An antibiotic composition of a penicillin, a β-lactamase inhibitor and an ion-chelating agent

Abstract

The present invention relates to an antimicrobial composition of a penicillin, a β-lactamase inhibitor and an ion-chelating agent. More particularly, the present invention relates to an antibiotic composition of piperacillin, avibactam and EDTA in a predefined ratio for the effective management of bacterial infections by decreasing the MIC and increasing the stability of the composition.

Description

[0001] “AN ANTIBIOTIC COMPOSITION OF A PENICILLIN, A p-LACTAMASE INHIBITOR AND AN ION-CHELATING AGENT”

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of antibiotics. More particularly, the present invention relates to an antibiotic composition comprising of a penicillin drug, a beta lactamase inhibitor and an ion-chelating agent for the management of bacterial infections.

[0004] BACKGROUND OF THE INVENTION

[0005] Beta lactam antibiotics show bactericidal effects by inhibiting cell wall synthesis through binding to penicillin binding proteins, which are involved in the crosslinking of peptidoglycans in cell wall synthesis in both gram negative and gram positive bacteria. Commonly used beta lactam antibiotics comprises of penicillins (piperacillin, ampicillin, temocillin etc.), carbapenems (imipenem, meropenem etc.), cephalosporins (ceftazimide, cefepime, cefotaximes etc) and monocyclic beta lactams (aztreonam, BAL30072). These drugs are ineffective against bacteria producing beta lactamases, which irreversibly binds to former and inactivate the drug. However, single beta lactamase inhibitor is not effective against all classes of beta lactamases. After years of research, synthetic non-p-lactam p- lactamase inhibitor, e.g. avibactam, with broader spectrum of activity than conventional clavulanic acid and the sulfone inhibitors are developed. Resistance to the p-lactams continues to increase, especially in gram-negative organisms. Hence, p-Lactam / p- lactamase inhibitor combinations (BLICs) are used to neutralize infections caused by beta lactamase producing bacteria. The intention of using BLICs is to protect the beta lactam antibiotic from beta lactamases. Beta lactamase inhibitor despite being structurally similar to penicillin, possess weak antibacterial activity, however, when administered along with beta lactam antibiotic, the resultant combination becomes highly potent against beta lactamase producing bacteria. Beta lactamase inhibitor irreversibly binds to beta lactamase, which in turn prevent the latter to inactivate the beta lactam antibiotic. The commonly used BLICs are amoxicillin / clavulanate, ticarcillin / clavulanate, ampicillin / sulbactam, and piperacillin / tazobactam. However, these BLICs are not active against all bacteria especially producing extended range of beta lactamases, e.g., AmpC p- lactamases, and carbapenemases. Hence, new combinations of p-Eactam / p-lactamase inhibitor are continuously explored.

[0006] US11559514B2 reveals an antibiotic composition against resistant Staphylococcus. It comprises of carbapenem or other p-lactam capable of binding the allosteric site of PBP2a, P-lactamase inhibitor; and p-lactam that binds the open configuration of the active site of PBP2a in the ratio of 1:1:1. However, the cited invention uses meropenem, tazobactam and amoxicillin in combination which are commonly used and does not neutralize wide range of bacteria especially producing different class of beta lactamases such as AmpC - lactamases, and carbapenemases.

[0007] WO2018025248A1 discloses the preparation of parenteral injections comprising Ethylene diamine tetra acetic acid (EDTA) and water. The formulation further comprises citric acid and diluent for improving the potency of antibiotics selected from Ceftriaxone sodium, Sulbactam Sodium, Cefoperazone sodium, Sulbactam Sodium, Cefepime Hydrochloride, Cefpirome Sulfate, Ceftazidime Sodium, Ceftalozone Sulfate, Cefotaxime Sodium, Tazobactam Sodium, Piperacillin Sodium, Avibactam Sodium, Piperacillin, Tazobactam or combination thereof. However, the present invention does not reveal any in vitro and in vivo experimental data regarding the antibacterial effect of the cited injectable composition.

[0008] CN107789355 discloses the pharmaceutical composition comprising piperacillin and avibactam for management of drug-resistant Streptococcus pneumonia. The weight ratio of piperacillin and avibactam is 5:1 to 15: 1 and the composition is formulated as lyophilized powder injection. However, the cited invention is focused on gram positive strain of bacteria and beta lactamase resistance is more prominent in gram negative bacteria.

[0009] AU2016307256A1 reveals the pharmaceutical composition comprising avibactam and antibacterial agent which is a beta-lactam antibiotic for use in the treatment and / or prevention of bacterial infections. However, the cited invention does not reveal the concentration or ratio of two drugs used in the study. Additionally, no in vitro and in vivo experimental data is provided. Zeiser et al, 2019, J Clinical Microbiology 57:10.1128 / jcm.00181-19, htps: / / doi.org / 10.1128 / jcm.00181-19 reveals the combinations of piperacillin- avibactam tested against a panel of ceftazidime-avibactam resistant Burkholderia cepacia complex and Burkholderia gladioli Cystic Fibrosis Isolates and only a single B. multivorans isolate AU28442 remained resistant to all agents tested in antibiotic susceptibility testing. MIC values are found in the range of 1 to 16 pg / ml excluding the resistant isolate AU28442 (MIC >128 pg / ml). However, the cited document does not provide any information regarding the concentration of avibactam and piperacillin used in the study.

[0010] Therefore, there is a need of a pharmaceutical composition that is active against a wide range of bacterial species with improved antibacterial efficacy (reduced MIC value) as well as increased stability of product to treat bacterial infections with minimal side effects.

[0011] OBJECT OF THE INVENTION

[0012] The main object of the present invention is to provide an antibiotic composition against bacterial infections.

[0013] Another object of the present invention is to provide an antibiotic composition comprising a penicillin, a beta lactamase inhibitor and an ion-chelating agent.

[0014] Yet another object of the present invention is to provide a method of preparation of the antibiotic composition.

[0015] Yet another object of the present invention is to provide an antibiotic composition with enhanced stability and improved antibacterial efficacy.

[0016] Yet another object of the present invention is to provide an antibiotic composition in the form of lyophilized powder.

[0017] Still another object of the present invention is to provide an antibiotic composition for prevention of bacterial infections in hospital settings.

[0018] SUMMARY OF THE INVENTION The present invention relates to an antibiotic composition against the bacterial species. More particularly, the present invention relates to an antibiotic composition comprising a penicillin, a beta lactamase inhibitor and an ion-chelating agent.

[0019] In an embodiment, the present invention provides an antibiotic composition comprising of a penicillin; a p-lactamase inhibitor; and an ion-chelating agent. Said penicillin is piperacillin in an amount ranging from 3600 mg to 4400 mg; said p-lactamase inhibitor is avibactam in an amount ranging from 450 mg to 550 mg; said ion-chelating agent is a salt of ethylene diamine tetraacetic acid (EDTA) in an amount ranging from 33.75 mg to 41.25 mg. The antibiotic composition is effective against bacterial infections. Preferably, the penicillin is essentially in an amount of 4000 mg, the p-lactamase inhibitor is essentially in an amount of 500 mg and the salt of EDTA is essentially disodium EDTA in an amount of 37.5 mg.

[0020] Here, said penicillin is piperacillin; said p-lactamase inhibitor is avibactam; and said ionchelating agent is a salt of ethylene diamine tetraacetic acid (EDTA). Further, said antibiotic composition further includes pharmaceutically acceptable excipients selected from a group of a diluent, a buffer, a stabilizer, a tonicity adjuster or a combination thereof. Essentially, said diluent is sterile water for injection (SWFI), said buffer is sodium bicarbonate, said tonicity adjuster is sodium chloride and said stabilizer is sodium citrate or a combination thereof.

[0021] The incorporation of EDTA in composition lowers the minimum inhibitory concentration (MIC) in the bacterial assay, which suggests the in vitro levels of susceptibility of bacteria towards the antibiotic composition. Furthermore, EDTA also helps in the disruption of extracellular polymeric substance (a major component of biofilm structure) thus leading to the breakdown of the biofilm.

[0022] The antibiotic composition of the present invention exhibits reduced minimum inhibitory concentration (MIC) value against bacteria.

[0023] The antibiotic composition is effective in controlling bacterial infections in hospital settings. The antibiotic composition of the present invention comprises a penicillin, a beta lactamase inhibitor and an ion-chelating agent. Here, the ion-chelating agent is present in a concentration of 3 mg / mL. 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] As used herein, “piperacillin” is a new generation of semisynthetic penicillin that has a broad spectrum of activity against gram-positive and gram-negative aerobic and anaerobic bacteria.

[0028] As used herein, “avibactam” is a non-p-lactam p-lactamase inhibitor taken in combination for the management of complicated intra-abdominal infections, complicated urinary tract infections and hospital or ventilator-acquired pneumonia. Avibactam inactivates some p- lactamases (Ambler class A p-lactamases, Ambler class C and some Ambler class D p- lactamases) by a unique covalent and reversible mechanism, and protects the antibiotic from degradation by certain p-lactamases.

[0029] The present invention relates to an antibiotic composition against the bacterial species. More particularly, the present invention relates to an antibiotic composition comprising of a penicillin, a beta lactamase inhibitor and an ion-chelating agent. In a preferred embodiment, the present invention provides an antibiotic composition comprising of: a penicillin; a p-lactamase inhibitor; and an ion-chelating agent. Here, said penicillin is piperacillin in an amount ranging from 3600 mg to 4400 mg; said p-lactamase inhibitor is avibactam in an amount ranging from 450 mg to 550 mg; said ion-chelating agent is a salt of ethylene diamine tetraacetic acid (EDTA) in an amount of 33.75 mg to 41.25 mg; and said antibiotic composition is effective against bacterial infections. Preferably, said penicillin is in an amount of 4000 mg, said p-lactamase inhibitor is in an amount of 500 mg and said salt of EDTA is essentially disodium EDTA in an amount of 37.5 mg.

[0030] Moreover, said salt of EDTA is selected from calcium disodium EDTA, diammonium EDTA, dipotassium EDTA, disodium EDTA, TEA-EDTA, tetrasodium EDTA, tripotassium EDTA or trisodium EDTA. The incorporation of EDTA in composition lowers the minimum inhibitory concentration (MIC) in the bacterial assay, which suggests the in vitro levels of susceptibility of bacteria towards the antibiotic composition. Furthermore, EDTA also helps in the disruption of extracellular polymeric substance (a major component of biofilm structure) thus leading to the breakdown of the biofilm.

[0031] The antibiotic composition of the present invention exhibits minimum inhibitory concentration (MIC) value of 3.0 pg / mL against P. aeruginosa,' in a range of 0.38 to 0.75 pg / mL against E. coli,' in a range of 2.0-3.0 pg / mL against Klebsiella pneumoniae,' and in a range of 0.19 to 0.25 pg / mL against S’, aureus. Further, said antibiotic composition exhibits zone of inhibition ranging from 27 mm - 32 mm.

[0032] Moreover, said antibiotic composition further includes pharmaceutically acceptable excipients selected from a group of a diluent, a buffer, a stabilizer, a tonicity adjuster or a combination thereof. Essentially, said diluent is sterile water for injection (SWFI), said buffer is sodium bicarbonate, said tonicity adjuster is sodium chloride, said stabilizer is sodium citrate or a combination thereof. The antibiotic composition is in the form of a powder or an injection. The antibiotic composition of the present invention is comprising of a penicillin and a beta lactamase inhibitor in a ratio of 8: 1 and an ion-chelating agent. Here, the ion-chelating agent is present in a concentration of 3 mg / mL. EXAMPLE 1

[0033] For Experimentation Details

[0034] Method of preparation of powder injection

[0035] Selection of ingredients or raw materials

[0036] The active pharmaceutical ingredients (API) includes: piperacillin sodium in an amount of 4 gm (4000 mg), avibactam sodium in an amount of 0.5 gm (500 mg) and disodium EDTA in an amount of 37.5 mg.

[0037] Disodium EDTA act as active as well as a chelating excipient that provides metal ion chelation (to enhance antibacterial activity and to prevent degradation) and hence, is a biofilm breaker. Besides the active ingredients, the antibiotic composition of the present invention further includes a pharmaceutically acceptable ingredient selected from a group of diluent, buffer, stabilizer, tonicity adjuster, or a combination thereof. A diluent or a solvent solvent is a substance that dissolves the API or other ingredients, creating a solution or other liquid form. Solvents are essential for preparing liquid dosage forms like solutions or injectable that help to ensure that the API is properly dispersed and can be administered effectively. The solvent, as used herein in the antibiotic composition is sterile water for injection (SWFI) taken as q.s. for reconstitution. A buffer is a solution employed to maintain a stable pH. Buffers are crucial for drug stability, solubility, and efficacy, as many drugs are pH-sensitive. Buffers resist changes in pH when small amounts of acid or base are added, preventing the degradation of drug components or altering the solubility. Buffers help to maintain the optimal pH for drug stability and activity. The pH buffer or stabilizer, as taken herein to maintain alkaline pH is, sodium bicarbonate while sodium citrate is taken as secondary buffer in q.s. to maintain pH of 6.5-7.0. The tonicity adjuster employed for regulating isotonicity for injection is sodium chloride in an amount of 10 mg. Nitrogen gas (q.s.) is employed during filling as inert gas to displace oxygen in vials and to prevent oxidation.

[0038] Sterilization and depyrogenation of primary packing material

[0039] 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. 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 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.

[0048] EXAMPLE 2

[0049] Method of Analysis of piperacillin and avibactam with EDTA for injection (4.5 gm)

[0050] Sampling

[0051] 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.

[0052] Total sample and label claim

[0053] The total number of samples taken was 60 vials. Each vial contains: piperacillin sodium (sterile) IP equivalent to anhydrous piperacillin = 4000 mg; avibactam sodium (sterile) IP equivalent to avibactam = 500 mg; and disodium EDTA (IP) equivalent to 37.5 mg. The samples were stored at a controlled room temperature.

[0054] Tests and method of analysis

[0055] Table 1 shows parameters taken for method of analysis.

[0056] Table 1: Parameters for method of analysis Description

[0057] A white to off white colored powder sample was filled in a 30 mL 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.

[0058] Identification by high performance liquid chromatography (HPLC)

[0059] The principal peak in the chromatogram obtained with test solution corresponded to the peak in chromatogram that was obtained with reference solution.

[0060] Average fill weight: ±2% of targeted fill weight

[0061] 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).

[0062] Average weight = (Sum of individual content of 20 vials) - 20 . (1)

[0063] Uniformity of fill weight: ±10% of average fill weight

[0064] The minimum and maximum %age was calculated by formula having equations (2) and (3):

[0065] Minimum %age = ((Minimum individual weight - Average fill weight) x 100) / Average fill weight . (2)

[0066] Maximum %age = ((Maximum individual weight - Average fill weight) x 100) / A verage fill weight . (3)

[0067] Bacterial endotoxins The bacterial endotoxins were in a range of not more than 0.1 endotoxin unit (EU) per mg.

[0068] Sterility

[0069] Media employed was as follows: soybeans casein digest media (30 gm for 1000 mL) and fluid thioglycolate media (29.75 gm for 1000 mL). 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. (3) The filtered purified water not more than 500 mL was taken for blank test.

[0077] (4) The sample was degassed sonicating for about 30 seconds.

[0078] (5) The sample was analyzed against the white and black background for black particle, white particle, fibre particle and any other visible particle.

[0079] 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.

[0080] Test preparation and analysis

[0081] 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.

[0082] Test sample quantity and preparation

[0083] The content of 10 units was combined in a clean container. The sample was degassed by sonicating for about 30 seconds.

[0084] Constituted solution

[0085] Completeness of solution: The solid was dissolved completely, leaving no visible residue as undissolved matter. 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.

[0086] 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 gm 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.

[0087] Assay of piperacillin by liquid chromatography

[0088] The solvent mixture was prepared by 25 volumes of acetonitrile and 75 volumes of 3.12% w / v solution of sodium dihydrogen phosphate. Test solution was prepared by dissolving equivalent to 100 mg of the substance under examination in 100.0 mT of the solvent mixture. The reference solution was prepared by dissolving equivalent to 100 mg of the substance under examination in 100.0 mT of the solvent mixture.

[0089] Chromatographic system

[0090] A stainless steel column of dimension 25 cm x 4.6 mm that is packed with octadecylsilane bonded to porous silica of size 5 gm was employed.

[0091] Preparation of mobile phase A: A mixture of 576 volumes of water, 200 volumes of a 3.12 % w / v solution of sodium dihydrogen phosphate and 24 volumes of an 8.0% w / v solution of tetrabutylammonium hydroxide, adjusted to pH=5.5 with dilute phosphoric acid or dilute sodium hydroxide solution, added 200 volumes of acetonitrile.

[0092] Preparation of mobile phase B: A mixture of 126 volumes of water, 200 volumes of a 3.12% w / v solution of sodium dihydrogen phosphate and 24 volumes of an 8.0% w / v solution of tetrabutylammonium hydroxide, adjusted to pH=5.5 with dilute phosphoric acid or dilute sodium hydroxide solution, added 650 volumes of acetonitrile.

[0093] Flow rate: 1 mF per minute; wavelength of 220 nm and injection volume of 20 gF was taken. Table 2 shows the adjustment of the mixture of the mobile phase A and B with respect to the time. The reference solution B was injected. The test was not valid unless the relative standard deviation for replicable injection was not more than 1.0%. The reference solution B and test solution B was injected.

[0094] Table 2: Mixture of mobile phase A and mobile phase B

[0095] Assay of Avibactam sodium

[0096] A phosphate buffer (pH = 4.0) was prepared as follows: 2.75 gm of potassium dihydrogen orthophosphate was dissolved in 500 mT of water and further was diluted to 1000 mT with water. The pH was adjusted to 4.0T0.05 by dilute orthophosphoric acid. Mobile phase was prepared by mixing 900 mT of the phosphate buffer and 100 mT of acetonitrile. The standard solution was prepared as follows: an amount equivalent to 40 mg of avibactam sodium working standard into a 50 mT volumetric flask was taken and diluted up to mark with mobile phase. The sample solution was prepared as follows: about 40 mg of sample was weighed into a 50 mT volumetric flask and diluted up to mark with mobile phase.

[0097] The chromatographic system included a C18 column with a dimension of 250 x 4.6 mm packed with octadecylsilane bonded to porous silica of size 5 pm. The flow rate was 1.0 mT per minute and detection was achieved at a wavelength of 230 nm. The injection volume was 10 pT. The column temperature was 25°C.

[0098] Assay of EDTA 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.

[0099] 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):

[0100] %age or concentration of analyte= (Volume x Factor x Normality x Avg. wt.) / (0.1 M x sample wt.) . (4)

[0101] 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.

[0102] EXAMPLE 3

[0103] Synergistic Effect of the pharmaceutical composition of piperacillin, avibactam and EDTA

[0104] Synergy testing by disk diffusion testing

[0105] 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 A of all the components (piperacillin, avibactam and EDTA), as depicted in Table 3.

[0106] Procedure

[0107] 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 mE 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.

[0108] Table 3: Synergy study report by disk diffusion assay

[0109] EXAMPLE 4

[0110] Method of antimicrobial susceptibility testing of MIC strips (LML Gradient Strip)

[0111] 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.

[0112] 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.

[0113] 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.

[0114] Handling

[0115] 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.

[0116] Procedure

[0117] 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).

[0118] 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:

[0119] • For bactericidal drugs (e.g. B-lactams), the MIC were read at the point of complete inhibition of growth, including hazes, microcolonies and isolated colonies.

[0120] • For bacteriostatic drugs, the endpoints were read at 80% inhibition, i.e. the initial point of significant inhibition.

[0121] • For bactericidal drugs, when macrocolonies were observed within the ellipse, all macrocolonies were read up to 3 mm from the strip.

[0122] • 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.

[0123] • 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.

[0124] • Hemolysis, swarming growth and a thin line of growth alongside the strip were ignored to read the values for MIC.

[0125] • When the ellipse of inhibition meets the strip between two different concentrations, the next upper value was read as MIC.

[0126] • 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.

[0127] • For B-lactamase inhibitor combinations, extrapolate the upper ellipse of inhibitor to read appropriate MIC.

[0128] Interpretation of Results

[0129] 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.

[0130] Quality Control

[0131] 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.

[0132] Table 4: Piperacillin / Avibactam / EDTA MIC at different concentrations (Media: MHA; Incubation at 37°C) EXAMPLE 5

[0133] Experimental analysis of minimum inhibitory concentration (MIC)

[0134] Microbial culture

[0135] Escherichia coli (NCIM 2065), Pseudomonas aeruginosa (NCIM 2200), Enterococcus faecalis (MTCC 439) and Staphylococcus aureus (NCIM 5345) were taken. NCIM stands for National collection of industrial microorganisms; and MTCC stands for Microbial type culture collection.

[0136] Media and diluent

[0137] Soybean casein digest agar (SCDA), Mueller hinton agar (MHA) and normal saline with a concentration of 0.9% were taken.

[0138] Accessories

[0139] Calibrated micropipette, sterile micropipette tips of 100 pL 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.

[0140] Instrument and equipment

[0141] 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.

[0142] Method for analysis of MIC

[0143] Step 1. Preparation of microbiological media

[0144] 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. Step 2. Preparation of inoculum

[0145] 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 SODA 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.

[0146] Step 3. MIC test procedure

[0147] 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 Ezy 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 Ezy MIC strip. The applicator along with attached Ezy 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 Ezy MIC strip was avoided. The Ezy MIC strip was adsorbed within 60 seconds and firmly adheres to the surface of agar. Care was taken that once the Ezy 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.

[0148] Interpretation 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. Observation

[0149] Table 5 shows values of the minimum inhibitory concentration obtained for the antibiotic composition of piperacillin, avibactam and EDTA by taking piperacillin / avibactam / EDTA MIC strips. Table 6 shows values of the minimum inhibitory concentration obtained for piperacillin by taking piperacillin 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 antibiotic composition of piperacillin, avibactam and EDTA.

[0150] Table 5: MIC of the antibiotic composition of piperacillin, avibactam and EDTA by taking piperacillin, avibactam and EDTA MIC strips

[0151] Table 6: MIC of piperacillin by taking piperacillin MIC strips

[0152] Table 7: Comparison of MIC of piperacillin MIC strips and piperacillin / avibactam / EDTA MIC strips (Media: MHA and incubation temperature: 37°C)

[0153] Therefore, the present invention [provides an antibiotic composition of piperacillin, avibactam and EDTA that is effective in controlling bacterial infections in hospital settings. The antibiotic composition effectively manages the bacterial infections like pneumonia, skin and soft tissue infection and urinary tract infection with reduced minimum inhibitory concentration.

[0154] 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 antibiotic composition comprising of: a penicillin; a p-lactamase inhibitor; and an ion-chelating agent; wherein, said penicillin is piperacillin in an amount ranging from 3600 mg to 4400 mg; said p-lactamase inhibitor is avibactam in an amount ranging from 450 mg to 550 mg; said ion-chelating agent is a salt of ethylene diamine tetraacetic acid (EDTA) in an amount ranging from 33.75 mg to 41.25 mg; and said antibiotic composition is effective against bacterial infections.

2. The antibiotic composition as claimed in claim 1, wherein said penicillin is essentially in an amount of 4000 mg.

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

4. The antibiotic composition as claimed in claim 1 , wherein said salt of EDTA is selected from calcium disodium EDTA, diammonium EDTA, dipotassium EDTA, disodium EDTA, TEA-EDTA, tetrasodium EDTA, tripotassium EDTA or trisodium EDTA.

5. The antibiotic composition as claimed in claim 1, wherein said salt of EDTA is essentially disodium EDTA in an amount of 37.5 mg.

6. The antibiotic composition as claimed in claim 1, wherein said antibiotic composition further includes pharmaceutically acceptable excipients selected from a group of a diluent, a buffer, a stabilizer, a tonicity adjuster or a combination thereof.

7. The antibiotic composition as claimed in claim 1, wherein said diluent is sterile water for injection (SWFI), said buffer is sodium bicarbonate, said tonicity adjuster is sodium chloride and said stabilizer is sodium citrate or a combination thereof.

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

9. The antibiotic composition as claimed in claim 1, wherein said antibiotic composition exhibits minimum inhibitory concentration (MIC) value of 3.0 pg / mL against P. aeruginosa.

10. The antibiotic composition as claimed in claim 1, wherein said antibiotic composition exhibits MIC value in a range of 0.38 to 0.75 pg / mT against E. coli.

11. The antibiotic composition as claimed in claim 1, wherein said antibiotic composition exhibits MIC value in a range of 2.0-3.0 pg / mT against Klebsiella pneumoniae.

12. The antibiotic composition as claimed in claim 1, wherein said antibiotic composition exhibits MIC value in a range of 0.19 to 0.25 pg / mT against S’. aureus.

13. The antibiotic composition as claimed in claim 1, wherein said antibiotic composition exhibits zone of inhibition ranging from 27 mm - 32 mm.

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