An antimicrobial composition for managing bacterial infections

Abstract

The present invention relates to an antimicrobial composition for managing bacterial infections. More particularly, the present invention relates to the antimicrobial composition comprising of a cephalosporin in combination with a beta lactamase inhibitor and a chelating agent. The antimicrobial composition of beta lactam in combination with beta lactamase inhibitor and a synergistic concentration of chelating agent with pharmaceutically acceptable ingredients is effective in management of bacterial infections in hospital settings.

Description

[0001] “AN ANTIMICROBIAL COMPOSITION FOR MANAGING BACTERIAL INFECTIONS”

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of pharmaceutical compositions. More particularly, the present invention relates to an antimicrobial composition comprising of a cephalosporin in combination with a beta lactamase inhibitor and a chelating agent for effectively managing bacterial infections.

[0004] BACKGROUND OF THE INVENTION

[0005] Multidrug resistance (MDR) is acquired by microorganisms as a response to lethal doses of antimicrobial drugs and is a leading concern in public health with many infectious diseases regressing back to the pre-antimicrobial drugs era. The MDR organisms which are most threatening to public health are MDR bacteria that develop by the accumulation of resistant genes on R plasmids, where each one of the genes corresponds to a single drug. There are different types of MDR bacteria depending on their degrees of resistance including extensively drug resistant (XDR) and pan drug resistant (PDR) wherein XDR refers to a single bacterium being resistant to all antibiotics except for one or two classes of antibiotics and PDR bacteria are non-susceptible to all classes of antibiotics.

[0006] Beta lactams are a class of antibiotics and have been utilized for the treatment and management of bacterial infections. This class further includes Penicillin, Cephalosporins, Carbapenems, Monobactams. The prior arts have discussed the usage of beta lactams for an efficient management of bacterial diseases. But the beta-lactamase enzymes in bacteria cleave the beta lactam rings of the antibiotics thus, inactivating them and building resistance. Corresponding to the increasing resistance to cephalosporins, more recent efforts have been focused on combining some cephalosporins with p-lactamase inhibitors to widen the antimicrobial activity.

[0007] US11559514B2 disclosed antibacterial compositions and methods of treating bacterial infections caused by resistant bacteria comprising at least one carbapenem or other p- lactam binding to allosteric site of PBP2a, at least one p-lactamase inhibitor, at least one P-lactam binding to active site of PBP2a, and one pharmaceutically acceptable excipient wherein the at least one -lactamase inhibitor is selected from the group consisting of clavulanic acid (clavulanate), sulbactam, tazobactam and avibactam, and the at least one P-lactam that binds the open configuration of the active site of PBP2a is selected from the group consisting of cefepime, cefozopran, cefpirome, cefquinome, ceftaroline, and ceftobiprole, and the excipient may be preservatives like antioxidants, such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate, citric acid, sodium citrate; chelators such as EDTA or EGTA; and antimicrobials, such as parabens, chlorobutanol, or phenol. However, the cited invention discloses a combination of piperacillin with tazobactam that manifests into adverse events like gastrointestinal issues such as diarrhea and skin reactions.

[0008] US11471422B2 disclosed a composition for targeted delivery of poorly bioavailable therapeutic agents comprising of stealth targeted nanoparticles, wherein said stealth targeted nanoparticles comprise of a non-lipidic polymer matrix, a non-protein targeting agent / ligand, one or more than one poorly bioavailable therapeutic agent wherein the therapeutic agent could be a drug which could be a combination of cephalosporin and one or more of co-drug non - beta lactamase inhibitors, wherein the said beta-lactam antibiotic is selected from the group consisting of ceftriaxone, cefepime, ceftazidime, cefuroxime, cefexime, cefpodoxime, cefaclor, cefoperazone, cefadroxil, ceftibuten, cefdinir, cefditoren, ceftizoxime, cefamandole, cefazolin, cefonicid, cefoxitin, cefprozil, cephalexin, cephapirin, ceftobiprole, ceftolozane, ceftaroline, cephradine, and pharmaceutically acceptable salts thereof and the said co-drug beta-lactamase inhibitor is selected from the group consisting of sulbactam, tazobactam, clavulanic acid, avibactam, relebactam, RPX709, NXL104, AVE1330A, and pharmaceutically acceptable salts (EDTA), isomers, and derivatives thereof. However, the cited document discloses a combination of ceftriaxone and avibactam that a short half-life which would require frequent administrations.

[0009] US20200297824A1 disclosed a method of protecting a patient's gastrointestinal microbiome, comprising, administering an effective amount of a pharmaceutical composition comprising a beta-lactamase to a patient, further comprising administering beta-lactame or in combination of beta-lactamase inhibitor in need thereof wherein the beta-lactam antibiotic is a cephalosporin which includes cefepime (5-200mg) and beta- lactamase inhibitor is one or more of clavulanic acid, tazobactam, sulbactam, avibactam, or EDTA. However, the cited invention fails to disclose an effective combination of cephalosporins with beta-lactamase inhibitors.

[0010] W02020227530A1 disclosed a pharmaceutical composition comprising a pyrimidine, an antimicrobial agent and / or an antiviral agent for non-chemotherapeutic use, or a pharmaceutically acceptable salt thereof and a pharmaceutical acceptable carrier wherein the antimicrobial agent includes but not limited to cefepime (2.0 gm), and pharmaceutically acceptable salts include but not limited to EDTA, and the composition also comprise of b-lactamase inhibitor which includes but is not limited to avibactam. However, the cited invention fails to disclose a stable working concentration of beta lactamase inhibitorfor a reduced MIC.

[0011] IN202341066970 disclosed a pharmaceutical composition comprising amorphous Avibactam Sodium of Formula (I) and its pharmaceutically acceptable salts and fimher comprising said B-lactamine antibiotic including but not limited to cefepime wherein the formulation may also contain a chelating agent like EDTA. However, the cited invention discloses a combination of Avibactam Sodium with Ceftazidime, which has adverse side effects.

[0012] Therapeutically, effective use of antibiotics, a cornerstone of modern medicine, is threatened by the increasing prevalence of antibiotic-resistant and / or antibiotic-tolerant microbes. The existing antibiotic compositions include several drawbacks such as low halflife that can be compensated by increased frequency of administration or by increasing the doses, which result in adverse side effects. Further, the ratio of cephalosporin to beta lactamase inhibitor is critical for the bactericidal effect of cephalosporin.

[0013] Therefore, a need exists for compositions that are stable, have reduced antibiotic MIC, have minimal side effects and / or improved approaches that can potentiate the efficacy of antimicrobial agents, both in vitro and in vivo.

[0014] OBJECT OF THE INVENTION

[0015] The main object of the present invention is to provide an antimicrobial composition and a method of preparation thereof. Another object of the present invention is to provide an antimicrobial composition comprising of a cephalosporin in combination with a beta lactamase inhibitor and a chelating agent.

[0016] Yet another object of the present invention is to provide an antimicrobial composition comprising of cefepime, avibactam and EDTA in the form with enhanced stability and decreased MIC.

[0017] Yet another object of the present invention is to provide an antimicrobial composition comprising of cefepime, avibactam and EDTA in the form of injection.

[0018] Still another object of the present invention is to provide an antimicrobial composition comprising of the cephalosporin in combination with the beta lactamase inhibitor and the chelating agent for management of bacterial infections.

[0019] SUMMARY OF THE INVENTION

[0020] The present invention relates to an antimicrobial composition for effective management of bacterial infections. More particularly, the present invention relates to the antimicrobial composition comprising of a cephalosporin in combination with a beta lactamase inhibitor and a chelating agent.

[0021] In an embodiment, the present invention provides an antimicrobial composition comprising of a cephalosporin; a p-lactamase inhibitor; and a chelating agent. Here, said cephalosporin is cefepime in an amount ranging from 1800 mg to 2000 mg; said betalactamase inhibitor is avibactam in an amount from 450 mg to 500 mg; and said chelating agent is ethylene diamine tetracetic acid (EDTA) in an amount ranging from 33.75 mg to 41.25 mg. More particularly, the cephalosporin is essentially in an amount of 2000 mg, beta-lactamase inhibitor is essentially in an amount of 500 mg and the chelating agent is essentially in an amount of 37.5 mg. Further, said antimicrobial composition further includes pharmaceutically acceptable excipients selected from a group of a solubilizing agent, a pH buffer, a stabilizer, a solvent or a combination thereof.

[0022] The antimicrobial composition of the present invention effectively controls bacterial infections like gastroenteritis, skin and soft tissue infections, urinary tract infections, bacterial pneumonia and sexually transmitted infections with reduced minimum inhibitory concentration in a range of 0.055-0.50 gg / mL. The present invention relates to an antimicrobial composition for the management of bacterial infections in hospital settings. The antimicrobial composition includes a cephalosporin in combination with a beta lactamase inhibitor in a ratio of 4:1 and a chelating agent. Here, the chelating agent is in a concentration of 3 mg / mT.

[0023] The above objects and advantages of the present invention will become apparent from the hereinafter set forth detailed description of the invention, and claims appended herewith.

[0024] DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention will now be described hereinafter with reference to the accompanying drawings in which a preferred embodiment of the invention is shown. This invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough, and will fully convey the scope of the invention to those skilled in the art.

[0026] The present invention now will be described hereinafter with reference to the detailed description, in which some, but not all embodiments of the invention are indicated. Indeed, the invention may be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout. The present invention is described fully herein with nonlimiting embodiments and exemplary experimentation.

[0027] The present invention relates to an antimicrobial composition for effective management of bacterial infections. More particularly, the present invention relates to the antimicrobial composition comprising of a cephalosporin in combination with a beta lactamase inhibitor and a chelating agent.

[0028] In a preferred embodiment, the present invention provides an antimicrobial composition comprising of a cephalosporin; a p-lactamase inhibitor; and a chelating agent.

[0029] Here, said cephalosporin is cefepime in an amount ranging from 1800 mg to 2200 mg; said beta-lactamase inhibitor is avibactam in an amount ranging from 450 mg to 550 mg; and said chelating agent is ethylene diamine tetraacetic acid (EDTA) in an amount of 33.75 mg to 41.25 mg. Preferably, said cephalosporin is in an amount of 2000 mg; said betalactamase inhibitor is in an amount of 500 mg; and said chelating agent is in an amount of 37.5 mg.

[0030] Further, the chelating agent is present in a synergistic amount. The cephalosporin and the non-beta lactamase inhibitor are in a defined ratio of 4: 1 and the chelating agent is present in a concentration of 3 mg / mL. The chelating agent (EDTA) in synergistic amount reduces the MIC of antibiotics (cefepime and avibactam as used herein) and breaks the biofilm of resistant bacteria. The antimicrobial action of EDTA primarily involves chelating essential divalent cations like magnesium and calcium, disrupting bacterial cell walls or biofilms that lead to increased membrane permeability, enhancement of the activity of other antimicrobial agents and potentially reducing bacterial viability. EDTA also inhibits biofilm formation and disrupt existing biofilms by disrupting the extracellular polymeric substance a major component of the biofilm matrix, and by enhancing the ability of the antimicrobial agents like cefepime, avibactam to penetrate and kill biofilm cells. Hence, EDTA enhances the efficacy of the antimicrobial agents like cefepime, avibactam by increasing the penetration into bacteria or by disrupting the resistance mechanisms. Therefore, EDTA is acting as a potentiating and sensitizing agent in combination with cefepime and avibactam.

[0031] Moreover, said antimicrobial composition further includes pharmaceutically acceptable excipients selected from a group of a solubilizing agent, a pH buffer, a stabilizer, a solvent or a combination thereof. Preferably, said solubilizing agent is L-arginine, said pH buffer is sodium bicarbonate, said stabilizer is sodium citrate, and said solvent is sterile water for injection or a combination thereof.

[0032] The antimicrobial composition of the present invention is in the form of a powder or an injection and is effective against bacterial infections. Said antimicrobial composition exhibits minimum inhibitory concentration (MIC) value of 0.50 pg / mL against P. aeruginosa, in a range of 0.047-0.064 pg / mL against E. colt, in a range of 1.0-1.5 pg / mL against 5. aureus,' and in a range of 0.125-0.19 pg / mL against K. pneumoniae. Additionally, said antimicrobial composition exhibits zone of inhibition ranging from 21 mm - 35 mm. Hence, the antimicrobial composition of cefepime, avibactam and EDTA effectively controls bacterial infections like gastroenteritis, skin and soft tissue infections, urinary tract infections, bacterial pneumonia and sexually transmitted infections with reduced minimum inhibitory concentration in a range of 0.055-0.50 pg / mL.

[0033] EXAMPLE 1

[0034] For Experimentation Details Method of preparation of powder injection

[0035] Selection of ingredients or raw materials

[0036] The active pharmaceutical ingredients (API) include: cefepime hydrochloride in an amount equivalent of 2 gm (2000 mg), avibactam sodium in an amount of 0.5 gm (500 mg) and disodium EDTA in an amount of 37.5 mg. The antimicrobial composition of cefepime, avibactam and disodium EDTA further includes pharmaceutical acceptable excipients, as given in Table 1. Disodium EDTA is a chelating agent that enhances antibacterial activity, prevents degradation and is a biofilm breaker. L-arginine is a solubilizing agent that is employed with beta-lactams and also act as pH buffer and stabilizer. Sodium citrate is a secondary buffer, while sodium bicarbonate is another pH buffer and stabilizer to maintain alkaline pH for cefepime stability. Sterile water for injection (SWFI) is taken for reconstitution. Nitrogen gas is an inert gas that is employed during filling of vials to displace oxygen and to prevent oxidation. L-arginine was taken in an amount of 150 mg; sodium citrate and sodium bicarbonate was taken as q.s. to pH 6.5- 7.0, sterile water for injection (SWFI) and nitrogen gas were also taken as q.s. in the antimicrobial composition.

[0037] Sterilization and depyrogenation of primary packing material

[0038] Before depyrogenation, equipments were cleaned. The glass vials were depyrogenated with the help of depyrogenation tunnel. The flip of seal and rubber plug were sterilized through autoclave.

[0039] Preparation and blending of API

[0040] Before preparation of blending, equipments were cleaned and the systematic process was followed for assessing the cleanliness and control of the manufacturing environment to ensure product safety and compliance with regulatory standards. The key factors involved were: air quality (particulate and microbial contamination), surface cleanliness (equipment, workbenches, walls and floors), personnel hygiene (gowning and behavior) and water quality taken for API production. The different raw materials were taken in required quantity, as per calculation and were transferred to a blender for blending. After blending, the raw material sample was collected by IPQA from the blender and was sent to QC for testing the uniformity content.

[0041] Filling raw material sample into glass vials

[0042] The equipments were cleaned and the surrounding environment was monitored before filling the sample into glass vial. The sterile powder sample was filled aseptically under controlled area (local aseptic processing) into a depyrogenated glass vial. After filling, the filled glass vials were closed with a stopper followed by sealing with the help of a sealing machine.

[0043] Visual inspection of the filled glass vials

[0044] The area and inspection table was cleaned before performing visual inspection. The visual inspection was performed by a qualified visual inspector. The filled and sealed glass vial were inspected visually to check any breakage of a filled glass vial, sealing quality, fill weight, etc.

[0045] Quality control testing

[0046] Sterility testing was done to ensure that no microbial contamination took place. Potency testing was performed to verify the correct drug dosage. Particulate testing was done to check for the absence of visible particles. The reconstitution testing confirmed that the powder sample was dissolved properly and retained potency. Average fill weight testing was done to confirm fill weight as per BMR limit and other test parameter was performed, as per the finished product specification.

[0047] Packaging and labelling of the sealed glass vials

[0048] The packing activity was performed, as per BPR. The area was cleaned followed by environment monitoring. The labelling of the filled and sealed glass vial was checked, as per BPR frequency. All parameters like batch number, manufacturing date and expiry date and related information was checked, as per BPR frequency. The product was packed, as per BPR and the finished product sample was collected and sent to QC for complete analysis. After receiving COA from QC, the batch was released.

[0049] EXAMPLE 2

[0050] Method of Analysis of cefepime and avibactam with EDTA for injection (2.5 gm)

[0051] Sampling

[0052] The sampling was done during initial filling, after regular interval of one and finally at the end of the filling. Equal quantity of sample was taken during initial filling, after regular interval of one and finally at the end of the filling. The samples were sent to micro lab and during packing, after completion of 20%, 50% and 80% of the process, a composite sample was collected specifically for chemical testing. The composite sample was placed in a poly bag labelled as “Sample for Analysis” along with a sample information label.

[0053] Total sample and label claim

[0054] The total number of samples taken was 60 vials. Each vial contains: cefepime hydrochloride (sterile) IP equivalent to anhydrous cefepime = 2000 mg; avibactam sodium (sterile) IP equivalent to anhydrous avibactam = 500 mg; and disodium EDTA (IP) equivalent to 37.5 mg. The samples were stored at a controlled room temperature not exceeding 30°C.

[0055] Tests and method of analysis

[0056] Table 1 shows parameters taken for method of analysis.

[0057] Table 1: Parameters for method of analysis

[0058] Description A white to pale yellow powder sample was filled in clear and colorless glass vial. An approximately 100 mg of sample was taken in a clean and dry petri dish, was spread uniformly and finally, the color and nature of the sample was observed visually.

[0059] Identification by high performance liquid chromatography (HPLC)

[0060] The retention time of the major peak of the sample solution corresponds to that of the standard solution as obtained in the assay.

[0061] Average fill weight: ±2% of targeted fill weight

[0062] Any adhering labels were removed from a container and the outside of the container was washed and dried. The container was opened and immediately weight of the container and weight of contents of the container was taken. The container was emptied as completely as possible by gentle tapping and rinsed initially with water and then with 95% ethanol and dried at a temperature of 100-105°C for 1 hour, if the nature of the container precluded such treatment to constant weight. The container was then cooled in desiccator and weighed. The difference between the weights represented the weights of the contents. The procedure was repeated with further 19 containers and the average weight was determined by the formula having equation (1).

[0063] Average weight = (Sum of individual content of 20 vials) - 20 . (1)

[0064] Uniformity of fill weight: ±10% of average fill weight

[0065] The minimum and maximum %age was calculated by formula having equations (2) and (3):

[0066] Minimum %age = ((Minimum individual weight - Average fill weight) x 100) / A verage fill weight . (2)

[0067] Maximum %age = ((Maximum individual weight - Average fill weight) x 100) / A verage fill weight . (3)

[0068] Sterility

[0069] Media employed was as follows: soybeans casein digest media (30 gm for 1000 mT) and fluid thioglycolate media (29.75 gm for 1000 mT). The apparatus includes a suitable unit consisting of a closed reservoir and a receptacle between which a properly supported membrane of appropriate porosity was placed. For sterility testing, the membrane (cellulose nitrate) of pore size was more than 0.45 micron and diameter of approximately 50 mm.

[0070] Membrane filtration method

[0071] 1 mL of stock solution of organism (S. aureus, B. subtilis and C. albicane) was inoculated into 99 mL sterile normal saline solution. The obtained solution was then filtered through 0.45 micron cellulose nitrate filter paper and the filtrate was collected into sterile flask, the filter paper was cut into two pieces and the filter paper was inoculated into tubes of thioglycolate and soybean casein digest medium. The same procedure was repeated for each of the organisms. The tubes were inoculated for 14 days and were observed daily and no growth was observed in filtrate up to 14 days.

[0072] Particulate matter

[0073] The particulate matter was analyzed by liquid particle counter. The sample was observed visually for checking the visible particulate matter and the constituted solution was essentially free from particle of foreign matter. For the sub visible particulate matter, it was observed that the particles of size >10 pm were present in a range of not more than 6000 particles per vial and particles of size >25 pm were present in a range of not more than 600 particles per vial.

[0074] Blank preparation and analysis

[0075] (1) Before analyzing a test sample, a blank test for system was suitably done.

[0076] (2) Glassware was thoroughly cleaned with non-ionic detergent and was made free from any residue of water.

[0077] (3) The filtered purified water not more than 500 mL was taken for blank test.

[0078] (4) The sample was degassed sonicating for about 30 seconds.

[0079] (5) The sample was analyzed against the white and black background for black particle, white particle, fibre particle and any other visible particle.

[0080] If any type of visible particles were present in the preparation, then the preparation was discarded and points (2 to 4) were repeated until a sample free from any visible particle was obtained. For testing of sub visible particle, the sample was put on the liquid particle counter’s stage. The sample syringe was put into the sample and put a magnetic stirrer into the sample. A bottle was taken for collection of waste and the waste generation syringe was put into the waste collection waste bottle.

[0081] Test preparation and analysis

[0082] After the blank analysis, again the system was flushed with water and proceeded for test sample analysis. The samples were put on the laminar air flow workbench provided for liquid particle count; the stickers and labels were removed by dipping into warm water. Each sample vial was reverted for approximately 20 times followed by addition of sterilized water of injection as directed in the labeling and each sample was analyzed against the white and black background for black particle, white particle, fiber particle and any other visible particle. After visual inspection, the outer surface of container opening was cleaned by a jet of particle free water and by avoiding any contamination of the content, the closure of the container was removed. Then, the test sample was analyzed, as per described in the section blank preparation and analysis.

[0083] Test sample quantity and preparation

[0084] The content of 10 units was combined in a clean container. The sample was degassed by sonicating for about 30 seconds.

[0085] Constituted solution

[0086] Completeness of solution: The solid was dissolved completely, leaving no visible residue as undissolved matter.

[0087] Clarity of solution: The constituted solution was not significantly less clear than an equal volume of the diluents and sterile water for injection contained in a similar vessel and examined similarly.

[0088] 5 sample vials and 1 blank vial were taken. Sterile water for injection was added as directed in the labelling and was filtered through 0.2 pm filter. The solution was mixed well and the sample was completely dissolved. The air bubbles were removed with the help of sonicator. Now, the sample vial with blank was allowed to stand and the sample vial was observed against black and white background to check the presence of any undissolved matter. The solution was as clear as the blank vial containing sterile water for injections in the sample vial and in visible particulate matter.

[0089] Assay of cefepime determined by liquid chromatography

[0090] The chemicals employed were monobasic potassium phosphate, phosphoric acid, acetonitrile, potassium hydroxide and water. Solution A was prepared by mixing 0.68 mg / mT of monobasic potassium phosphate in water. Solution B was prepared by mixing acetonitrile and solution S in a ratio of 1 :9 followed by adjusting with 2% phosphoric acid or 2% potassium hydroxide to a pH of 5.0. Solution C was prepared by mixing acetonitrile and solution A in a ratio of 1:1 followed by adjusting with 2% phosphoric acid or 2% potassium hydroxide to a pH of 5.0. Table 2 depicts the concentration of solution B and solution C in the mobile phase with respect to time.

[0091] The standard solution was prepared as follows: weighed 185 mg of cefepime hydrochloride working standard and 28 mg of avibactam sodium working standard in a 100 mT volumetric flask and diluted up to mark with solution B. Further, 5 mF of the obtained solution was diluted in a 50 mL volumetric flask up to mark with solution B.

[0092] The sample solution was prepared as follows: Weighed 215 mg of the sample in a 100 mL volumetric flask and diluted up to mark with solution B.

[0093] The parameters involved in the chromatographic system were as follows: mode=LC; UV detector of wavelength 254 nm; column od dimension 4.6 mm x 25 cm and 5 pm packing LI; flow rate of ImL / minute; injection volume of 10 pL; system suitability sample: standard solution; suitability requirements: tailing factor of NMT 1.5, relative standard deviation of NMT 2.0%; and analysis samples: standard solution and sample solution.

[0094] Table 2: Mobile phase

[0095] Assay: EDTA

[0096] The reagents and chemicals were 2 M hydrochloric acid (HC1), 0.1 M lead nitrate, hexamine, xylenol orange triturate. The solution of HC1 was taken by diluting 17.0 mT of HC1 with 100 mT of purified water.

[0097] An amount equivalent to 0.35 gm of EDTA (13 gm of test powder) was dissolved in sufficient water to produce 300 mT and sonicated for 10-15 minutes in sonicator, added 2 gm of hexamine and 2 mT of 2M HC1. Titrated with 0.1 M lead nitrate using 50 mg of xylenol orange triturate as indicator. A blank titration was performed. 1 mT of 0.1 M lead nitrate was equivalent to 0.03362 gm of Ci0Hi4N2Na2O8.2H2O. The calculation was done by the formula having equation (4):

[0098] %age or concentration of analyte= (Volume x Factor x Normality x Avg. wt.) / (0.1 M x sample wt.) . (4)

[0099] Where, the product of Volume, Factor and Normality gave the number of equivalents (or moles, after adjusting for stoichiometry) of titrant used in the reaction. Multiplication by average weight converted the equivalents into a mass (or a comparable measure) of the analyte. The obtained product divided by 0.1 M (the normalization constant) and sample weight adjusted the calculated mass relative to both the standardized titrant concentration and the amount of sample taken. The final result was often expressed as a percentage or concentration of the analyte in the sample and essentially, depicted that how much of the substance was present based on how much titrant was required for complete reaction.

[0100] EXAMPLE 3

[0101] Synergistic Effect of the antimicrobial composition of cefepime, avibactam and EDTA Synergy testing by disk diffusion testing

[0102] Qualitative measurement of the drug interaction was performed by the disk diffusion method. The advantage of the disk diffusion method was ease of performance in a clinical laboratory and utilization of commercially available antimicrobial-impregnated disks and media. The technique utilized the same inoculum and Mueller-Hinton agar as routine Bauer-Kirby susceptibility testing. Disks impregnated with individual antimicrobial agents were placed at a distance equal to the sum of the zone radii of inhibition of drugs when tested separately. After overnight incubation, the interface of zone of inhibition was examined. Synergism showed an enhancement or bridging at or near the junction of the two zones of inhibition, or inhibition of growth only due to the combined effects in compound D of all the components (cefepime, avibactam and EDTA), as depicted in Table 3. In conclusion, the results suggest that the combination of cefepime and avibacatam with EDTA exhibits enhanced antimicrobial effect in effective management of bacterial infections.

[0103] Procedure

[0104] The agar and antimicrobial disks were allowed to warm to room temperature. Then, by a sterile loop or swab, three to five colonies of the bacterial isolate was transferred to 5 mL of sterile CSMHB and incubated at a temperature of 35°C until a turbidity that was equivalent to a 0.5 McFarland standard was achieved (1.5 x 108CFU / mL). Next step was the inoculation of agar plate by dipping a sterile cotton-tipped swab into the inoculum within 15 min after adjusting the turbidity of the inoculum suspension and rotating for several times. The excess inoculum was removed by pressing the swab against the wall of the tube above the liquid. The swab was steaked over the entire agar surface three times, the plate was rotated approximately 60° each time to ensure even distribution of inoculum. Finally, the rim of the agar was swabbed. The inoculated plate was allowed to stand for 3-15 minutes before the application of the disks. Further, the disks were applied to inoculated agar plates by a sterile forceps where the disks were separated by a distance equal to the sum of the zone of radii for each disk tested separately. Each disk was pressed down to ensure complete contact with the agar surface. The disk was not moved once a contact was made with the agar surface. Next, the inoculated plates were incubated at a temperature of 35°C ± 2°C for 16-18 hours in an ambient air incubator. The plates were inverted (lids facing down), and care was taken that the stack was not more than five high. In the end, the plates were examined for a confluent lawn of growth and the interface of the zones of inhibition was observed.

[0105] Table 3: Synergy study report by disk diffusion assay

[0106] EXAMPLE 4

[0107] Method of antimicrobial susceptibility testing of MIC strips (LML Gradient Strip)

[0108] Intended Use The LML gradient strip was intended for the quantitative determination of the antimicrobial susceptibility of microorganisms, i.e., to determine the Minimum Inhibitory Concentration (MIC) of antimicrobial agent in pg / mL. The strip was coated with exponentially increasing concentration of antimicrobial agent on a predefined scale.

[0109] Principle The working principle of LML gradient strip was based on a combination of dilution and diffusion concepts in antimicrobial susceptibility testing. Although the methodology was similar to disk diffusion method, the gradient strip was not based on diffusion principles; the strip generates stable and increasing concentration of antimicrobial agent (as defined on the scale) immediately after having a contact with culture medium. With gradient strip the MIC was perceived as, where the ellipse of inhibition meets the drug concentration on the strip as pg / mL.

[0110] Storage All packages were stored either at controlled room temperature range of 18-22°C, in a refrigerator (at a temperature range of 4-8°C) or freezer (< -20°C) as specified on the product label, until the given expiry date. All LML strips immediately after opening the package were utilized. If any strips were left over from an opened package, the strips were either re-sealed or placed in a desiccated airtight storage container, and stored at the temperature stated on the label or at < -20°C. The strips were utilized until the expiry date if properly stored and handled. The presence of moisture exhibited a high level of detrimental effect on the performance quality of antimicrobial gradient strip.

[0111] Handling

[0112] The package was allowed to reach room temperature and waited until the complete evaporation of water condensation on package surfaces. It was made sure that the package was intact before opening the gradient strips. The package was opened through tear notch and the strip was handled only at top logo area with the help of clean forceps. The exposure of the active surface (the side that does not contain any labelling) of the strip with any medium other than the seeded culture medium was avoided.

[0113] Procedure

[0114] Two or three morphologically similar colonies preferably from the non-selective culture medium were selected. Normal saline was inoculated and turbidity was adjusted to 0.5 McFarland standard. A lawn culture was prepared on Mueller Hinton Agar (MHA) within 15 minutes of inoculum preparation. Within 15 minutes, the lower end (with the lowest concentration of antibiotic) of the gradient strip was carefully placed and then the remaining portion of the strip was rolled onto the agar surface; care was taken that the active side of the entire strip exhibited a good contact with the medium. Care was taken that no adjustment was done on the position of gradient strip once a contact with the medium was established. One or two strips were placed on 90 mm plate and up to 6 strips were placed on 150 mm plate. The culture plates were incubated in an inverted position at a temperature of 35±2°C. Unless specifically indicated, the results were always read between 16-18 hours of incubation (for all non-fastidious organisms).

[0115] Reading the MIC After incubation, the values obtained for minimum inhibitory concentration (MIC) were read from where the ellipse of inhibition meets the drug concentration on the strip. The test was repeated in case of contamination, too light or too heavy or uneven inoculum / growth. The gradient strip MIC endpoints were usually unambiguous; however, the reading rules mentioned below herein were followed:

[0116] • For bactericidal drugs (e.g. E-lactams), the MIC were read at the point of complete inhibition of growth, including hazes, microcolonies and isolated colonies.

[0117] • For bacteriostatic drugs, the endpoints were read at 80% inhibition, i.e. the initial point of significant inhibition.

[0118] • For bactericidal drugs, when macrocolonies were observed within the ellipse, all macrocolonies were read up to 3 mm from the strip.

[0119] • When there was no ellipse of inhibition, i.e. , growth occurred along the entire strip, the MIC was read as > the highest concentration on the MIC scale.

[0120] • When the ellipse of inhibition is below the strip (does not meet the strip), the MIC was read as < the lowest value on the MIC scale.

[0121] • Hemolysis, swarming growth and a thin line of growth alongside the strip were ignored to read the values for MIC.

[0122] • When the ellipse of inhibition meets the strip between two different concentrations, the next upper value was read as MIC.

[0123] • When the ellipse of inhibition is uneven on both sides of the strip, i.e., indicating two different values, the upper value was read as MIC; further, when the deviation is greater than one fold repeat the test.

[0124] • For E-lactamase inhibitor combinations, extrapolate the upper ellipse of inhibitor to read appropriate MIC.

[0125] Interpretation of Results

[0126] Fatest available CLSI or EUCAST susceptibility breakpoints were employed for the interpretation of MIC results. In case, the gradient MIC value was between the standard two-fold dilutions, the value was rounded to the next upper two fold value for categorization as Susceptible, Intermediate, or Resistant.

[0127] Quality Control

[0128] The gradient strip and test procedure were considered satisfactory when the observed MIC values fall within the acceptable MIC range provided for quality control (QC) strains. The patient results were not reported in the case of QC failure.

[0129] Table 4: Cefepime / Avibactam / EDTA concentration gradient observed MIC (Media: MHA and incubation temperature: 37°C) (EXAMPLE 5

[0130] Experimental analysis of minimum inhibitory concentration MIC)

[0131] Microbial culture Escherichia coli (NCIM 2065), Pseudomonas aeruginosa (NCIM 2200), Klebsiella, pneumoniae (NCIM 5663) and Staphylococcus aureus (NCIM 5345) were taken. NCIM stands for National collection of industrial microorganisms; and MTCC stands for Microbial type culture collection.

[0132] Media and diluent

[0133] Soybean casein digest agar (SCDA), Mueller hinton agar (MHA) and normal saline with a concentration of 0.9% were taken.

[0134] Accessories

[0135] Calibrated micropipette, sterile micropipette tips of 100 pT and 1.0 mL, sterile cotton swab, sterile disposable petri plates of dimension 15 mm x 100 mm, Bunsen burner, inoculating loop, conical flask, sterile scissors, sterile forceps, sterile disposable gloves, borosilicate glass tube of dimension 15 mm x 180 mm, vortex, 70% of filtered isopropyl alcohol (IP A) and test tube stand.

[0136] Instrument and equipment

[0137] A weighing balance, pH meter, horizontal autoclave, dynamic pass box, biosafety cabinet, Biological oxygen demand (BOD) incubator 37°C, colony counter, refrigerator, micropipette of capacity range 10-1000 pl and vertical autoclave were taken.

[0138] Method for analysis of MIC

[0139] Step 1. Preparation of microbiological media

[0140] The media and diluent were prepared and sterilized. The sterilized molten medium was cooled to 45-50°C, poured in sterile, dry petri plates and was allowed to solidify. The petri plates containing media was avoided from drying in laminar flow and was immediately taken for swabbing.

[0141] Step 2. Preparation of inoculum

[0142] Microorganisms were sub-cultured from working cultures stored at 2-8°C. Once microorganism was sub-cultured, the organism was avoided from passing more than three times. This means that the total passages does not exceed five times. The growth of slant of SCDA or equivalent with the help of sterile normal saline was harvested. The culture suspension was prepared for all the mentioned microbial cultures. The concentration of the microbial inoculum was confirmed by comparing the turbidity of inoculum with the standard 0.5 McFarland. The alternate method for standardizing the inoculum is optical method of 0.08-0.13 OD turbid suspension at 620 nm.

[0143] Step 3. MIC test procedure

[0144] The media plates of Mueller hinton agar for rapidly growing aerobic organisms were prepared as mentioned above. The inoculum swab was made ready by dipping a sterile non-toxic cotton swab into the standardized inoculum tube and the soaked swab was rotated firmly against the upper inside wall of the tube to remove excess fluid. The swab was avoided from dripping wet. The entire agar surface of Mueller hinton agar plate was inoculated with the swab three times and turning the plate at 60 degree angle between each streaking. The Cefepime / avibactam / EDTA MIC strip container was taken from cold storage at -20°C. Before opening, the strip was kept at room temperature for 15 minutes. Thereafter, an applicator was taken and the broader sticky side of applicator was gently pressed on the centre of Cefepime / avibactam / EDTA MIC strip. The applicator along with attached Cefepime / avibactam / EDTA MIC strip was lifted and the strip was placed at a desired position on agar plate swabbed with test culture. The applicator was gently turned clockwise with fingers so that the applicator was detached from the strip. The pressing of Cefepime / avibactam / EDTA MIC strip was avoided. The Cefepime / avibactam / EDTA MIC strip was adsorbed within 60 seconds and firmly adheres to the surface of agar. Care was taken that once the Cefepime / avibactam / EDTA MIC strip was in contact with the agar surface, the strip was not moved or repositioned even if the disk was not in the proper location because the drug begins to diffuse immediately upon contact with the agar. After all the strips were in place, the lid of the plates was replaced. The plates were inverted and were placed in an incubator at 37°C for 16-18 hours.

[0145] Interpretation

[0146] After the completion of incubation, the MIC was interpreted by observing that where the ellipse was intersecting the MIC scale on the strip. The isolated colonies, micro-colonies and haze appearing in the zone of inhibition were indicative of hetero nature of the culture having resistant subpopulation. In case the hetero nature of the culture having resistant subpopulation was observed, the reading for MIC determination at a point on the scale above which no resistant colonies was observed close to MIC strip within a distance in a range of 1-3 mm. When the growth occurs along the entire strip, the MIC was reported as highest values on the MIC strip. When the inhibition ellipse was below the strip and does not intersect the strip, the MIC was reported as lowest value on the MIC scale.

[0147] Observation

[0148] Table 5 shows values of the minimum inhibitory concentration obtained for the antimicrobial composition of cefepime, avibactam and EDTA by taking cefepime / avibactam / EDTA MIC strips. Table 6 shows values of the minimum inhibitory concentration obtained for piperacillin by taking cefepime MIC strips. Table 7 shows the comparison of values obtained in Table 5 and Table 6. Table 7 represent the reduction in MIC that is achieved with the antimicrobial composition of cefepime, avibactam and EDTA. Table 5: MIC of the antibiotic composition of cefepime, avibactam and EDTA by taking cefepime, avibactam and EDTA MIC strips

[0149] Table 6: MIC of cefepime by taking cefepime MIC strips

[0150] Table 7: Comparison of MIC of cefepime MIC strips and cefepime / avibactam / EDTA MIC strips (Media: MHA and incubation temperature:

[0151] 37°C)

[0152] Therefore, the present invention provides an antimicrobial composition of cefepime, avibactam and EDTA that effectively controls bacterial infections with reduced minimum inhibitory concentration in a range of 0.055-0.50 pg / mL. Many modifications and other embodiments of the invention set forth herein will readily occur to one skilled in the art to which the invention pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

CLAIMSWe claim:

1. An antimicrobial composition comprising of: a cephalosporin; a p-lactamase inhibitor; and a chelating agent; wherein, said cephalosporin is cefepime in an amount ranging from 1800 mg to 2200 mg; said beta-lactamase inhibitor is avibactam in an amount ranging from 450 mg to 550 mg; and said chelating agent is ethylene diamine tetraacetic acid (EDTA) in an amount ranging from 33.75 mg to 41.25 mg.

2. The antimicrobial composition as claimed in claim 1 , wherein said cephalosporin is essentially in an amount of 2000 mg.

3. The antimicrobial composition as claimed in claim 1 , wherein said beta-lactamase inhibitor is essentially in an amount of 500 mg.

4. The antimicrobial composition as claimed in claim 1 , wherein said chelating agent is essentially in an amount of 37.5 mg.

5. The antimicrobial composition as claimed in claim 1 , wherein said antimicrobial composition further includes pharmaceutically acceptable excipients selected from a group of a solubilizing agent, a pH buffer, a stabilizer, a solvent or a combination thereof.

6. The antimicrobial composition as claimed in claim 1, wherein said solubilizing agent is L-arginine, said pH buffer is sodium bicarbonate, said stabilizer is sodium citrate and said solvent is sterile water for injection or a combination thereof.

7. The antimicrobial composition as claimed in claim 1, wherein said antimicrobial composition is in the form of a powder or an injection.

8. The antimicrobial composition as claimed in claim 1, wherein said antimicrobial composition exhibits minimum inhibitory concentration (MIC) value of 0.50 pg / mb against P. aeruginosa.

9. The antimicrobial composition as claimed in claim 1, wherein said antimicrobial composition exhibits MIC value in a range of 0.047-0.064 pg / mT against E. coli.

10. The antimicrobial composition as claimed in claim 1, wherein said antimicrobial composition exhibits MIC value in a range of 1.0- 1.5 pg / mL against 5. aureus.

11. The antimicrobial composition as claimed in claim 1, wherein said antimicrobial composition exhibits MIC value in a range of 0.125-0.19 pg / mL against K. pneumoniae.

12. The antimicrobial composition as claimed in claim 1, wherein said antimicrobial composition exhibits zone of inhibition ranging from 21 mm - 35 mm.

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