WO2002030469A2 - Beta-lactam antibiotic-polysaccharide complex - Google Patents

Abstract

The present invention provides novel Beta-lactam antibiotic-polysaccharide complexes having a lower dose of active moiety resulting in equal pharmacological and microbiological response compared to respective parent drugs, leading to lesser toxicity and reduced cost of treatment for the patients and also, 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.

Description

ENHANCED THERAPEUTIC EFFICACY OF NOVEL BETA-LACTAM ANTIBIOTIC-POLYSACCHARIDE COMPLEX

TECHNICAL FIELD The present invention relates to novel Beta-lactam antibiotic-Polysaccharide complexes having improved therapeutic efficacy. The novel complexes represent an improvement in the therapeutic efficacy compared to uncomplexed Beta-lactam antibiotics for example, Cephalosporin antibiotics. The present novel complexes facilitate optimization in drug delivery with an objective to enhance the efficacy of the therapeutic moieties with reduced toxicity.

The present invention also relates to a novel process for the preparation of Beta- lactam antibiotic-Polysaccharide complexes having improved therapeutic efficacy. BACKGROUND ART

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 by both parenteral and oral route depending on their physico-chemical characteristics, pharmaco-dynamic and pharmaco-kinetic profile. The choice of antibiotics depends on the type of infection, the dose and route of administration based on the severity of the infection.

The injectible cephalosporins are employed for systemic use for prompt action and bioavailability of these dosage forms is found to be 100 percent. The pharmaco kinetic studies show that injectible cephalosporins are plasma-bound by about 60 percent and remaining portion of cephalosporins, being highly wat r 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 upto about 85 percent depending on the molecule.

Beta-lactam 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 an 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 the novel complex that utilizes less amount of the active ingredient to achieve the same therapeutic efficacy were developed, it would significantly reduce the cost of treatment.

The applicant's invention encompasses the optimum usage of the therapeutic moiety, preferably injectible cephalosporins, with polysaccharides to result in a novel complex which provides efficacious, less toxic and more cost-effective drug delivery intended for treating infections in mammals, particularly humans. Polysaccharides are carbohydrates derived from plant and microbial sources. Polysaccharides are used for many pharmaceutical and industrial applications. 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 cost-intensive 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 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 optimize the efficacy of Beta-lactam antibiotic employing any polysaccharides. The applicants believe that any invention which optimises the efficacy of this antibiotic group has significant positive benefits for the society. DISCLOSURE OF THE INVENTION

The main object of the invention is to develop antibiotic polysaccharide complexes, which optimize the proportion of active moiety (antibiotic) which is required for therapeutic response for a particular indication with benefits of toxicity reduction and economy in treatment costs. By virtue of applicability to a range of antibiotics the invention has potential to benefit a huge population of patients requiring antibiotic treatment. Another object of the invention is to provide a novel composition containing Beta- lactam complexes, and useful in the treatment of microbial infections.

Yet another object is to provide novel Beta-lactam complexes with polysaccharides.

Still another object is to provide novel compositions useful in the treatment of bacterial infections in lower dosage.

Yet another object of the invention is to provide a process for the production of novel composition containing Beta-lactam antibiotics complexed with polysaccharides.

Yet 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 present invention provides novel Beta-lactam antibiotic- polysaccharide complexes having a lower dose of active moiety resulting in equal pharmacological and microbiological response compared to respective parent drugs, leading to lesser toxicity and reduced cost of treatment for the patients.

Also, 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

In accordance with the above and other objects, the invention provides therapeutically effective pharmaceutical compositions comprising Beta-lactam ring containing antibiotics, together with polysaccharides and useful in the treatment of microbial infections. The term 'Beta-lactam' compounds as used herein denotes compounds essentially consisting of a Beta-lactam ring, which may be fused with other rings such as 5-member thiazolidine ring systems (6APA) derived products and 6-member cephalosporin ring systems (6ADCA/7-ACA derived products). Typical examples of such compounds are antibiotics such as penicillin, cephalosporins, monocyclic beta lactams, their derivatives etc. Particular cephalosporin antibiotics that may be used for practice of the invention are such as cefazolin sodium, ceftriaxone sodium, cefoperazone sodium, cefotaxime sodium, cefuroxime sodium, cephradine sodium etc.

Generally, the carrier with which the Beta-lactam antibiotic may be complexed are linear or straight chain polysaccharides such as arabinose, agarose, low molecular weight dextran (mol wt 5000), activated dextran, inulin, chitin, chitosan, mannan, lichenin and their derivatives.

The term 'effective amount' in the composition, the applicant means an amount- that would be sufficient to evoke antimicrobial or antibacterial effect in spite of using lower amount of antibiotic compared to parent antibiotic. The amount will vary depending upon the antibiotic in question and the treatment regime. A person skilled in the art can readily determine the amount of antibiotic in the composition. For ease of practice of the invention, the applicants recommend that the pharmaceutical composition may contain Beta-lactam ring containing antibiotics in the range of about 20 - 70%. The proportion of antibiotics to the polysaccharides depends on the polysaccharides and the antibiotic used.

Take the case of Cefazolin complex formed according to the practice of the invention, Cefazolin can be present in the range of 40 to 70% and the polysaccharide may constitute the balance of the composition, which is achieved in stoichiometric proportions in aqueous solution at a definite pH through aseptic lyophilisation process. Tests conducted with various ratios revealed 60:40 to be the optimum ratio.

The Applicants have found that Beta-lactam antibiotics when complexed with polysaccharides exhibit unexpected synergistic properties and possess a useful and advantageous profile of activity. The applicants have also found that the complex can be effectively administered as a drug in lower concentrations to achieve best results. The applicants have selected polysaccharides, having linear structure, as these are water soluble and good carriers of drugs. In addition, these 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 optimized drug delivery. The ideal polysaccharides are those provided with polar hydroxyl groups, so that they can attach with the active moiety (antibiotic) as a part of the complexation process through hydrogen bonding or non-covalent non-ionic bonding. In other words, the drug-polysaccharide complex in present invention involves an altogether different chemical combination, which is much simpler and economically viable. The pharmaceutical composition of the present invention can be prepared by a process having at least the steps of: i) reacting antibiotics containing Beta-lactam ring containing with polysaccharides in the presence of a solvent, and ii) lyophilizing the antibiotic-polysaccharide solution under vacuum to obtain antibiotic-Polysaccharide Complex as a dry solid.

In an embodiment of the present invention, the Beta-lactam polysaccharide complex formation is activated by use of cynogen bromide.

In an embodiment, the Beta-lactam ring containing antibiotics are selected from the group comprising cephalosporins, cefazolin, cephradine, ceftriaxone, cefoperazone, cefotaxime, other cephlosporins, betalactums and their derivatives.

In . another embodiment, the polysaccharides are selected from the group comprising Arabinose, Agarose, low molecular weight dextran, activated dextrans, inulin, chitin, chitosan, mannan, lichenin, other polysaccharides and their derivatives.

In still another embodiment, the proportion of Beta-lactam compounds to polysaccharides ranges between 20 - 70%.

In yet another embodiment, the proportion of polysaccharides in the composition ranges from 80 - 30%.

In an embodiment, the reaction is effected at a pH of about 4.5 to 7.5 and 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 -40°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 lyophilisation. In the present process, the antibiotic polysaccharide complex solution so obtained is to be filtered through 0.2-micron filter. The temperature selected for this lyophilisation are -40°C, -10°C, +10°C and +35°C under a vacuum of about 2.2 x IO^"2 mbar.

An aspect of the invention also involves addition of suitable pH adjustment agents such as Sodium bicarbonate, activation of the polysaccharides for complexation process through cyanogen bromide treatment, in appropriate cases.

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 fused 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, which is preferably sterile. They can be selected for example, from the group comprising cefazolin, ceftriaxone, cephradine, cefoperazone, cefotaxime, other cephlosporins, betalactums 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, lichenin, other polysaccharides 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 recommended pH for conducting the reaction is in the range of 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 70: 30 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 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 40 to 70% 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 presenfin the composition in comparison to polysaccharides may range between 20 - 70%. The portion of polysaccharides will vary from 80 - 30%. The solvent used in the process is water. The temperatures selected for this lyophilisation or freeze-drying are about - 0°C to 35°C.

The Applicants have conducted in-vivo test protocols, which indicate that the novel composition of the invention may be formulated in various physical forms, and adapted for oral use, or as injectibles. Oral forms include tablets, syrups, capsules, coated delivery system etc. Also envisaged herein would be coated microcapsules or beadlet form suspended in a liquid polysaccharide or any other inert carrier, which may thereafter, be encapsulated. In the preparation of these tablets or forms suited for oral use, suitable additives such as thickening agents, etc. as known in the art are employed. Although it is suitable to formulate the composition of the invention as tablets, the preferred method for administration of antibiotics is in the form of injectibles.

The solubility of complex involving cephalosporins and polysaccharides makes it appropriate for parenteral application. The polysaccharides are rapidly excreted in urine without accumulation in the tissues. This invention optimises the proportion of active moiety (antibiotic) 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, 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 novel Beta-lactam antibiotic- Polysaccharide complexes and preferably, Sterile Cephalosporin-Polysaccharide complexes having a lower dose of active moiety, resulting in equal/similar phamacological and microbiological response compared to uncomplexed drug leading to lesser toxicity and reduced cost of treatment for the patients.

Brief Description of the accompanying Drawings The invention is further illustrated by the following drawings wherein: Fig.l : 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 of Cefazolin complex

Fig.7 : represents the Multi plot of polymer, cefazolin standard and cefazolin complex Fig.8 : represents the DSC thermogram of pure polymer Fig.9 : represents the DSC thermogram of pure Cefazolin sodium Fig.10: represents the DSC thermogram of Cefazolin complex Fig.ll: represents the DSC thermogram of pure Ceftriaxone sodium Fig.12: represents the DSC thermogram of Ceftriaxone complex Fig.13: represents the IR spectrum of pure polymer Fig.14: represents the IR spectrum pattern of pure Cefazolin sodium Fig.15: represents the IR spectrum pattern of Cefazolin complex Fig.16: represents the IR spectrum of pure Ceftriaxone sodium Fig.17: represents the IR spectrum of Ceftriaxone complex

Fig.18: represents the efficacy study of Cefazolin complex in S. aureus infected Swiss

Albino mice (males) Fig.19: represents the efficacy study of Cefazolin complex in S. aureus infected Swiss

Albino mice (females) Fig.20: represents the efficacy study of Ceftriaxone complex in S. aureus infected Swiss

Albino mice (males) Fig.21: represents the efficacy study of Ceftriaxone complex in S. aureus infected Swiss

Albino mice (females)

This invention optimizes 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 injectible.

This invention optimizes 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 will 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. Narious changes and variations may be made to the present invention without departing from the spirit and scope of the invention.

The composition envisaged in the present invention are based on the fact that Cephalosporin antibiotics such as Cefazolin Sodium, Ceftriaxone Sodium, Cefoperazone Sodium, Cefotaxime Sodium, Cefuroxime Sodium, and a number of other Cephalosporin derivatives of various generations are a therapeutically more popular group of Beta-lactam antibiotics and have better activity profile and spectrum compared to other Beta-lactam antibiotics. Cephalosporin group also offers a significantly wider range of antibiotic products. The invention is discussed in detail hereinafter with reference to non-limiting examples, which relate to preparation of the composition, X-ray diffraction studies, differential scanning calorimetry, test for antimicrobial effects and test on animal models (mice). 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 will 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. Narious changes and variations may be made to the present invention without departing from the spirit and scope of the invention.

EXAMPLES Preparation of the Composition EXAMPLE 1 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 may be added to adjust the pH. The drug-polysaccharide complex solution thus obtained 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 IO^"2 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 bicarbonate may be added to adjust the pH. The drug-polysaccharide complex solution thus obtained 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 IO^"2 mb.ir. 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 may be added to adjust the pH. The drug-polysaccharide complex solution thus obtained 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 IO^"2 mbar. EXAMPLE 4

The produce 1 gm of Cefuroxime - Polysaccharide complex, 0.6 gms of Cefuroxime and 0.4 gms of Polysaccharide are mixed with water under the pH condition between 5.5 to 6.5. Sodium bicarbonate may be added to adjust the pH. The drug- polysaccharide complex solution thus obtained is filtered through 0.2 micron 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 of about 2.2 x 10^"2 mbar. EXAMPLE 5 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 may be added to adjust the pH. The drug- polysaccharide complex solution thus obtained is filtered through 0.2 micion 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 IO^"2 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. The invention embraces within its scope blends of antibiotics with polysaccharides in different forms.

The antibiotic compositions, prepared .by using the process of the invention, have been analyzed for antibiotic contents and other physico-chemical properties like water content and pH of aqueous solution. Further characterisation of the composition has been done using various analytical techniques Vhich include X-ray Powder Diffraction Studies, Differential Scanning Calorimetry and Infra -Red Spectroscopy. These analytical techniques confirmed the formation of Complexes between the Antibiotic and the Polymer. Toxicity studies of the pharmaceutical composition of the invention have been carried out. Acute and subacute toxicity studies reveal a wide safety margin in the therapeutic use of compositions containing Cefazolin complex and Ceftriaxone. Various tests as outlined herein below have been conducted to test the efficacy of the composition. EXAMPLE 6 (X-ray Powder Diffraction Studies)

X-ray Powder Diffraction Studies were performed using Shimadzu XRD system. The powder diffraction pattern as shown in figures 1 to 7, reveal that the powder diffraction pattern of the antibiotic-polymer complex manufactured by this technology is different from that of pure parent antibiotic and that of pure parent compound. 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:

Ceftriaxone Complex

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 the graphs shown in

Figures 8 to 12. The following were observed:-

(i) Cefazolin DSC thermograms of Cefazolin shows sharp endothermic peak at 95.8°C and an exothermic peak at 199.1°C. 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 indicates 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.

The IR spectrum of the complexes is depicted in figures 13 to 17.

EXAMPLE 8: Testing the in-vitro antimicrobial effect of the 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 anti microbial agent. Test substances used were carried out on a number of microorganisms.

1. Cefazolin - Standard

2. Cefazolin - Complex

3. Ceftriaxone - Standard 4. Ceftriaxone - Complex

The protocol used the results and conclusions are discussed herein below. Preparation of Test Substances:

The test substances were prepared fresh prior to testing. 5 mg of the test substance was weighed accurately and dissolved in 10 ml of Mueller -

Hinton broth (Oxoid). The final concentration of the drug was 500 μg/ml. This solution as filtered through a 0.22 μ syringe filter (Millipore) prior to use.

MICRO-ORGANISMS:

Standard strains of following organisms from ATCC were used for testing against the test substances:

1. Staph.aureus ATCC # 25923

2 Strept. pyogenes ATCC # 19615 3 Strept. pneumoniae ATCC # 6303 4 H. influenzae ATCC # 49247 5, Proteus mirabilis ATCC # 25933 6 E.coli ATCC # 8739 7 Serratia marsescens ATCC # 8100 8 Neisseria gonorrhoeae ATCC # 25934

Preparation of inoculum:

The microorganism to be tested is inoculated in Mueller - Hinton broth and incubated for 16 - 18 hrs. at 35+ 1°C. The turbid broth is diluted prior to testing to contain 10⁵ to 10° organisms / ml.

PROCEDURE:

1. 14 clear, sterile, cotton - plugged tubes (13 x 100 mm) are labeled from 1 to 14.

2. Using aseptic technique, pipette 0.5 ml of MHB (Mueller - Hinton Broth) into tubes 2 through 13. 3. Add 0.5 ml of the test substance (500 μg/ml) into tube 1, 2 and 14. Mix the contents of tube 2 well and transfer to tube 3. Mix well and transfer 0.5 ml to tube 4. Continue this procedure till tube 12. Discard 0.5 ml from tube 12. Tube 13 receives no drug. Tube 13 and 14 serves as controls. 4. To all the tubes add 0.5 ml of the inoculum containing IO⁵ to IO⁶ organisms/ml. 5. The final volume in each tube is 1.0 ml. The following table shows the concentration of drug in each tube. This is the double dilution or serial two-fold dilution. 6. The tubes are incubated at 35+ 1°C for 16-18 hours, the tubes are then examined macroscopically for evidence of growth. 7. The lowest concentration of drug in the series showing no growth is taken as the

MIC and is usually expressed as micrograms / ml. Experimental Outline: Serial tube dilution for MIC determination

* Discard 0.5 ml from tube 12.

Since the main aim of this work is to compare the MIC values of the standard drug with the complex, narrow range dilution was carried to determine the actual value.

Based on the MIC values obtained from the double dilution method, the concentration of the drug was further narrowed to the ranges obtained.

RESULTS:

The tables A and B show the results obtained for the standard as well as the complex. The actual amount of drug in the complex and the increased percentage of activity of the complex in vitro are shown.

CONCLUSION:

Cefazolin complex is active against a wide range of organisms. The complex shows an enhanced in vitro activity of 25.94% to 38.89%, compared to standard Cefazolin, against various groups of organisms.

Ceftriaxone complex has anti-microbial effect against various groups of organisms. The complex shows as enhanced in vitro activity of 11.11% - 43.52% compared to standard

Ceftriaxone against different groups of microbes.

Project No.: CPLN/MIC/2000

Comparison & Determination of susceptibility of bacteria to cephalosporin - Standard &

Complex^ Table A: Comparative MIC data for Cefazolin standard and Cefazolin complex

Project No.: CPLN/MIC/2000

Comparison & Determination of susceptibility of bacteria to cephalosporin - Standard &

Complex

Table B: Comparative MIC data for Ceftriaxone standard and Ceftriaxone complex

EXAMPLE 9

Antibiotic Efficacy Studies of Cefazolin complex and Ceftriaxone complex, using mouse model were conducted. Intramuscular administration of antibiotic complex exhibit bactericidal activity in S. aureus infected^' mice. The in vivo efficacy of antibiotic complexes in clearing the infection of S. aureus has been found to be comparable with that of the respective parent antibiotics. The experiment was designed as per the method of Nagano et al. (1997), Magni et al., (1990), Takenouchi et al. (1994), Kundsen et al, (1997), Sauve et al., (1996), and Kaur et al., (2000).

Six groups of Swiss albino mice (Mus musculus) comprising of 10 males and 10 females per group were used for the study. 3 groups of mice were infected with S. aureus (experimental group) and 3 groups were maintained uninfected (control group). The infected groups were designed as: Group II (untreated), Group V (treated with cefazolin reference drug) and Group VI (treated with the cefazolin complex). The uninfected control groups were classified as: Group I (untreated), Group III (treated with cefazolin reference drug) and Group IN (treated with cefazolin complex). The treatment with drug was started 60 - 75 min after the introduction of infection and continued for five days.

Each mouse was observed twice daily for mortality and signs of toxicity. Group mean body weight was monitored daily. All surviving animals in the infected group were sacrificed on day 8 of the study and the bacterial load on selected organs and tissue fluids was determined. Animals that died during the course of treatment were also evaluated for bacterial load on the same organs and body fluids.

The results of the efficacy study are summarized hereinbelow and depicted in Figs.

18 to 21.

The important findings of the study comparing the in vivo efficacy of cefazolin complex with cefazolin standard are summarized herein below. Mortality

No treatment related mortality was observed during the course of the study. 60% of animals in the infected - untreated group (Grp II) died during the course of the study. The recorded mortalities were considered as the result of S.aureus infection. Clinical Observations No treatment related clinical symptoms were obseryed in any treatment group during the experimental period. Mice from group II (infected -untreated) exhibited diarrhea, lethargy, abstention from food and water. Body

Body weights of treated animals were found to be comparable with their respective controls. Gross Pathological changes

All animals in group II, V & VI that either died of infection or from which S.aureus was isolated on day 8, showed ballooning of the intestine, greenish coloration of the viscera, and abscess formation Bacteriological findings

Intramuscular administration of Cefazolin standard drug and cefazolin complex, in S.aureus infected mice exhibited 94.73% and 94.44% clearance of S.aureus respectively. Conclusion

Intramuscular administration of cefazolin complex exhibited bactericidal activity in S.aureus infected mice. The in - vivo efficacy of cefazolin complex was found to be comparable with that of standard cefazolin in clearing infection by S.aureus Test Procedure

The objective of this study was to evaluate the 'Efficacy of Cefazolin polysaccharide Complex in S. aureus infected Swiss albino mice'. The study was conducted following the procedure of Nagano et at., (1997), Magni etal, (1990), Takenouchi et al, (1994), Knudsen et al.,(1991), Sauve etal.,(1996), andKaureM., (2000).

Mouse was selected as a test system because it is a readily available rodent species.

It has been historically shown to be a suitable model for efficacy testing of antibiotics and is recommended by the regulatory authorities. The intra - muscular route of drug administration represents a possible route in humans. The results of the study are believed to be of value in predicting the efficacy of cefazolin polymer complex in human.

The study was performed by the Department of Microbiology, Dr.A.L.M Postgraduate Institute of Basic Medical Sciences, Chennai; and all the data generated and recorded during this study will be stored in the archives of Orchid Health Care, Chennai. STUDY SCHEDULE

Study Initiation: - Dec. 3, 1999

Acclimatization Start: - July 16, 2000.

Study Start: - July 21,2000.

Study Completion:- Oct. 5, 2000

Animal & Animal Husbandry:

A total of 60 male & 60 female mice (Mus musculus) of Swiss Albino strain obtained from King Institute of Preventive Medicine, Guindy, Chennai, were used for the study. The mice were received into the experimental procedure room, kept under group feeding and allowed to acclimatize for 5 days. The mice were randomly allocated to groups of ten by sex to ensure similarity in mean body weight between the groups. Body weights of the mice ranged between 31 - 36 gm for males, and 21 -28 gm for females at the start of the study.

Individual mice were identified with number painted at the base of the tail and cage card showing project number, study, group, sex and mouse number. Numbers were given serially starting with group I (control). At the start of the study the mice were approximately seven weeks old.

The mice were housed sex wise in-groups of five in clean, sterilized, solid floor polypropylene cages. Clean rice (paddy) husk was used as bedding material. The cages were kept on a five-tier rack. The mice were fed with pellet feed supplied by Kamadhenu Enterprises, Bangalore and water filtered through Aquaguard water filtration system was provided in clean polypropylene bottles. Both feed and water were provided to all mice ad libitum. Fresh feed was provided thrice a week and water bottles were refilled daily.

The animals were maintained in an air-conditioned room at a temperature of 22 +

3°C with relative humidity of 50-70%.

Treatment Groups:

The study consisted of three infected and three uninfected groups:

GROUP I: Uninfected - Untreated control GROUP II: Infected - Untreated control.

GROUP ul: Uninfected - Treated with Cefazolin Standard reference GROUP IV: Uninfected - Treated with Cefazolin complex. GROUP V: Infected - Treated with Cefazolin Standard reference drug. GROUP VI: Infected - Treated with Cefazolin complex. Experimental outline:

Preparation of culture media

Culture media were prepared by weighing and mixing the required quantity in distilled water and autoclaved for 20 min at 121 °C at 15 lbs pressure.

Infective Organism:

S.aureus Smith strain was used as the infecting organism. Cultures were demonstrated to be free from contamination by bacteriologic culture under aerobic, anaerobic & microaerophilic atmospheres at 37°C, prior to initiation of study. Gram stain & biochemical assays were performed to confirm the bacterial identity.

Isolate was confirmed as S.aureus since they were aerobic, catalase positive, DNase positive, coagulase positive, gram positive cocci in clusters.

Preparation of Infective suspension

The infective organism was grown overnight in BHI broth at 36°C + I°C. Overnight broth culture of S.aureus was centrifuged at 2500 rpm for 30 min. The supernatant was decanted & the deposit was resuspended in sterile physiological saline & matched with MacFarland standard II (6 x 10⁸ / ml). 0.5 ml of this solution was used for intraperitoneal (ip) injection. Preparation of antibiotic solution:

The required quantity of the test substance (Cefazolin standard / Cefazolin Complex) was weighed and dissolved in WFI to give a final concentration of lOmg/ml. The solution was sterilized by syringe filtration (Millipore, 0.22μ.) method prior to administering in mice. Route of Administration:

S.aureus infected and uninfected animals were treated with the drug by the intra muscular

(im) route.

Concentration & Frequency of Administration

The^rug (standard or complex) was administered at a concentration of 100 mg/ kg body weight, once daily for a period of 5 consecutive days. Fresh solution of drug was prepared everyday prior to dosing. The mice from control were administered saline alone.

OBSERVATIONS: Clinical Symptoms: Animals were observed for all visible signs of reaction to the treatment such as skin & fur changes, eyes & mucous membrane changes, respiratory, circulatory, autonomic & central nervous system, somatomotor activity, behavior pattern & general changes twice a day.

Body weights:

Group mean body- weights was recorded on the day of commencement of dosing, and once daily thereafter.

Mortality:

All animals were observed twice daily for mortality or morbidity during the period of study.

Grass Pathological changes:

Visceral gross pathological changes were observed and noted in those animals, which underwent post-mortem or sacrificed on day 8.

Estimation of Bacterial load:

All animals dying during the course of treatment (i.e. before the end of the study) underwent post - mortem. Peritoneal fluid, Liver, Heart and kidney were cultured for the presence of S.aureus in Nutrient agar. All the surviving animals from Group II, V & VI were sacrificed 48 hours after the last treatment by cervical dislocation. The mice were examined carefully for external and internal abnormalities and portions of Liver, Heart and kidney were collected aseptically and chopped to small pieces and inoculated in Nutrient broth. Later they were plated in Nutrient agar.

Peritoneal fluid was directly plated in Nutrient agar. It was incubated aerobically at 37°C for 24 hrs. The number of bacteria on each plate was counted manually at the end of the incubation period, and CFU was calculated for each organ.

Treatment of Result:

Raw data were processed to give group means and standard deviations with significance between the control and treated groups. The results were statistically analyzed using Chi

Square analysis. Values of p < 0.05 were considered to be statistically significant.

Archives:

All raw data and a copy of final report will be retained in the archives at Orchid Healthcare for a period of 5 years.

RESULTS & DISCUSSION :

Clinical Symptoms:

Males: {Table 1: Summary of Clinical signs}

Administration of Cefazolin standard or complex for five consecutive days did not exhibit any signs of reaction to the treatment. However, from group II one male (5%) exhibited diarrhoea, while two males (10%) showed lethargy & abstained from food & water. All these symptoms were considered as a result of S.aureus infection.

Females: (Table I: Summary of Clinical signs]

No treatment related clinical signs were recorded in animals of any treatment group during the course of study. From females in-group II, diarrhoea was recorded in five females

(25%), whereas lethargy and abstention from food and water was noted in eight females

(40%). All these were considered as signs of S.aureus infection.

Body Weights:

Males Average body weights of treated animals were found to be comparable with the respective controls.

Females:

No treatment related alterations were observed in body weight of treated animals.

Gross Pathological changes: All animals in group II, V & VI that either died of infection or S.aureus isolated on day 8 showed gross pathological changes as evidenced by Ballooning of the intestine, greenish coloration of the viscera, foul smell and abscess formation in the kidney and liver.

Mortality:

Males : TTable 2: Summary of mortality & S.aureus load.1 40% of the animals died in-group π due to infection with S.aureus. One animal each from

Group I & VI died due to excessive bleeding. Animals in all other groups survived throughout the study period.

Females: [Table 2; Summary of mortality &, S.aureus load] Mice from S.aureus infected (group II) exhibited 80% mortalities on day 2 of the infection.

One animal each from Group I, m, V & VI died due to excessive bleeding. All other animals survived till the end of the study.

Females were more susceptible to infection & showed a higher mortality rate than males

(50% females Vs 25% males). Estimation of Bacterial Load: [Table 3: Estimation of bacterial load]

Animals that died during the course of treatment were autopsied and bacterial culture of specific organs and body fluid were examined.

All mice in Group II, group V & VI which survived up to day 8 were also sacrificed and- organ culture was done. Systemic infection was achieved within a few hours after inoculation as seen by the death of 60% animals by 72 hrs of induction of infection in group II. S.aureus was isolated from peritoneal fluid, liver, kidney & heart in all these animals.

50% (4/8) animals in Group II (Infected-untreated) sacrificed on day 8 showed presence of

S.aureus. Among the treated groups, 94.73% (18 / 19) animals in group V (treated with Cefazolin standard) exhibited clearance of S.aureus. This was found to be statistically significant compared to the untreated control (group II). 94.44% (17/18) animals in group VI

(treated with Cefazolin complex) exhibited clearance of S.aureus. This is Statistically significant compared to untreated control (group II) and Statistically Not Significant compared to group V (treated with Cefazolin control).

CONCLUSION :

Intramuscular administration of cefazolin complex exhibited bactericidal activity in S.aureus infected mice. The in - vivo efficacy of cefazolin complex was found to be comparable with . that of standard cefazolin in clearing infection by S.aureus. Efficacy study of Cefazolin complex in S.aureus infected Swiss Albino mice

Table 1 : Summary of Clinical Signs

Efficacy study of Cefazolin complex in S.aureus infected Swiss Albino mice Table 2: Summary of Mortality & S.aureus Load

Efficacy study of Cefazolin complex in S.aureus infected Swiss Albino mice Table 3 : Summary of Estimation of Bacterial Load

As discussed above, the in vivo efficacy of Ceftriaxone complex was also compared with Ceftriaxone standard in S.aureus infected Swiss albino mice. The study protocol was identical to that of the protocol used for cefazolin. The results are discussed hereinbelow.

TREATMENT OF RESULT

Raw data were processed to give group means and standard deviations with significance between the control and treated groups. The results were statistically analyzed using Chi Square analysis. Values of p<0.05 were considered to be statistically significant.

ARCHIVES

All raw data and a copy of final report will be retained in the archives at Orchid Healthcare for a period of 5 years.

RESULTS & DISCUSSIONS

Clinical Symptoms:

Male: [Table 4: Summary of Clinical signs,]

Administration of Ceftriaxone standard or complex for five consecutive days did not exhibit any signs of reaction to the treatment. However, from group II four males (40%) exhibited diarrhea, while two males (20%) showed lethargy & abstained from food & water. All these symptoms were considered as a result of S.aureus infection..

Females: [Table 4: Summary of Clinical signs]

No treatment related clinical signs were recorded in animals of any treatment group during the course of study. From females in group II, diarrhoea was recorded in ten females (100%), whereas lethargy and abstention from food and water was noted in eight females (80%). All these were considered as signs of S.aureus infection.

Mortality:

Males: TTable 5. Summary of mortality & S.aureus load! 20% of the animals died in group II, and 10% in group VI due to infection with S.aureus.

Animals in all other groups survived throughout the study period

Females: fTable 5 Summary of mortality & S.aureus load]

Mice from S.aureus infected (group II) exhibited 70% mortalities on day 2 of the infection. All other animals survived till the end of the study.

Females were more susceptible to infection and showed a higher mortality rate than males

(70% females Vs 20% males).

Body Weights:

Males: Average body weights of treated animals were found to be comparable with the respective controls.

Females:

No treatment related alterations were observed in body weight of treated anim als.

Gross Pathological changes All animals in group II, & VI that either died of infection or from which S.aureus was isolated on day 8, showed gross pathological changes as evidenced by Ballooning of the intestine, greenish coloration of the viscera, foul smell and abscess formation in the kidney and liver.

Estimation of Bacterial Load : [Table 6 Estimation of bacterial load] Animals that died during the course of treatment were autopsied and bacterial culture of specific organs and body fluid were examined.

All mice in Group II, group V & VI which survived up to day 8 were also sacrificed and organ culture was done.

Systemic infection was achieved within a few hours after inoculation as seen by the death of 45% animals by 72 hrs of induction of infection in-group II. S.aureus was isolated from peritoneal fluid, liver, kidney & hearrin all these animals.

72.7% (8 / 11) animals In-Group II (Infected - untreated) sacrificed on day 8 showed presence of S.aureus. Among the treated groups, 100% (20/20) animals in group V (treated with

Ceftriaxone standard) exhibited clearance of S.aureus. This was found to be statistically significant compared to the untreated control (group II), 90% (18 / 20) animals in group VI

(treated with Ceftriaxone complex) exhibited clearance of S.aureus. This is Statistically significant compared to untreated control (group II) and Statistically Not Significant compared to group V (treated with Ceftriaxone control). CO NCLUSIO N :

Intramuscular administration of ceftriaxone complex exhibited bactericidal activity in S.aureus infected mice. The in - vivo efficacy of ceftriaxone complex was found to be comparable with standard ceftriaxone in clearing infection by S.aureus.

Efficacy study of Ceftriaxone complex in S.aureus infected Swiss Albino mice

Table 4 : Summary of Clinical Signs

Efficacy study of Ceftriaxone complex in S.aureus infected Swiss Albino mice Table 5: Summary of Mortality & S.aureus Load

Efficacy study of Ceftriaxone complex in S.aureus infected Swiss Albino mice Table 6 : Summary of Estimation of Bacterial Load

EXAMPLE 10 (Toxicity studies) (i) Acute Toxicity:

Studies in mouse have shown that Ceftriaxone complex and Cefazolin complexes produce less toxicity as compared with the Ceftriaxone sodium and Cefazolin sodium respectively. The intramuscular and intravenous median lethal dose (LD 50) of each of the complexes is greater than 5000mg/kg - body weight. The toxic effects observed are typical of cephalosporin i.e. decreased locomotor activity and ataxia. These effects are qualitatively and quantitatively similar to those observed in toxicity studies of Ceftriaxone sodium and Cefazolin sodium and may be attributed to high peak plasma concentration of the active agent. (ii) Subacute Toxicity

The Sprague Dawley rats and Swiss albino mice were administered the compounds at doses ranging from 30 - 900 mg/kg/day for four weeks through intramuscular and intravenous route respectively. There was no mortality in test system (rat/mice) of any treatment groups. Some signs of reaction to the treatment such as decreased locomotor activity, body weight etc. were recorded at the higher doses. The observed effects are attributable to the active constituent of the complex and are characteristic of cephalosporin. No significant treatment and dose related changes were observed in feed consumption,' haematology, urianalysis and organs weights. Marginal increased activity of alkaline phosphatase and serum glutamic pyruvic transminase were reported in rats of high dose group. However, compound treated mice did not exhibit any treatment-related alterations in parameters of clinical chemistry. Except minimal to slight hepatopathy, no treatment related pathological changes were identified in animals of compound treated groups. The observed alteration was recorded at the highest dose level (900 mg/kg/day).

The No Observed Effect Level (NOEL) for the Cefazolin complex was found to be ^• 100 mg/kg body weight and 30 mg/kg body weight for the Ceftriaxone complex. The above results indicate that the repeated dose toxicity studies in rat and mice revealed a wide safety margin in the therapeutic use of Cefazolin complex and Ceftriaxone complex.

CEFTRIAXONE-POLYSACCHARIDE COMPLEX 1. Acute Intravenous Toxicity Study

Swiss albino mice, approximately 7 weeks of age at the time of initiation of the study, were given doses of 0 or 5000 mg/kg body weight of ceftriaxone complex. Animals were randomly assigned to treatment groups and scheduled for necropsy following 14 days observation period. The drug was administered through the intravenous route. At the end of the study body weight and macroscopic pathology were determined. Administration of the dose of the composition caused reduced locomotor activity and ataxia with onset at 10 minutes after dosing and persisted up to 2 days after giving the dose of the composition. The median lethal dosage (LD₅₀) was greater than 5000 mg/kg body weight.

2.Acute Intra-muscular Toxicity Study

In another study, Swiss albino mice, approximately 7 weeks of age at the time of initiation of the study, were given doses of 0 or 5000 mg/kg body weight of ceftriaxone complex. Animals were randomly assigned to treatment groups and scheduled for necropsy following 14 days observation period. The drug was administered by the intramuscular route. At the end of the study body weight and macroscopic pathology were determined. Administration of the dose of the composition caused reduced locomotor activity and ataxia with onset at 10 minutes after dosing and persisted up to 4 days after giving the dose of the composition. The median lethal dosage (LD₅0) was greater than 5000 mg/kg body weight.

3.Sub-chronic Intravenous Toxicity Study

In another study, Swiss albino mice, approximately 8 weeks of age at the time of initiation of the study, were given doses of 0; 30; 100; 300 or 900-mg/kg body weight of Ceftriaxone Sodium Complex. Animals were randomly assigned to treatment groups and scheduled for necropsy following 28 days observation period. The drug was administered by the intravenous route. At the end of the study body weight, food consumption, urinalysis, hematology, clinical chemistry, organ weight and gross and macroscopic pathology were studied. The administration of the dose of the composition caused reduced locomotor activity and ataxia and decreased body weights were observed in mice given 300 or 900 mg/kg day. Mice from 900 mg/kg body weight treated group also evoked mild hepatopathy. The phenomenon was transient and species specific. No Observable Effect Level (NOEL) was about 100 mg/kg/day.

4.Sub-chronic Intramuscular Toxicity Study

In another study, Sprague Dawley albino rat, approximately 8 weeks of age at the time of initiation of the study, were given doses of 0; 30; 100; 300 or 900 mg/kg body weight of Ceftriaxone Sodium Complex. Animals were randomly assigned to treatment groups and scheduled for necropsy following 28 days observation period. The drug was administered by the intramuscular route. At the end of the study body weight, food consumption, urinalysis, hematology, clinical- chemistry, organ weight and gross and macroscopic pathology were studied. Reduced locomotor activity, ataxia decreased body weight were observed in mice given 100; 300 or 900 mg/kg/day. In addition to this rats from 900 mg/kg body weight treated group evoked decreased body weight. Marginal increased relative weight of kidney in males and decreased relative weight of liver in females of 900 mg/kg body weights was also observed. No treatment-related microscopic alterations were detected. The changes were not associated with morphologic representative of a systemic toxicologic response. The No Observable Effect Level (NOEL) was about 30 mg/kg/day. CEFAZOLIN-POLYSACCHARIDE COMPLEX: 1. Acute Intravenous Toxicity study

In another study, Swiss albino mice, approximately 7 weeks of age at the time of initiation of the study, were given doses of 0 or 5000 mg kg body weight of Cefazolin Sodium Complex. Animals were randomly assigned to treatment groups and scheduled for necropsy following 14 days observation period. The drug was administered by the intravenous route. At the end of the study body weight and macroscopic pathology were determined. The administration of the dose of the composition caused reduced locomotor activity and ataxia with onset at 10 minutes after dosing and persisted up to 4 days after dosing. The Median lethal Dose (LD₅₀) was about greater than 5000-mg/kg-body weight. 2. Acute Intramuscular Toxicity Study

In another study, Swiss albino mice, approximately 7 weeks of age at the time of initiation of the study, were given doses of 0 or 5000-mg/kg body weight of Cefazolin Sodium Complex. Animals were randomly assigned to treatment groups and scheduled for necropsy following 14 days observation period. The drug was administered by the intramuscular route. At the end of the study body weight and macroscopic pathology were determined. Administration of the dose of the composition caused reduced locomotor activity and ataxia with onset at 10 minutes after dosing and persisted up to 4 days after dosing. The Median lethal Dose (LD₅₀) was about greater than 5000-mg/kg-body weight. 3.Sub-chronic Intravenous Toxicity Study

In another study, Swiss albino mice, approximately 8 weeks of age at the time of initiation of the study, were given doses of 0; 100; 300 or 900-mg/kg body weight of Cefazolin Sodium Complex. Animals were randomly assigned to treatment groups and scheduled for necropsy following 28 days observation period. The drug was administered by the intravenous route. At the end of the study body weight, food consumption, urinalysis, hematology, clinical chemistry, organ weight, and gross and macroscopic pathology were determined. Reduced locomotor activity and ataxia and decreased body weight gain were observed in mice given 300 or 900 mg/kg day. In addition to this, female- rats from 900 mg/kg body weight treated group exhibited increased relative weight of adrenal. Mice from 900 mg/kg body weight treated group also evoked mild hepatopathy which was transient and species specific. The No Observable Effect Level (NOEL) was about 100 mg/kg/day. 4.Sub-chronic Intramuscular Toxicity Study

In another study, Sprague Dawley albino rat, approximately $ weeks of age at the time of initiation of the study, were given doses of 0; 100; 300 or 900-mg/kg body weight of Cefazolin Sodium Complex. Animals were randomly assigned to treatment groups and scheduled for necropsy following 28 days observation period. The drug was administered by the intramuscular route. At the end of the study body weight, food consumption, urine analysis, hematology, clinical chemistry, organ weight and gross and microscopic pathology were studied. Rats given 300 to 900 mg/kg bodyweight exhibited reduced locomotor activity, ataxia, decreased body weight and increased serum alkaline phosphate level. This increase in serum alkaline phosphatase values for treated rats were not associated with morphologic changes representative of a systemic toxicologic response pathological examination did not revealed any treatment related changes as compared with the control. The No Observable Effect Level (NOEL) was about 100 mg/kg/day.

Claims

CLAIMS:

1. A therapeutically effective pharmaceutical composition comprising Beta-lactam antibiotics and polysaccharides, and useful in the treatment of microbial infections.

2. A composition as claimed in claim 1 wherein, the Beta-lactam ring containing antibiotics are selected from the group comprising Cephalosporins such as Cefazolin

Sodium, Ceftriaxone Sodium, Cefoperazone Sodium, Cefotaxime Sodium, Cefuroxime Sodium, other cephlosporins and betalactums and their derivatives.

3. A composition as claimed in claim 1 wherein, the amount of Beta-lactam antibiotics is in the range of 20 to 70%.

4. A composition as claimed in claim 1 wherein, the polysaccharides are selected from the group comprising Arabinose, Agarose, Low Molecular Weight dextran, Activated clinical dextrans, Inulin, Chitin, Chitosan, Mannan, Lichenin and their derivatives / variants.

5. A composition as claimed in claim 1 wherein, the composition can be formulated in physical forms such as injectibles, tablets, or other forms.

6. A composition as claimed in claim 1 wherein, the composition exhibits enhanced in- vitro activity as determined by minimum inhibitory concentration assays.

7. A composition as claimed in claim 1 wherein, the composition has in vitro activity comparable to the parent drug in animal models.

8. A composition as claimed in claim 1, which is non-toxic in animals and humans.

9. A process for the production of 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 the antibiotic-polysaccharides solution under vacuum to obtain antibiotic polysaccharides complex as a dry solid.

10. A process as claimed in claim 9 wherein, the Beta-lactam polysaccharide complex formation is activated by use of cynogen bromide.

11. A process as claimed in claim 9 wherein the Beta-lactam rjng containing antibiotics arc selected from the group comprising cephalosporins, cefazolin, cephradine, ceftriaxone, cefoperazone, cefotaxime, other cephlosporins and betalactums and their derivatives.

12. A process as claimed in claim 9 wherein the polysaccharides are selected from the group comprising Arabinose, Agarose, low molecular weight dextran, activated dextrans, inulin, chitin, chitosan, mannan, lichenin, other polysaccharides and their derivatives.

13. A process as claimed in claim 9 wherein the proportion of Beta-lactam compounds to polysaccharides ranges between 20 - 70%.

14. A process as claimed in claim 9 wherein the proportion of polysaccharides in the composition ranges from 80 - 30%.

15. A process as claimed in claim 9 wherein the reaction is effected a pH of about 4.5 to 7.5.

16. A process as claimed in claim 9 wherein the solvent is water.

17. A process as claimed in claim 9 wherein the buffer added to the reaction mixture is sodium bicarbonate, sodium carbonate and triethanolamine.

18. A process as claimed in claim 9 wherein the complex is obtained by freeze drying under temperature ranging between -40°C to +35°C.

19. A process as claimed in claim 9 wherein which results in the complexes which have enhanced in vitro activity as determined by minimum inhibitory concentration assays.

20. A process as claimed in claim 9 which results in the complexes, which have comparable in vivo activity to that of parent drug in animal models.

21. A process as claimed in claim 9 wherein, the complex is obtained through lyphilization.