics which treat bacterial infections fall altogether in a different class of compounds compared to anticancer or any other therapeutic group of drugs. The disease conditions and treatment profile for which Beta-lactum antibotics are intended are far more widely prevalent. There is no reported attempt so far made to optimise the efficacy of Beta-lactum antibiotic employing any polysaccharides. There has been no reported process disclosing the complexing of Beta-lactums, preferably cephalosporin injectables, with polysaccharides in order to enhance the therapeutic efficacy of the basic molecule. The applicant believes that any process invention which optimises the efficacy of this antibiotic group has significant positive benefits for the society. DESCRIPTION OF THE INVENTION The present .invention provides a process for producing antibiotic complexes comprising an effective therapeutic amount of Beta-lactum and polysaccharide as a complexing ligand in each complex. The therapeutic quantum of Beta-lactum may vary fi-om 50 to 75% by weight in the complex depending on the active therapeutic moiety. The invention for which this patent application is being made involves complexation of Beta-lactums, preferably sterile cephalosporins, with appropriate polysaccharides to achieve optimum drug delivery for therapeutic efficacy and toxicity control. The preferred polysaccharides that can be employed are Arabinose, Agarose, Low molecular weight dextran, Activated clinical dextrans, Inulin, Chitin, Chitosan, Mannan, Lichenin, and their derivatives / variants. The applicant's preferred polysaccharides, having linear structure, are water soluble and good carriers of drug. In addition, the polysaccharide carriers are pharmacologically and toxicologically inert and hence, these carriers are suitable to impart properties to link the drugs through the ligand attachments involving the polysaccharides for optimised drug delivery. The polysaccharides should be provided with polar hydroxyl groups, so that they can attach with the active moiety as a part of the complexation process through hydrogen bonding. In other words, the present process involves altogether a different chemical reaction which is much simpler and economically viable. The solubility of complex involving cephalosporins and p>olysaccharides as injectables makes it appropriate for parenteral application. The polysaccharides are rapidly excreted in urine without accumulation in the tissues. The sterile Cephalosporins for which this approach is applicable comprise Cefazolin Sodium, Ceftriaxone Sodium, Cefoperazone Sodium, Cefotaxime Sodium, Cefliroxime Sodium and a number of other cephalosporin derivatives of various generations. In the case of Cefazolin complex which is being cited here as an example, Cefazolin can be present in the range of 55 to 65% and the polysaccharide constitutmg the balance, which is achieved in stoichiometric proportions in ^ aqueous solution at a definite pH through aseptic lyophilisation process. The preferred proportion of Cefazolin to the polysaccharide is 60:40. hi the present process, the antibiotic polysaccharide complex solution so obtained is to be filtered through 0.2 micron filter. The temperatures selected for this lyophilisation are -40° C, -10° C, + 10° C and +35° C under a vacuum of about 2.2 x 10'^ mbar. This invention optimises the proportion of active moiety which is required for therapeutic response for a particular indication leading to toxicity reduction and also economy in treatment cost. By virtue of its applicability to a range of sterile Cephalosporins preferably, and other Beta-Iactums, administered in the parenteral route, the invention has potential to benefit a huge population of patients requiring parenteral Cephalosporin and other Beta-lactum antibiotic treatment. Therefore, the present invention provides a process for producing novel Beta-lactum antibiotic-Polysaccharide complexes and preferably. Sterile Cephalosporin-Polysaccharide complexes having a lower dosage of active moiety, resulting in equal phamacological and microbiological response compared to uncomplexed drug leading to lesser toxicity and reduced cost of treatment for the patients. The present invention produces a synergistic composition having enhanced and surprising properties which has not been envisaged. Some embodiments of the invention also involve addition of suitable pH adjustment agents such as Sodium bicarbonate, activation of the polysaccharides for complexation process through cyanogen bromide treatment. EXAMPLE To produce 1 gm of Cefazolin - Polysaccharide complex, 0.6 gms of Cefazolin and 0.4 gms of Polysaccharide are mixed with water under the pH condition between 5.5 to 6.5. Sodium bicarbonate is added to adjust the pH. The polysaccharide complex solution thus obtained is filtered through 0.2micron filter and transferred into a sterilized vial. Finally, the vial containing the solution is aseptically lyophilised under ^0°C, -10°C, + 10" C and +35° C under a vacuum ofabout2.2xl0'^mbar. CEFAZOLIN - POLYSACCHARIDE COMPLEX UNDER MINIMUM INHIBITORY CONCENTRATION (MIC) STUDIES The above MICs are lower than the reported MICs of Cefazolin towards the above organisms (i.e., 16 meg and less / ml). The above Table clearly establishes the fact that the complexed Cefazolin-Polysaccharide antibiotic ( 60 : 40 ratio) provides equivalent or better response on the microorganisms cited above compared to uncomplexed Cefazolin antibiotic (100%). Animal studies were conducted, for example, on Swiss Albino mice. Thirty mice were taken for evaluation with the following treatment pattern : ten with complexed drug (Cefazolin complex), ten with uncomplexed drug (Cefazolin) and ten untreated. Staph.aureus and Proteus Mirabilis were the selected microorganisms against a designed bum-caused infection. The rate of healing for the complexed drug confirms to the desired response in equivalent pattem to that of uncomplexed drug. The above example is given as a mere illustration and this should not be construed in any manner to limit the scope of the present invention. The applicant's preferred Cephalosporin antibiotic - Polysaccharide complexes in the specification is based on the fact that Cephalosporin antibiotics such as Cefazolin Sodium, Ceftriaxone Sodium, Cefoperazone Sodium, Cefatoxime Sodium, Cefiiroxime Sodium, and a number of other Cephalosporin derivatives of various generations are therapeutically the more popular group of Beta-lactum antibiotics and have better activity profile and spectrum compared to other Beta-lactum antibiotics. Cephalosporin group also offers a significantly wider range of antibiotic products. 1 ' Focus on Cephalosporin antibiotics as a preferred antibiotic group for polysaccharide complexing is relevant and appropriate, but does not in any manner detract the applicability of the concept to other Beta-lactums through similar complexing of polysaccharides under the applicant's invention. FIELD OF INVENTION The present invention relates to a novel process for the preparation of Beta-lactam antibiotic-Polysaccharide complexes having improved therapeutic efficacy. The novel complex produced by the present process imparts an improvement in the therapeutic efficacy compared parent (known) Beta-lactam antibiotics, for example. Cephalosporin antibiotics. The present novel process results in complexes that facilitate optimisation in drug delivery with an objective to enhance the efficacy of the therapeutic moieties, with reduced toxicity. BACKGROUND OF THE INVENTION Beta-lactam antibiotic compounds are derived from 6-APA (6 amino penicillanic acid), 7-ADCA (7 amino desacetoxy cephalosporanic acid) or 7-ACA (7 amino cephalosporanic acid). Beta-lactam antibiotics are widely used for treatment of infections caused by both gram-positive and gram-negative organisms. Beta-lactam antibiotic acts through inhibition of cell wall synthesis by the inhibition of the peptido-glycan synthesis and teichoic acid metabolism of the organism. It also inhibits the cross-linking of N-acetyl muramic acid with N-acetyl glucosamine, thereby preventing the integrity of the bacterial cell wall. Among the commonly known Beta-lactam antibiotics which are used for wide range of bacterial infections. Cephalosporin antibiotics have emerged as a preferred group of antibiotics. Cephalosporin antibiotics have broad spectrum activity covering both gram-positive and gram-negative organisms. These are administered in both parenteral and oral routes depending on their physico-chemical characteristics, pharmaco-dynamic and pharmaco¬kinetic profile. The choice of antibiotics depends on the type of infection and the dose and route of administration based on the severity of the infection. The injectable cephalosporins are employed for systemic use for prompt action and bio¬availability of these dosage forms is found to be 100 per cent. The pharmaco-kinetic stuaies snow that injectable cephalosporins are plasma-bound by about 60 per cent and remaining portion of cephalopsorins, being highly water soluble gets excreted through urinary route which does not elicit any therapeutic response at the target site. In case of other Beta-lactams, the plasma-protein binding may range up to about 85 per cent depending on the molecule. Polysaccharides are carbohydrates derived from plant and microbial sources. Polysaccharides are used for many industrial applications. Beta-Iactam antibiotics (including Cephalosporins) being life-saving antibiotics, any invention which enhances the therapeutic efficacy of the products while at the same time reducing the toxicity is a highly desirable objective. Usually the patients affected by infections tend to have altered and weakened physiological systems. Also, generally, patients tend to have multiple pathological conditions which cannot bear the risk of high toxicity burden. In this context, if the invention achieves an attendant lower dosage of active moiety, it reduces the risk of higher dose exposure which otherwise would be inevitable with only the conventional moiety. Moreover, these antibiotics are costly. If a complex that utilises less amount of the active ingredient to achieve the same therapeutic efficacy is developed, it would significantly reduce the cost of treatment. Conventionally, Beta-Iactam antibiotics are synthesized from 6-APA, 7-ADCA and 7-ACA depending upon the molecule. More specifically, Cephalosporin antibiotics are synthesized from 7-ADCA or 7-ACA. The process of synthesis is such that the end product would be the active pharmaceutical ingredient of the required cephalosporin representing 100% of the active moiety. The applicant's invention encompasses the complexing of an active moiety such as Beta-Iactam antibiotics, with polysaccharide to obtain optimum usage of the therapeutic moiety. This process results in a novel lyophilised complex which provides efficacious, less toxic and more cost-effective drug delivery intended for treating infections in mammals, particularly humans. PRIOR ART There had been some earlier studies conducted for optimisation of anti-cancer drugs, namely Mitomycin and Methotrexate for the control of toxicity as the above drugs are highly toxic. This had been achieved through complexation involving polysaccharides. In the case of Mitomycin and Methotrexate the attachment of the polysaccharides to the molecule had been mediated through artificially synthesized spacer arm. Such synthesis is complex, time consuming and costly in nature since it requires the activation of the polysaccharide to link with this pre-synthesized spacer arm for the establishment of the final linkage with the active moiety viz.. Mitomycin and Methotrexate. However, complexation of Beta-lactam antibiotics with polysaccharides has so far not been reported. Beta-lactam antibiotics which treat bacterial infections fall altogether in a different class of compounds as compared to anticancer or any other therapeutic group of drugs. The disease conditions and treatment profile for which Beta-lactam antibiotics are intended are far more widely prevalent. There is no reported attempt so far made to optimise the efficacy of Beta-lactam antibiotic employing any polysaccharides. There has been no reported process disclosing the complexing of Beta-lactams, preferably cephalosporin injectibles, with polysaccharides in order to enhance the therapeutic efficacy of the basic molecule. The applicant believes that any process invention which optimises the efficacy of this antibiotic group has significant positive benefits for the society. OBJECTS OF THE INVENTION The main object of the invention is to provide a process for the production of novel composition containing Beta-lactam antibiotics complexed with polysaccharides. Still another object is to provide a process which is easy and quick to practice. Yet another object is to provide a process resulting in novel composition and useful for treatment of microbial infections at lower dosage. SUMMARY OF THE INVENTION Accordingly, the invention provides a process for the production of novel compositions containing Beta-lactam antibiotics, said process comprising the steps of reacting Beta-lactam ring containing antibiotics with polysaccharides in the presence of a solvent, and lyophilisation of the complex under vacuum. DETAILED DESCRIPTION OF THE INVENTION A process for the production of Beta-lactam antibiotic polysaccharide complex comprising the steps of: i) reacting a Beta-lactam ring containing antibiotic compound such as hereindescribed with a polysaccharide in the presence of a solvent at room temperature, i i) adding a buffer such as hereindescribed to adjust the pH, and i i i) recovering Beta-lactam antibiotic-polysaccharide complex from the solution as a dry solid by lyophilization at a temperature ranging between -40°C to 35°C. In accordance with the above and other objectives, the invention provides novel Beta-lactam complexes, said process comprising the steps of: i) reacting Beta-lactam ring containing antibiotics with polysaccharides in the presence of a solvent, and ii) lyophilising antibiotic-polysaccharides solution under vacuum to obtain antibiotic polysaccharides complex as a dry solid. In an embodiment, the Beta-lactam ring containing antibiotics are selected from the group comprising cephalosporins, cefazolin, cephradine, ceftriaxone, cefoperazone and cefotaxime. In another embodiment, the polysaccharides are selected from the group comprising Arabinose, Agarose, low molecular weight dextran, activated dextrans, insulin, chitin, chitosan, mannan, lichenin and their derivatives. In still another embodiment, the proportion of Beta-lactam compounds to polysaccharides ranges between 20 - 60%. In yet another embodiment, the proportion of polysaccharides in the composition ranges from 80 - 40%. In an embodiment, the reaction is effected a pH of about 4.5 to 7.5. The solvent is water. In yet another embodiment, the buffer added to the reaction mixture is sodium bicarbonate, sodium carbonate and triethanolamine. In another embodiment, the complex is obtained by freeze drying under temperature ranging between ^0°C to +35°C. In an embodiment the process results in the complexes which have enhanced in vitro activity as determined by minimum inhibitory concentration assays. In another embodiment, the process results in the complexes which have comparable in vivo activity to that of parent drug in animal models. In yet another embodiment, the complex is obtained through lyphilization. To discuss in detail, the applicants envisage that compounds having Beta-lactam ring such as cephalosporins are best suited as starting materials for the process. Such compounds may have 'Beta-lactam' ring as the central structure, or may be fiised with other ring systems, such as 5-member thiazolidine ring systems (-6APA derived products) and 6 member cephalosporin ring systems (-6ADCA/7-ACA derived products). Examples of Beta-lactam antibiotics include synthetic and semisynthetic penicillins, cephalosporins, and other beta lactam compounds or their derivatives. The Beta-lactam compounds best suited for the reaction are cephalosporins, it is preferred should be sterile. They can be selected for example, from the group comprising cefazolin. cettnaxone, cephradine, cefoperazone, cefotaxime and other similar molecules of various generations. The polysaccharides best suited for the reaction are those having a linear structure and being water soluble. Such polysaccharides tend to act as effective inert carriers and therefore, the best candidates for this reaction. Such polysaccharides may be selected from the group comprising Arabinose, Agarose, low molecular weight dextran (mol wt 5000), activated dextrans, inulin, chitin, chitosan, mannan, Uchenin and their derivatives. While most polysaccharides would lend to initiation of the process, some may require activation. This is achieved by addition of activating agents such as cyanogen bromide etc. The reaction is effected in the presence of inert solvents, such as water. The most appropriate pH recommended for conducting the reaction is in the neighbourhood of alkaline pH, for example 4.5 to 7.5. For advantageous and smooth conduct of the reaction, a pH range of 5.5 to 6.5 is recommended. As known in the art, appropriate buffers may be added to reaction mixture to adjust the pH to the range suggested above. Typically, sodium bicarbonate is added. Other buffers such as sodium carbonate and triethanolamine may also be considered. The composition that results by the process of the invention is a Beta-lactam -polysaccharide complex formed by interaction between the compounds based on non-covalent and non-ionic bonds. This complex so formed is obtained through lyophilization. In the present process, the antibiotic-polysaccharide complex solution so obtained by appropriate proportion, is to be filtered. The temperature selected for lyophilisation vary from -40°C to +35°C under vacuum conditions. The final product appears as a porous solid material. The present invention provides a process for producing antibiotic complexes comprising an effective therapeutic amount of Beta-lactam and polysaccharide as a complexing ligand in each complex. The proportion of the antibiotic polysaccharide ratio may vary from 20 : 80 to 60 : 40 by wt. depending on the antibiotic chosen. The invention for which this patent application is being made involves complexation of Beta-lactam, preferably cephalosporins, with appropriate polysaccharides to achieve optimum drug delivery for therapeutic efficacy and toxicity control. The applicant's prefer polysaccharides, having linear structure, since these are water soluble and good carriers of drug. In addition, the polysaccharide carriers are pharmacologically and toxicologically inert and hence, these carriers are suitable to impart properties to link the drugs through the ligand attachments involving the polysaccharides for optimised drug delivery. The polysaccharides should be provided with polar hydroxyl groups, so that they can attach with the active moiety as a part of the complexation process through hydrogen bonding. In other words, the present process involves altogether a different chemical reaction which is much simpler and economically viable. The solubility of complex involving cephalosporins and polysaccharides as injectibles makes it appropriate also for parenteral application. The polysaccharides are rapidly excreted in urine without accumulation in the tissues. The Cephalosporins for which this approach is applicable comprise Cefazolin Sodium, Ceftriaxone Sodium, Cefoperazone Sodium, Cefotaxime Sodium, Cefuroxime Sodium, cephradine and a number of other cephalosporin derivatives of various generations. In the case of Cefazolin complex which is being cited here as an example, Cefazolin can be present in the range of 55 to 65% and the polysaccharide constituting the balance. which is achieved in stoichiometric proportions in aqueous solution at a definite pH through aseptic lyophilisation process. The proportion of Beta-lactam antibiotics that may be present in the composition in comparison to polysaccharides may range between 20 - 60%. The portion of polysaccharides, will vary fi^om 80 - 40%. The solvent used in the process is water. The temperatures selected for this lyophilisation or fi-eeze drying are about -40°C to 35°C. The products obtained by the process of the invention have been subjected to X-ray crystallograph and DSC studies. The results of the studies are discussed herein below and illustrated by the following figures wherein : Fig. 1 : represents the powder diffraction pattern of pure polymer Fig.2 : represents the powder diffraction pattern of Ceftriaxone sodium Fig. 3 : represents the powder diffraction pattern of Ceftriaxone complex Fig.4 : represents Multi plot of polymer, ceftriaxone standard and ceftriaxone complex Fig.5 : represents the powder diffraction pattern of Cefazolin sodium Fig.6 : represents the powder diffraction pattern Cefazolin complex Fig. 7 : represents the Muhi plot of polymer, cefazolin standard and cefazolin complex Fig. 8 : represents the powder diffraction pattern of Cephradine complex Fig. 9 : represents the powder diffraction pattern of Cephradine standard Fig. 10: represents the Multi plot of polymer, Cephradine standard and Cephradine complex Fig. 11: represents the DSC thermogram of pure polymer Fig. 12: represents the DSC thermogram of pure Cefazolin sodium Fig. 13: represents the DSC thermogram Cefazolin complex Fig. 14: represents the DSC thermogram of pure Ceftriaxone sodium Fig. 15: represents the DSC thermogram of Ceftriaxone complex Fig. 16: represents the DSC thermogram of pure Cephradine Fig. 17: represents the DSC thermogram of Cephradine complex This invention optimises the proportion of active moiety which is required for therapeutic response for a particular indication leading to toxicity reduction and also economy in treatment cost. By virtue of its applicability to a range of sterile Cephalosporins preferably, and other Beta-lactams, administered in the parenteral route, the invention has potential to benefit a huge population of patients requiring parenteral Cephalosporin and other Beta-lactam antibiotic treatment. Therefore, the present invention provides a process for producing novel Beta-lactam antibiotic-Polysaccharide complexes and preferably. Sterile Cephalosporin-polysaccharide complexes having a lower dosage of active moiety, resulting in equivalent pharmacological and microbiological response compared to parent drug leading to lesser toxicity and reduced cost of treatment for the patients. Focus on Cephalosporin antibiotics as a preferred antibiotic group for polysaccharide complexing is relevant and appropriate, but does not in any manner detract the applicability of the concept to other Beta-lactam through similar complexing of polysaccharides under the applicant's invention. The Applicants have conducted in-vivo test protocols which indicate that the novel composition produced by the process of the invention may be formulated in various physical forms and adapted for oral use, or as injectables. This invention optimises the proportion of active moiety (antibiotic) which is required for equivalent therapeutic response compared to parent drug for a particular indication leading to toxicity reduction and also economy in treatment cost. By virtue of its applicability to a range of sterile Cephalosporins, and other Beta-lactams, administered in the parenteral route, the invention has potential to benefit a huge population of patients requiring parenteral Cephalosporin and other Beta-lactam antibiotic treatment. Therefore, the present invention provides a process for producing novel Beta-lactam antibiotic-Polysaccharide complexes and preferably, Sterile Cephalosporin-Polysaccharide complexes, having a lower dosage of active moiety, resulting in equivalent phamacological and microbiological response compared to parent drug leading to lesser toxicity and reduced cost of treatment for the patients. It should be understood that the scope of the present invention is not limited to the specific compositions or methods as described herein and that any composition or method having steps equivalent to those described herein fall within the scope of the present invention. Preparation routes of the composition of the invention and the method for combating microbial infections by the administration of the composition is merely exemplary so as to enable one of ordinary skill in the art to use the teachings of the invention, such as the composition of the invention and use it according to the described process and its equivalents. It vAll also be understood that although the form of the invention shown and described herein constitutes preferred embodiments of the invention, it is not intended to illustrate all possible forms of the invention. The words used are words of description rather than of limitation. Various changes and variations may be made to the present invention without departing from the spirit and scope of the invention. EXAMPLE 1 (preparation of the composition) To produce 1 gm of Cefazolin - Polysaccharide complex, 0.6 gms of Cefazolin and 0.4 gms of Polysaccharide are mixed with water under the pH condition between 5.5 to 6.5. Sodium bicarbonate is added to adjust the pH. The polysaccharide complex solution thus obtained is fihered through 0.2 micron fiher and transferred into a sterilized vial. Finally, the vial containing the solution is aseptically lyophilised under -40°C, -10°C, +10°C and +35°C under a vacuum of about 2.2 x 10'^ mbar. EXAMPLE 2 To produce 1 gm of Ceftriaxone- Polysaccharide complex, 0.6 gms of Ceftriaxone and 0.4 gms of Polysaccharide are mixed with water under the pH condition between 5.0 to 6.5. Sodium carbonate is added to adjust the pH. The polysaccharide complex solution thus obtained is filtered through 0.2 micron fdter and transferred into a sterilized vial. Finally, the vial containing the solution is aseptically lyophilised under -40°C, -10°C, +10°C and +35°C under a vacuum of about 2.2 x 10"^ mbar. EXAMPLE 3 To produce 1 gm of Cephradine - Polysaccharide complex, 0.6 gms of Cephradine and 0.4 gms of Polysaccharide are mixed with water under the pH condition between 5.5 to 6.5. Sodium bicarbonate is added to adjust the pH. The polysaccharide complex solution thus obtained is filtered through 0.2 micron fiher and transferred into a sterilized vial. Finally, the vial containing the solution is aseptically lyophilised under -40°C, -lO^C, +10°C and +35°C under a vacuum of about 2.2 x 10'^ mbar. EXAMPLE 4 The produce 1 gm of Cefuroxime - Polysaccharide complex, 0.6 gms of Cefiiroxime and 0.4 gms of Polysaccharide are mixed with water under the pH condition between 5.5 to 6.5. Sodium bicarbonate is added to adjust the pH. The polysaccharide complex solution thus obtmned is filtered through 0.2 micron filter and transferred into a sterilized vial. Finally, the vial containing the solution is aseptically lyophilised under -40°C, -10°C, +10°C and +35°C under a vacuum of about 2.2 x 10'^ mbar. EXAMPLES The produce 1 gm of Cefoperazone - Polysaccharide complex, 0.6 gms of Cefoperazone and 0.4 gms of Polysaccharide are mixed with water under the pH condition between 5.5 to 6.5. Sodium bicarbonate is added to adjust the pH. The polysaccharide complex solution thus obtained is filtered through 0.2 micron fiher and transferred into a sterilized vial. Finally, the vial containing the solution is aseptically lyophilised under -40*'C, -10°C, +10°C and +35°C under a vacuum of about 2.2 x 10'^ mbar. As illustrated above, the method can be used for preparation of different types compositions within the teachings of the invention and containing antibiotic - polysaccharide complexes. The process involves addition of relevant antibiotic and polysaccharide in stoichiometric proportions in aqueous solution at a definite pH. The antibiotic complexes so obtained are aseptically lyophilized at selected temperatures and vacuum. EXAMPLE 6 (X-ray Powder DifTraction Studies) X-ray Powder Diffraction Studies were performed using Shimadzu XRD system. The powder diffraction pattern reveals that the powder diffraction pattern of the antibiotic-polymer complex manufactured by this technology is different fi-om that of pure parent antibiotic and that of pure. The major difference in the 2 theta values of the 3 major peaks of the polymer, parent antibiotic and that of the complex can be summarized as follows: EXAMPLE 7 [Differential Scanning Calorimetry (DSC)] DSC is a technique of thermal analysis, whereby the energy added to or subtracted from the sample, so as to maintain the sample and reference at the same temperature, is recorded. The output is a plot of heat applied to the same (MW) Vs temperature. DSC studies were done using Mettler Toledo DSC system. DSC thermograms of pure polymer, parent antibiotic and the antibiotic-polymer complex are shown in Figures 11 to 18. The following were observed :-(i) Cefazolin DSC thermograms of Cefazolin shows sharp endothermic peak at 95.8°C and an exothermic peak at 199. TC. It can be observed from the DSC thermogram of Polymer, Cefazolin and Cefazolin Complex that an additional exothermic peak has appeared at about 240°C in the complex which is not there in case of polymer. Also the narrow peak at 95°C does not appear instead a broad peak can be observed in the range of 90 - 100°C. This suggest changes in thermal behaviour of polymer and Cefazolin complexation. (ii) Ceftriaxone DSC thermograms of standard Ceftriaxone reveal presence of a well defined narrow exothermic peak at about 262°C which is not seen in case of Ceftriaxone complex. Further the DSC thermogram of complex show 2 additional exothermic peaks in the range of 260°C to 295°C which are not present either in polymer or standard Ceftriaxone. The changes in the thermal nature of the complex from that of standard indicate formation of complex between the antibiotic and polymer. (iii) Cephradine DSC thermograms of standard Cephradine show narrow exothermic peaks at about 206°C, 257°C and 284^ and a broad endothermic peak at about 116°C. Appearance of broad peaks at about 189°C, 221'C and 250°C in DSC thermogram of complex suggest a change in the thermal behaviour, which signifies formation of complex between Cephradine and the polymer on lyophilisation. These confirm that there is a formation of a complex between the antibiotic-polymer prepared using the lyophilisation technology. EXAMPLE 8 : Testing the antimicrobial effect of tlie composition The susceptibility of a microorganism to an antimicrobial agent is ascertained by dilution tests. In the tube dilution method, specific amounts of the antibiotic, prepared in decreasing concentrations in broth by serial dilution technique, are inoculated with a broth culture of the bacterium to be tested. The susceptibility of any organism is determined, after a suitable period of incubation, by macroscopic observation of the presence or absence of growth in the varying concentration of the antimicrobial agent. The end point is a measure of the bacteriostatic effect of the agent on the bacterium and is commonly referred to as the Minimum Inhibitory Concentration (MIC). The amount of drug in the tube containing the least amount of the antibiotic with no observable growth is the MIC. The tube dilution method is considered to the most accurate for determination of susceptibility to measured amounts of an antimicrobial agent. Test substances used were carried out on a number of microorganisms. 1. Cefazolin - Standard 2. Cefazolin- Complex 3. Ceftriaxone - Standard 4. Ceftriaxone - Complex 5. Cephradine - Standard 6. Cephradine - Complex The protocol used, the results and conclusions are discussed herein below. Minimum Inhibitory Concentration values for Cefazolin complex and Ceftriaxone complex have been determined and compared with those of the respective parent drugs. The data shows a significant increase in the activity of the antibiotic in presence of the polysaccharide as complex. The above MICs are lower than the reported MICs of Cefazolin towards the above organisms (i.e., 16 meg and less / ml). The above Table clearly establishes the fact that the complexed Cefazolin-Polysaccharide antibiotic (60 : 40 ratio) provides equivalent or better response on the microorganisms cited above compared to uncomplexed Cefazolin antibiotic (100%). Staphyloccus aureus and Proteus mirabilis were the selected microorganisms against a designed burn caused infection. The rate of healing for the complexed drug confirms to the desired response in equivalent pattern to that of parent (known) drug. We claim: 1. A process for preparing Beta-lactam antibiotic polysaccharide complex comprising the steps of: i) reacting a Beta-lactam ring containing antibiotic compound such as hereindescribed with a polysaccharide in the presence of a solvent at room temperature, ii) adding a buffer such as hereindescribed to adjust the pH, and iii) recovering Beta-lactam antibiotic-polysaccharide complex from the solution as a dry solid by lyophilization at a temperature ranging between -40°C to 35°C. 2. A process as claimed in claim 1 wherein the Beta-lactam ring containing antibiotic compound is selected from the group consisting of cephalosporins, cefazolin, cephradine, ceftriaxone, cefoperazone and cefotaxime. 3. A process as claimed in claim 1 wherein the polysaccharide is selected from the group consisting of arabinose, agarose, low molecular weight dextran, activated dextrans, insulin, chitin, chitosan, mannan and lichenin. 4. A process as claimed in claim 1 wherein the proportion of Beta-lactam ring containing antibiotic compound to the polysaccharide ranges between 20 to 60%. 5. A process as claimed in claim 1 wherein the proportion of polysaccharides in the antibiotic-polysaccharide complex ranges between 40 to 80%. 6. A process as claimed in claim 1 wherein the solvent is water. 7. A process as claimed in claim 1 wherein the buffer is selected from sodium bicarbonate, sodium carbonate and triethanolamine. 8. A process as claimed in claim 1 wherein the reaction is effected at a pH of about 4.5 to 7.5 A process for the production of Beta-lactam antibiotic polysaccharide complex substantially as hereindescribed with reference to the examples and the accompanying drawings.

Full Text

FIELD OF INVENTION
The present invention relates to a novel process for the preparation of Beta-lactum antibiotic-Polysaccharide complexes having improved therapeutic efficacy. The novel complex produced by the present process imparts an improvement in the therapeutic efficacy compared to uncomplexed Beta-lactum antibiotics, for example, Cephalosporin antibiotics. The present novel process results in complexes that facilitate optimisation in drug delivery with an objective to enhance the efficacy of the therapeutic moieties, with reduced toxicity. Preferably, the novel process is especially suitable to produce injectable Beta-lactum antibiotics complexes involving polysaccharides.
BACKGROUND OF THE INVENTION
Beta-lactum antibiotic compounds are derived from 6-APA (6 amino penicillanic acid), 7-ADCA (7 amino desacetoxy cephalosporanic acid) or 7-ACA (7 amino cephalosporanic acid). Beta-lactum antibiotics are widely used for treatment of infections caused by both gram-positive and gram-negative organisms.
Beta-lactum antibiotic acts through inhibition of cell wall synthesis by the inhibition of the peptido-glycan synthesis and teichoic acid metabolism of the organism. It also inhibits the cross-linking of N-acetyl muramic acid with N-acetyl glucosamine, thereby preventing the integrity of the bacterial cell wall. Among the commonly known Beta-lactum antibiotics which are used for wide range of bacterial infections. Cephalosporin antibiotics have emerged as a preferred group of antibiotics.

Cephalosporin antibiotics have broad spectrum activity covering both gram-positive and gram-negative organisms. These are administered in both parenteral and oral routes depending on their physico-chemical characteristics, pharmaco¬dynamic and pharmaco-kinetic profile. The choice of antibiotics depends on the type of infection and the dose and route of administration based on the severity of the infection.
The injectable cephalosporins are employed for systemic use for prompt action and bio-availability of these dosage forms is found to be 100 percent. The pharmaco-kinetic studies show that injectable cephalosporins are plasma-bound by about 60 percent and remaining portion of cephalopsorins, being highly water soluble gets excreted through urinary route which does not elicit any therapeutic response at the target site. In case of other Beta-lactums, the plasma-protein binding may range upto about 85 percent depending on the molecule.
Polysaccharides are carbohydrates derived from plant and microbial sources. Polysaccharides are used for many industrial applications.
Beta-lactum antibiotics (including Cephalosporins) being life-saving antibiotics, any invention which enhances the therapeutic efficacy of the products while at the same time reducing the toxicity is a highly desirable objective. Usually the patients affected by infections tend to have altered and weakened physiological systems. Also, generally, patients tend to have multiple pathological conditions which cannot bear the risk of high toxicity burden. In this context, if the invention achieves an attendant lower dosage of active moiety, it reduces the risk of higher dose exposure which otherwise would be inevitable with onlv the conventional moiety.

Moreover, these antibiotics are costly. If the novel complex utilises less amount of the active ingredient to achieve the same therapeutic efficacy, it would significantly reduce the cost of treatment.
Conventionally, Beta-lactum antibiotics are synthesised from 6-APA, 7-ADCA and 7-ACA depending upon the molecule. More specifically, Cephalosporin antibiotics are synthesised from 7-ADCA or 7-ACA. The process of synthesis is such that the end product would be the active pharmaceutical ingredient of the required cephalosporin representing 100% of the active moiety.
The applicant's invention encompasses the complexing of the active moiety, preferably injectable cephalosporins, with polysaccharide to obtain optimum usage of the therapeutic moiety. This process results in a novel lyophilised complex which provides efficacious, less toxic and more cost-effective drug delivery intended for freating infections in mammals, particularly humans.
PRIOR ART
There had been some earlier studies conducted for optimisation of anti-cancer drugs, namely Mitomycin and Methotrexate for the confrol of toxicity as the above drugs are highly toxic. This had been achieved through complexation involving polysaccharides. In the case of Mitomycin and Methotrexate the attachment of the polysaccharides to the molecule had been mediated through artificially synthesised spacer arm. Such synthesis is complex, time consuming and costly in nature since it requires the activation of the polysaccharide to link with this pre-synthesised spacer arm for the establishment of the fmal linkage with the active moiety viz.. Mitomycin and Methotrexate.

However, complexation of Beta-lactum antibiotics with polysaccharides has so far not been reported. Beta-lactum antibiotics which treat bacterial infections fall altogether in a different class of compounds compared to anticancer or any other therapeutic group of drugs. The disease conditions and treatment profile for which Beta-lactum antibotics are intended are far more widely prevalent. There is no reported attempt so far made to optimise the efficacy of Beta-lactum antibiotic employing any polysaccharides.
There has been no reported process disclosing the complexing of Beta-lactums, preferably cephalosporin injectables, with polysaccharides in order to enhance the therapeutic efficacy of the basic molecule.
The applicant believes that any process invention which optimises the efficacy of this antibiotic group has significant positive benefits for the society.
DESCRIPTION OF THE INVENTION
The present .invention provides a process for producing antibiotic complexes comprising an effective therapeutic amount of Beta-lactum and polysaccharide as a complexing ligand in each complex. The therapeutic quantum of Beta-lactum may vary fi-om 50 to 75% by weight in the complex depending on the active therapeutic moiety.
The invention for which this patent application is being made involves complexation of Beta-lactums, preferably sterile cephalosporins, with appropriate polysaccharides to achieve optimum drug delivery for therapeutic efficacy and

toxicity control. The preferred polysaccharides that can be employed are Arabinose, Agarose, Low molecular weight dextran, Activated clinical dextrans, Inulin, Chitin, Chitosan, Mannan, Lichenin, and their derivatives / variants.
The applicant's preferred polysaccharides, having linear structure, are water soluble and good carriers of drug. In addition, the polysaccharide carriers are pharmacologically and toxicologically inert and hence, these carriers are suitable to impart properties to link the drugs through the ligand attachments involving the polysaccharides for optimised drug delivery. The polysaccharides should be provided with polar hydroxyl groups, so that they can attach with the active moiety as a part of the complexation process through hydrogen bonding. In other words, the present process involves altogether a different chemical reaction which is much simpler and economically viable.
The solubility of complex involving cephalosporins and p>olysaccharides as injectables makes it appropriate for parenteral application. The polysaccharides are rapidly excreted in urine without accumulation in the tissues.
The sterile Cephalosporins for which this approach is applicable comprise Cefazolin Sodium, Ceftriaxone Sodium, Cefoperazone Sodium, Cefotaxime Sodium, Cefliroxime Sodium and a number of other cephalosporin derivatives of various generations.
In the case of Cefazolin complex which is being cited here as an example, Cefazolin can be present in the range of 55 to 65% and the polysaccharide constitutmg the balance, which is achieved in stoichiometric proportions in

^
aqueous solution at a definite pH through aseptic lyophilisation process. The preferred proportion of Cefazolin to the polysaccharide is 60:40.
hi the present process, the antibiotic polysaccharide complex solution so obtained is to be filtered through 0.2 micron filter.
The temperatures selected for this lyophilisation are -40° C, -10° C, + 10° C and +35° C under a vacuum of about 2.2 x 10'^ mbar.
This invention optimises the proportion of active moiety which is required for therapeutic response for a particular indication leading to toxicity reduction and also economy in treatment cost. By virtue of its applicability to a range of sterile Cephalosporins preferably, and other Beta-Iactums, administered in the parenteral route, the invention has potential to benefit a huge population of patients requiring parenteral Cephalosporin and other Beta-lactum antibiotic treatment.
Therefore, the present invention provides a process for producing novel Beta-lactum antibiotic-Polysaccharide complexes and preferably. Sterile Cephalosporin-Polysaccharide complexes having a lower dosage of active moiety, resulting in equal phamacological and microbiological response compared to uncomplexed drug leading to lesser toxicity and reduced cost of treatment for the patients.
The present invention produces a synergistic composition having enhanced and surprising properties which has not been envisaged.

Some embodiments of the invention also involve addition of suitable pH adjustment agents such as Sodium bicarbonate, activation of the polysaccharides for complexation process through cyanogen bromide treatment.
EXAMPLE
To produce 1 gm of Cefazolin - Polysaccharide complex, 0.6 gms of Cefazolin and 0.4 gms of Polysaccharide are mixed with water under the pH condition between 5.5 to 6.5. Sodium bicarbonate is added to adjust the pH. The polysaccharide complex solution thus obtained is filtered through 0.2micron filter and transferred into a sterilized vial. Finally, the vial containing the solution is aseptically lyophilised under ^0°C, -10°C, + 10" C and +35° C under a vacuum ofabout2.2xl0'^mbar.
CEFAZOLIN - POLYSACCHARIDE COMPLEX UNDER MINIMUM INHIBITORY CONCENTRATION (MIC) STUDIES

The above MICs are lower than the reported MICs of Cefazolin towards the above organisms (i.e., 16 meg and less / ml).

The above Table clearly establishes the fact that the complexed Cefazolin-Polysaccharide antibiotic ( 60 : 40 ratio) provides equivalent or better response on the microorganisms cited above compared to uncomplexed Cefazolin antibiotic (100%).
Animal studies were conducted, for example, on Swiss Albino mice. Thirty mice were taken for evaluation with the following treatment pattern : ten with complexed drug (Cefazolin complex), ten with uncomplexed drug (Cefazolin) and ten untreated.
Staph.aureus and Proteus Mirabilis were the selected microorganisms against a designed bum-caused infection. The rate of healing for the complexed drug confirms to the desired response in equivalent pattem to that of uncomplexed drug.
The above example is given as a mere illustration and this should not be construed in any manner to limit the scope of the present invention. The applicant's preferred Cephalosporin antibiotic - Polysaccharide complexes in the specification is based on the fact that Cephalosporin antibiotics such as Cefazolin Sodium, Ceftriaxone Sodium, Cefoperazone Sodium, Cefatoxime Sodium, Cefiiroxime Sodium, and a number of other Cephalosporin derivatives of various generations are therapeutically the more popular group of Beta-lactum antibiotics and have better activity profile and spectrum compared to other Beta-lactum antibiotics. Cephalosporin group also offers a significantly wider range of antibiotic products.

1 '
Focus on Cephalosporin antibiotics as a preferred antibiotic group for polysaccharide complexing is relevant and appropriate, but does not in any manner detract the applicability of the concept to other Beta-lactums through similar complexing of polysaccharides under the applicant's invention.

FIELD OF INVENTION
The present invention relates to a novel process for the preparation of Beta-lactam antibiotic-Polysaccharide complexes having improved therapeutic efficacy. The novel complex produced by the present process imparts an improvement in the therapeutic efficacy compared parent (known) Beta-lactam antibiotics, for example. Cephalosporin antibiotics. The present novel process results in complexes that facilitate optimisation in drug delivery with an objective to enhance the efficacy of the therapeutic moieties, with reduced toxicity.
BACKGROUND OF THE INVENTION
Beta-lactam antibiotic compounds are derived from 6-APA (6 amino penicillanic acid), 7-ADCA (7 amino desacetoxy cephalosporanic acid) or 7-ACA (7 amino cephalosporanic acid). Beta-lactam antibiotics are widely used for treatment of infections caused by both gram-positive and gram-negative organisms.
Beta-lactam antibiotic acts through inhibition of cell wall synthesis by the inhibition of the peptido-glycan synthesis and teichoic acid metabolism of the organism. It also inhibits the cross-linking of N-acetyl muramic acid with N-acetyl glucosamine, thereby preventing the integrity of the bacterial cell wall. Among the commonly known Beta-lactam antibiotics which are used for wide range of bacterial infections. Cephalosporin antibiotics have emerged as a preferred group of antibiotics.
Cephalosporin antibiotics have broad spectrum activity covering both gram-positive and gram-negative organisms. These are administered in both parenteral and oral routes depending on their physico-chemical characteristics, pharmaco-dynamic and pharmaco¬kinetic profile. The choice of antibiotics depends on the type of infection and the dose and route of administration based on the severity of the infection.
The injectable cephalosporins are employed for systemic use for prompt action and bio¬availability of these dosage forms is found to be 100 per cent. The pharmaco-kinetic

stuaies snow that injectable cephalosporins are plasma-bound by about 60 per cent and remaining portion of cephalopsorins, being highly water soluble gets excreted through urinary route which does not elicit any therapeutic response at the target site. In case of other Beta-lactams, the plasma-protein binding may range up to about 85 per cent depending on the molecule.
Polysaccharides are carbohydrates derived from plant and microbial sources. Polysaccharides are used for many industrial applications.
Beta-Iactam antibiotics (including Cephalosporins) being life-saving antibiotics, any invention which enhances the therapeutic efficacy of the products while at the same time reducing the toxicity is a highly desirable objective. Usually the patients affected by infections tend to have altered and weakened physiological systems. Also, generally, patients tend to have multiple pathological conditions which cannot bear the risk of high toxicity burden. In this context, if the invention achieves an attendant lower dosage of active moiety, it reduces the risk of higher dose exposure which otherwise would be inevitable with only the conventional moiety.
Moreover, these antibiotics are costly. If a complex that utilises less amount of the active ingredient to achieve the same therapeutic efficacy is developed, it would significantly reduce the cost of treatment.
Conventionally, Beta-Iactam antibiotics are synthesized from 6-APA, 7-ADCA and 7-ACA depending upon the molecule. More specifically, Cephalosporin antibiotics are synthesized from 7-ADCA or 7-ACA. The process of synthesis is such that the end product would be the active pharmaceutical ingredient of the required cephalosporin representing 100% of the active moiety.
The applicant's invention encompasses the complexing of an active moiety such as Beta-Iactam antibiotics, with polysaccharide to obtain optimum usage of the therapeutic moiety. This process results in a novel lyophilised complex which provides efficacious,

less toxic and more cost-effective drug delivery intended for treating infections in mammals, particularly humans.
PRIOR ART
There had been some earlier studies conducted for optimisation of anti-cancer drugs, namely Mitomycin and Methotrexate for the control of toxicity as the above drugs are highly toxic. This had been achieved through complexation involving polysaccharides. In the case of Mitomycin and Methotrexate the attachment of the polysaccharides to the molecule had been mediated through artificially synthesized spacer arm. Such synthesis is complex, time consuming and costly in nature since it requires the activation of the polysaccharide to link with this pre-synthesized spacer arm for the establishment of the final linkage with the active moiety viz.. Mitomycin and Methotrexate.
However, complexation of Beta-lactam antibiotics with polysaccharides has so far not been reported. Beta-lactam antibiotics which treat bacterial infections fall altogether in a different class of compounds as compared to anticancer or any other therapeutic group of drugs. The disease conditions and treatment profile for which Beta-lactam antibiotics are intended are far more widely prevalent. There is no reported attempt so far made to optimise the efficacy of Beta-lactam antibiotic employing any polysaccharides.
There has been no reported process disclosing the complexing of Beta-lactams, preferably cephalosporin injectibles, with polysaccharides in order to enhance the therapeutic efficacy of the basic molecule.
The applicant believes that any process invention which optimises the efficacy of this antibiotic group has significant positive benefits for the society.
OBJECTS OF THE INVENTION
The main object of the invention is to provide a process for the production of novel composition containing Beta-lactam antibiotics complexed with polysaccharides.

Still another object is to provide a process which is easy and quick to practice.
Yet another object is to provide a process resulting in novel composition and useful for treatment of microbial infections at lower dosage.
SUMMARY OF THE INVENTION
Accordingly, the invention provides a process for the production of novel compositions containing Beta-lactam antibiotics, said process comprising the steps of reacting Beta-lactam ring containing antibiotics with polysaccharides in the presence of a solvent, and lyophilisation of the complex under vacuum. DETAILED DESCRIPTION OF THE INVENTION
A process for the production of Beta-lactam antibiotic polysaccharide complex comprising the steps of: i) reacting a Beta-lactam ring containing antibiotic compound such as hereindescribed with a
polysaccharide in the presence of a solvent at room temperature, i i) adding a buffer such as hereindescribed to adjust the pH, and i i i) recovering Beta-lactam antibiotic-polysaccharide complex from the
solution as a dry solid by lyophilization at a temperature ranging between -40°C to 35°C.
In accordance with the above and other objectives, the invention provides novel Beta-lactam
complexes, said process comprising the steps of:
i) reacting Beta-lactam ring containing antibiotics with polysaccharides in the presence of a
solvent, and
ii) lyophilising antibiotic-polysaccharides solution under vacuum to obtain antibiotic
polysaccharides complex as a dry solid.
In an embodiment, the Beta-lactam ring containing antibiotics are selected from the group comprising cephalosporins, cefazolin, cephradine, ceftriaxone, cefoperazone and cefotaxime.
In another embodiment, the polysaccharides are selected from the group comprising Arabinose, Agarose, low molecular weight dextran, activated dextrans, insulin, chitin, chitosan, mannan, lichenin and their derivatives.
In still another embodiment, the proportion of Beta-lactam compounds to polysaccharides ranges between 20 - 60%.

In yet another embodiment, the proportion of polysaccharides in the composition ranges from 80 - 40%.
In an embodiment, the reaction is effected a pH of about 4.5 to 7.5.
The solvent is water.
In yet another embodiment, the buffer added to the reaction mixture is sodium bicarbonate, sodium carbonate and triethanolamine.
In another embodiment, the complex is obtained by freeze drying under temperature ranging between ^0°C to +35°C.
In an embodiment the process results in the complexes which have enhanced in vitro activity as determined by minimum inhibitory concentration assays.
In another embodiment, the process results in the complexes which have comparable in vivo activity to that of parent drug in animal models.
In yet another embodiment, the complex is obtained through lyphilization.
To discuss in detail, the applicants envisage that compounds having Beta-lactam ring such as cephalosporins are best suited as starting materials for the process. Such compounds may have 'Beta-lactam' ring as the central structure, or may be fiised with other ring systems, such as 5-member thiazolidine ring systems (-6APA derived products) and 6 member cephalosporin ring systems (-6ADCA/7-ACA derived products). Examples of Beta-lactam antibiotics include synthetic and semisynthetic penicillins, cephalosporins, and other beta lactam compounds or their derivatives. The Beta-lactam compounds best suited for the reaction are cephalosporins, it is preferred should be sterile. They can be selected for example, from the group comprising cefazolin.

cettnaxone, cephradine, cefoperazone, cefotaxime and other similar molecules of various generations.
The polysaccharides best suited for the reaction are those having a linear structure and being water soluble. Such polysaccharides tend to act as effective inert carriers and therefore, the best candidates for this reaction. Such polysaccharides may be selected from the group comprising Arabinose, Agarose, low molecular weight dextran (mol wt 5000), activated dextrans, inulin, chitin, chitosan, mannan, Uchenin and their derivatives. While most polysaccharides would lend to initiation of the process, some may require activation. This is achieved by addition of activating agents such as cyanogen bromide etc.
The reaction is effected in the presence of inert solvents, such as water. The most appropriate pH recommended for conducting the reaction is in the neighbourhood of alkaline pH, for example 4.5 to 7.5. For advantageous and smooth conduct of the reaction, a pH range of 5.5 to 6.5 is recommended. As known in the art, appropriate buffers may be added to reaction mixture to adjust the pH to the range suggested above. Typically, sodium bicarbonate is added. Other buffers such as sodium carbonate and triethanolamine may also be considered.
The composition that results by the process of the invention is a Beta-lactam -polysaccharide complex formed by interaction between the compounds based on non-covalent and non-ionic bonds. This complex so formed is obtained through lyophilization.
In the present process, the antibiotic-polysaccharide complex solution so obtained by appropriate proportion, is to be filtered.
The temperature selected for lyophilisation vary from -40°C to +35°C under vacuum conditions. The final product appears as a porous solid material.

The present invention provides a process for producing antibiotic complexes comprising an effective therapeutic amount of Beta-lactam and polysaccharide as a complexing ligand in each complex. The proportion of the antibiotic polysaccharide ratio may vary from 20 : 80 to 60 : 40 by wt. depending on the antibiotic chosen.
The invention for which this patent application is being made involves complexation of Beta-lactam, preferably cephalosporins, with appropriate polysaccharides to achieve optimum drug delivery for therapeutic efficacy and toxicity control.
The applicant's prefer polysaccharides, having linear structure, since these are water soluble and good carriers of drug. In addition, the polysaccharide carriers are pharmacologically and toxicologically inert and hence, these carriers are suitable to impart properties to link the drugs through the ligand attachments involving the polysaccharides for optimised drug delivery. The polysaccharides should be provided with polar hydroxyl groups, so that they can attach with the active moiety as a part of the complexation process through hydrogen bonding. In other words, the present process involves altogether a different chemical reaction which is much simpler and economically viable.
The solubility of complex involving cephalosporins and polysaccharides as injectibles makes it appropriate also for parenteral application. The polysaccharides are rapidly excreted in urine without accumulation in the tissues.
The Cephalosporins for which this approach is applicable comprise Cefazolin Sodium, Ceftriaxone Sodium, Cefoperazone Sodium, Cefotaxime Sodium, Cefuroxime Sodium, cephradine and a number of other cephalosporin derivatives of various generations.
In the case of Cefazolin complex which is being cited here as an example, Cefazolin can be present in the range of 55 to 65% and the polysaccharide constituting the balance.

which is achieved in stoichiometric proportions in aqueous solution at a definite pH through aseptic lyophilisation process.
The proportion of Beta-lactam antibiotics that may be present in the composition in comparison to polysaccharides may range between 20 - 60%. The portion of polysaccharides, will vary fi^om 80 - 40%. The solvent used in the process is water.
The temperatures selected for this lyophilisation or fi-eeze drying are about -40°C to 35°C.
The products obtained by the process of the invention have been subjected to X-ray crystallograph and DSC studies. The results of the studies are discussed herein below and illustrated by the following figures wherein :
Fig. 1 : represents the powder diffraction pattern of pure polymer
Fig.2 : represents the powder diffraction pattern of Ceftriaxone sodium
Fig. 3 : represents the powder diffraction pattern of Ceftriaxone complex
Fig.4 : represents Multi plot of polymer, ceftriaxone standard and ceftriaxone complex
Fig.5 : represents the powder diffraction pattern of Cefazolin sodium
Fig.6 : represents the powder diffraction pattern Cefazolin complex
Fig. 7 : represents the Muhi plot of polymer, cefazolin standard and cefazolin complex
Fig. 8 : represents the powder diffraction pattern of Cephradine complex
Fig. 9 : represents the powder diffraction pattern of Cephradine standard
Fig. 10: represents the Multi plot of polymer, Cephradine standard and Cephradine
complex
Fig. 11: represents the DSC thermogram of pure polymer
Fig. 12: represents the DSC thermogram of pure Cefazolin sodium
Fig. 13: represents the DSC thermogram Cefazolin complex
Fig. 14: represents the DSC thermogram of pure Ceftriaxone sodium
Fig. 15: represents the DSC thermogram of Ceftriaxone complex
Fig. 16: represents the DSC thermogram of pure Cephradine

Fig. 17: represents the DSC thermogram of Cephradine complex
This invention optimises the proportion of active moiety which is required for therapeutic response for a particular indication leading to toxicity reduction and also economy in treatment cost. By virtue of its applicability to a range of sterile Cephalosporins preferably, and other Beta-lactams, administered in the parenteral route, the invention has potential to benefit a huge population of patients requiring parenteral Cephalosporin and other Beta-lactam antibiotic treatment.
Therefore, the present invention provides a process for producing novel Beta-lactam antibiotic-Polysaccharide complexes and preferably. Sterile Cephalosporin-polysaccharide complexes having a lower dosage of active moiety, resulting in equivalent pharmacological and microbiological response compared to parent drug leading to lesser toxicity and reduced cost of treatment for the patients.
Focus on Cephalosporin antibiotics as a preferred antibiotic group for polysaccharide complexing is relevant and appropriate, but does not in any manner detract the applicability of the concept to other Beta-lactam through similar complexing of polysaccharides under the applicant's invention.
The Applicants have conducted in-vivo test protocols which indicate that the novel composition produced by the process of the invention may be formulated in various physical forms and adapted for oral use, or as injectables.
This invention optimises the proportion of active moiety (antibiotic) which is required for equivalent therapeutic response compared to parent drug for a particular indication leading to toxicity reduction and also economy in treatment cost. By virtue of its applicability to a range of sterile Cephalosporins, and other Beta-lactams, administered in the parenteral route, the invention has potential to benefit a huge population of patients requiring parenteral Cephalosporin and other Beta-lactam antibiotic treatment.

Therefore, the present invention provides a process for producing novel Beta-lactam antibiotic-Polysaccharide complexes and preferably, Sterile Cephalosporin-Polysaccharide complexes, having a lower dosage of active moiety, resulting in equivalent phamacological and microbiological response compared to parent drug leading to lesser toxicity and reduced cost of treatment for the patients.
It should be understood that the scope of the present invention is not limited to the specific compositions or methods as described herein and that any composition or method having steps equivalent to those described herein fall within the scope of the present invention. Preparation routes of the composition of the invention and the method for combating microbial infections by the administration of the composition is merely exemplary so as to enable one of ordinary skill in the art to use the teachings of the invention, such as the composition of the invention and use it according to the described process and its equivalents. It vAll also be understood that although the form of the invention shown and described herein constitutes preferred embodiments of the invention, it is not intended to illustrate all possible forms of the invention. The words used are words of description rather than of limitation. Various changes and variations may be made to the present invention without departing from the spirit and scope of the invention.
EXAMPLE 1 (preparation of the composition)
To produce 1 gm of Cefazolin - Polysaccharide complex, 0.6 gms of Cefazolin and 0.4 gms of Polysaccharide are mixed with water under the pH condition between 5.5 to 6.5. Sodium bicarbonate is added to adjust the pH. The polysaccharide complex solution thus obtained is fihered through 0.2 micron fiher and transferred into a sterilized vial. Finally, the vial containing the solution is aseptically lyophilised under -40°C, -10°C, +10°C and +35°C under a vacuum of about 2.2 x 10'^ mbar.
EXAMPLE 2
To produce 1 gm of Ceftriaxone- Polysaccharide complex, 0.6 gms of Ceftriaxone and 0.4 gms of Polysaccharide are mixed with water under the pH condition between 5.0 to

6.5. Sodium carbonate is added to adjust the pH. The polysaccharide complex solution thus obtained is filtered through 0.2 micron fdter and transferred into a sterilized vial. Finally, the vial containing the solution is aseptically lyophilised under -40°C, -10°C, +10°C and +35°C under a vacuum of about 2.2 x 10"^ mbar.
EXAMPLE 3
To produce 1 gm of Cephradine - Polysaccharide complex, 0.6 gms of Cephradine and 0.4 gms of Polysaccharide are mixed with water under the pH condition between 5.5 to 6.5. Sodium bicarbonate is added to adjust the pH. The polysaccharide complex solution thus obtained is filtered through 0.2 micron fiher and transferred into a sterilized vial. Finally, the vial containing the solution is aseptically lyophilised under -40°C, -lO^C, +10°C and +35°C under a vacuum of about 2.2 x 10'^ mbar.
EXAMPLE 4
The produce 1 gm of Cefuroxime - Polysaccharide complex, 0.6 gms of Cefiiroxime and 0.4 gms of Polysaccharide are mixed with water under the pH condition between 5.5 to 6.5. Sodium bicarbonate is added to adjust the pH. The polysaccharide complex solution thus obtmned is filtered through 0.2 micron filter and transferred into a sterilized vial. Finally, the vial containing the solution is aseptically lyophilised under -40°C, -10°C, +10°C and +35°C under a vacuum of about 2.2 x 10'^ mbar.
EXAMPLES
The produce 1 gm of Cefoperazone - Polysaccharide complex, 0.6 gms of Cefoperazone and 0.4 gms of Polysaccharide are mixed with water under the pH condition between 5.5 to 6.5. Sodium bicarbonate is added to adjust the pH. The polysaccharide complex solution thus obtained is filtered through 0.2 micron fiher and transferred into a sterilized vial. Finally, the vial containing the solution is aseptically lyophilised under -40*'C, -10°C, +10°C and +35°C under a vacuum of about 2.2 x 10'^ mbar.
As illustrated above, the method can be used for preparation of different types compositions within the teachings of the invention and containing antibiotic -

polysaccharide complexes. The process involves addition of relevant antibiotic and polysaccharide in stoichiometric proportions in aqueous solution at a definite pH. The antibiotic complexes so obtained are aseptically lyophilized at selected temperatures and vacuum.
EXAMPLE 6 (X-ray Powder DifTraction Studies)
X-ray Powder Diffraction Studies were performed using Shimadzu XRD system. The powder diffraction pattern reveals that the powder diffraction pattern of the antibiotic-polymer complex manufactured by this technology is different fi-om that of pure parent antibiotic and that of pure. The major difference in the 2 theta values of the 3 major peaks of the polymer, parent antibiotic and that of the complex can be summarized as follows:

EXAMPLE 7 [Differential Scanning Calorimetry (DSC)]
DSC is a technique of thermal analysis, whereby the energy added to or subtracted from the sample, so as to maintain the sample and reference at the same temperature, is recorded. The output is a plot of heat applied to the same (MW) Vs temperature. DSC studies were done using Mettler Toledo DSC system. DSC thermograms of pure polymer, parent antibiotic and the antibiotic-polymer complex are shown in Figures 11 to 18. The following were observed :-(i) Cefazolin
DSC thermograms of Cefazolin shows sharp endothermic peak at 95.8°C and an exothermic peak at 199. TC. It can be observed from the DSC thermogram of Polymer, Cefazolin and Cefazolin Complex that an additional exothermic peak has appeared at about 240°C in the complex which is not there in case of polymer. Also the narrow peak at 95°C does not appear instead a broad peak can be observed in the range of 90 - 100°C. This suggest changes in thermal behaviour of polymer and Cefazolin complexation.
(ii) Ceftriaxone
DSC thermograms of standard Ceftriaxone reveal presence of a well defined narrow exothermic peak at about 262°C which is not seen in case of Ceftriaxone complex. Further the DSC thermogram of complex show 2 additional exothermic peaks in the range of 260°C to 295°C which are not present either in polymer or standard Ceftriaxone. The changes in the thermal nature of the complex from that of standard indicate formation of complex between the antibiotic and polymer.
(iii) Cephradine
DSC thermograms of standard Cephradine show narrow exothermic peaks at about 206°C, 257°C and 284^ and a broad endothermic peak at about 116°C. Appearance of broad peaks at about 189°C, 221'C and 250°C in DSC thermogram of complex suggest a change in the thermal behaviour, which signifies formation of complex between Cephradine and the polymer on lyophilisation.

These confirm that there is a formation of a complex between the antibiotic-polymer prepared using the lyophilisation technology.
EXAMPLE 8 : Testing the antimicrobial effect of tlie composition
The susceptibility of a microorganism to an antimicrobial agent is ascertained by dilution tests. In the tube dilution method, specific amounts of the antibiotic, prepared in decreasing concentrations in broth by serial dilution technique, are inoculated with a broth culture of the bacterium to be tested. The susceptibility of any organism is determined, after a suitable period of incubation, by macroscopic observation of the presence or absence of growth in the varying concentration of the antimicrobial agent. The end point is a measure of the bacteriostatic effect of the agent on the bacterium and is commonly referred to as the Minimum Inhibitory Concentration (MIC). The amount of drug in the tube containing the least amount of the antibiotic with no observable growth is the MIC.
The tube dilution method is considered to the most accurate for determination of susceptibility to measured amounts of an antimicrobial agent. Test substances used were carried out on a number of microorganisms.
1. Cefazolin - Standard
2. Cefazolin- Complex
3. Ceftriaxone - Standard
4. Ceftriaxone - Complex
5. Cephradine - Standard
6. Cephradine - Complex
The protocol used, the results and conclusions are discussed herein below.

Minimum Inhibitory Concentration values for Cefazolin complex and Ceftriaxone complex have been determined and compared with those of the respective parent drugs. The data shows a significant increase in the activity of the antibiotic in presence of the polysaccharide as complex.

The above MICs are lower than the reported MICs of Cefazolin towards the above organisms (i.e., 16 meg and less / ml).
The above Table clearly establishes the fact that the complexed Cefazolin-Polysaccharide antibiotic (60 : 40 ratio) provides equivalent or better response on the microorganisms cited above compared to uncomplexed Cefazolin antibiotic (100%).
Staphyloccus aureus and Proteus mirabilis were the selected microorganisms against a designed burn caused infection. The rate of healing for the complexed drug confirms to the desired response in equivalent pattern to that of parent (known) drug.

We claim:
1. A process for preparing Beta-lactam antibiotic polysaccharide
complex comprising the steps of:
i) reacting a Beta-lactam ring containing antibiotic
compound such as hereindescribed with a polysaccharide in the presence of a solvent at room temperature,
ii) adding a buffer such as hereindescribed to adjust the pH, and
iii) recovering Beta-lactam antibiotic-polysaccharide complex from the solution as a dry solid by lyophilization at a temperature ranging between -40°C to 35°C.
2. A process as claimed in claim 1 wherein the Beta-lactam ring containing antibiotic compound is selected from the group consisting of cephalosporins, cefazolin, cephradine, ceftriaxone, cefoperazone and cefotaxime.
3. A process as claimed in claim 1 wherein the polysaccharide is selected from the group consisting of arabinose, agarose, low molecular weight dextran, activated dextrans, insulin, chitin, chitosan, mannan and lichenin.
4. A process as claimed in claim 1 wherein the proportion of Beta-lactam ring containing antibiotic compound to the polysaccharide ranges between 20 to 60%.
5. A process as claimed in claim 1 wherein the proportion of polysaccharides in the antibiotic-polysaccharide complex ranges between 40 to 80%.
6. A process as claimed in claim 1 wherein the solvent is water.
7. A process as claimed in claim 1 wherein the buffer is
selected from sodium bicarbonate, sodium carbonate and triethanolamine.
8. A process as claimed in claim 1 wherein the reaction is
effected at a pH of about 4.5 to 7.5

A process for the production of Beta-lactam antibiotic
polysaccharide complex substantially as hereindescribed with reference to the examples and the accompanying drawings.

Documents:

0730-mas-1999 abstract.pdf

0730-mas-1999 claims.pdf

0730-mas-1999 correspondence-others.pdf

0730-mas-1999 correspondence-po.pdf

0730-mas-1999 description(complete).pdf

0730-mas-1999 drawings.pdf

0730-mas-1999 form-1.pdf

0730-mas-1999 form-13.pdf

0730-mas-1999 form-26.pdf

0730-mas-1999 form-3.pdf

0730-mas-1999 form-4.pdf

0730-mas-1999 form-5.pdf

0730-mas-1999 form-9.pdf

0730-mas-1999 petition.pdf

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Patent Number

193267

Indian Patent Application Number

730/MAS/1999

PG Journal Number

30/2009

Publication Date

24-Jul-2009

Grant Date

21-Apr-2005

Date of Filing

12-Jul-1999

Name of Patentee

M/S. ORCHID CHEMICALS & PHARMACEUTICALS LTD

Applicant Address

NO. 1, 6TH FLOOR, CROWN COURT, 34, CATHEDRAL ROAD, CHENNAI 600 086

Inventors:

#	Inventor's Name	Inventor's Address
1	PRASANTA KUMAR CHAKRABARTI	ORCHID CHEMICALS & PHARMACEUTICALS LIMITED, NO. 1, 6TH FLOOR, CROWN COURT, 34, CATHEDRAL ROAD, CHENNAI 600 086
2	CANAKAPALLI BHAKTAVATSALA RAO	ORCHID CHEMICALS & PHARMACEUTICALS LIMITED, NO. 1, 6TH FLOOR, CROWN COURT, 34, CATHEDRAL ROAD, CHENNAI 600 086

PCT International Classification Number

A61K31/545

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA