Comparative in-vitro Quality Evaluation of Cephalexin

Comparative in-vitro Quality Evaluation of Cephalexin A COMPARATIVE IN-VITRO QUALITY EVALUATION OF DIFFERENT BRANDS OF COMMERCIALLY AVAILABLE CEPHALEXIN IN KARACHI (PAKISTAN) SHAHBAZ AHMED NOOR Abstract: Approximately 600 generic drug applications (ANDAs) approved each year, 65 % of prescriptions are for generic indication from oncology to insomnia without repeating clinical trials .Every generic molecule references a previously approved drug. Cephalexin Monohydrates was first sold by Eli Lilly in 1972. The availability of many brands of cephalexin 500 mg capsules in Pakistan market today place healthcare providers in a difficult situation of choice of a suitable brand or alternate use possibility .The objectives of this study was to carry out pharmaceutical equivalence studies on different brands of cephalexin 500mg capsules available in local market of Karachi. The invitro parameters include all pharmacopeia and non-pharmacopeia tests that were dissolution rate( in 0.1 N Hydrochloric Acid at 37 à ¢Ã‚ Ã‚ °C ) ,weight uniformity , disintegration time , moisture content ( with the help of suitable Karl Fischer Titrator ) , appearance (observed physically before and after opening of the shell) , Identification ( compare manually retention time in HPLC chromatogram of sample with chromatogram of reference standard ) and HPLC assay of cephalexin (as describe below based on HPLC assay method AM1172) were also analyzed. Along with the inappropriate use of antimicrobials drugs, substandard antibiotic medicines may also contribute in resistance development against susceptible organism. The study will be helpful to evaluate the efficacy and justification of generic substitution of cephalexin 500 mg capsule brands in Karachi (Pakistan) INTRODUCTION: Antibiotic, being the wonder drugs, are widely prescribed in the developing countries. In reality, 25 to 40 % prescriptions contain one or more antibiotic (Public Sector studies, 1990-1993) Antibiotic resistance can be developed through its wide and inappropriate use without following any standard guidelines. This Alarming situation has been addressed in several scientific journals (Glass et al.,1980; Farrar , 1985; Saha et al., 2003;Lina et al.,2007;Hassan et al.,2009) Post –marketing surveillance include complete procedures undertaken to obtain more data and information about a product after it had been granted marketing authorization and made available for public use, this data used for product improvement ,development standard and regulations. Regulatory agencies rely on limited information get from clinical trials and from scientific literature as guide to granting to marketing authorization of medicine for public use. Post-marketing surveillance of approved dosage forms is to assess the quality, effectiveness and safe use of medicine to large population. To Assess the standard of a product, invitro dissolution test is widely used because , for any solid dosage forms, gastrointestinal absorption first requires dissolution of the tablet or capsules that librates the drug into solution (Goodman Gilman`s –The Pharmacology basis of Therapeutics,2006) The dissolution characteristics of a drug from the dosage form depend on many factors including its formulation and manufacturing process (Augsburger et al., 1983). Cephalexin Chemically, Cephalexin is {(6R, 7R)-7-[(R)-2-Amino-2-Phenylacetamido]-3-Methyl-8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid monohydrate} is a first generation cephalosporins for oral administration which is bactericidal, and mainly used in the treatment of various bacterial infections caused by gram +ve and gram -ve microorganisms. .( Ralph R et.al 1970).Excellent oral absorption and lack of serum binding of cephalexin compensates significantly for the lower in vitro activity.( Warren E. Wick et.al 1967). Antibacterial Spectrum of Cephalexin: Cephalexin appears to be a highly effective antibiotic. It possesses the qualities of rapid oral absorption, production of high drug levels in the blood and urine, and near absence of side effects. It is effective against infections due to gram-positive cocci infections, with the exception of Enterococcus, and for most infections caused by E. coli and Klebsiella. It is useful in the treatment of a wide variety of infections in infants and children, and particularly valuable with susceptible infections in patients who do not require parenteral antimicrobial therapy. (R.C. Rudoy, M.D.1977) The first-generation cephalosporins, epitomized by cephalothin and cefazolin, have good activity against gram-positive bacteria and relatively modest activity against gram-negative microorganisms. Most gram-positive cocci (with the exception of enterococci, methicillin-resistant S. aureus, and S. epidermidis) are susceptible. Most oral cavity anaerobes are sensitive, but the B. fragilis group is resistant. Activity against Moraxella catarrhalis, E. coli, K. pneumoniae, and P. mirabilis is good (Goodman Gilman`s –The Pharmacology basis of Therapeutics,2006) Literature Survey: A ) Helio et.al in 2007 In this study, they observed the antibacterial spectrum of the orally prescribe cephalosporin (cefaclor, cefdinir, cefpodoxime, cefprozil, cefuroxime axetil, cephalexin) and amoxicillin/clavulanate, as these drugs were widely prescribed for community acquired respiratory tract infections. Narrowest zone was of Cefaclor and Cephalexin against evaluated pathogens. Overall, the 3rd Generation of cephalosporin is more useful in respiratory tracts and other infections when administered orally, as compared with older generation or amoxicillin alone. B ) Chalermporn et.al in 2005 ,they studied for rapid and sensitive identification of cephalosporins drugs such as cefoxitin , cefazolin , cephalexin, cefadroxil , cefaclor and cefoperazone, a flow injection (FI) method was used. This method was applied successfully for the identification of cefoxitin, cefazolin, cephalexin, cefadroxil , cefaclor and cefoperazone. The method was successfully applicable for the identification of cefoxitin ,cefazolin , cephalexin and cefadroxil in the pharmaceutical formulation with a sample throughput of 90h−1. C ) Rist et.al in 2002, they studied on Oral Cephalexin compared with mupirocin calcium cream with respect to safety and efficacy in secondarily infected eczema treatment. The adverse effect most common , they observed was diarrhea and nausea, effective clinical dose of mupirocin cream was apply three times daily as compared to cephalexin orally four times daily in secondarily infected eczma as topical regimen was preferred by the patient that enhance patient compliance. D ) Lennart Venemalm et.al in 2001, worked for the diagnosis of allergy against ÃŽ ²-lactams, prazinone-polylysine conjugate was able to bind with antibodies IgE in sera from patient with suspected allergy to cephalosporin’s. This information may be used for diagnosis of allergy against ÃŽ ²-lactams. E ) Yagnesh P. Patel et.al in 1998 They studied, modern data on the isocratic assay of five antimicrobial drugs ,five different assay were performed to one single report, antibiotic used in this study were cephalexin and cefaclor (cephalosporin’s), isoniazid and pyrazinamide (anti-tubercular drugs) and minocycline (tetracycline series) . Overall they concluded that any one of the drug could be used as internal standard for other four drugs. F ) Godfried Owusu-Ababio et.al in 1995 Sustained release of cephalexin ciprofloxacin, was evaluated in open chemostat sytem. They concluded that chemostat system is convenient for sustained release drug testing as a function of formulation parameter. G ) Anne H. et.al in 1992 In this study, the Cefaclor uptake was observed concluded that it was sodium-independent, but proton-dependent and also the energy dependent , dipeptides inhibited uptake of ÃŽ ²-lactam antibiotic but amino acid had no effect on uptake , the overall data from this study concluded that a single proton-dependent dipeptide transport carrier support the orally absorbed antibiotic uptake into Caco-2 cell shows correlation for the use of Caco-2 cell for cellular model of the intestinal proton-dependent dipeptides transporters for study purpose. H ) R. Wise et.al in 1991 Cefibuten, invitro activity was studied against 572 bacterial strain and was compared with orally administered ÃŽ ²-lactams. Cefibuten being more active against Enterobacteriaceae. There was less activity of cefibuten cefexime against Staphylococci and reduce activity against streptococcus pneumonia. I ) Luis J et.al in 1982 They studied, Acid degradation of cephalexin in presence of formaldehyde, produce 2-Hydroxy-3-phenyl-6-methylpyrazine.In 5M HCL this product gives well defined reduction wavelength with half wave potential of -0.45 vs. SCE. The wave was diffusion –controlled irreversible, linear relation with the cephalexin conc. was shown with diffusion current and cephalexin in plasma can be determined through it. J ) Ralph R et.al in 1970 They performed to detect various crystal forms of compounds, the employment of solubility versus solvent composition diagram. This appears to be a convenient sensitive method for detecting new crystalline phase. Aim of Project: The objective of this study is to carry out pharmaceutical equivalence studies on different brands of cephalexin 500mg capsules available in local market of Karachi. The invitro parameters include all pharmacopeia and non-pharmacopeia tests that are dissolution rate, weight uniformity, disintegration time, moisture content, appearance, Identification and HPLC assay of cephalexin will also be analyzed. The susceptibility test of drug will also be evaluate by ICLS ( Formerly NCCLS ) Reference Disk Diffusion (Kirby-Bauer) method against different clinical isolates of Staphylococcus aureus (03), E. coli (03), Klebsiella pneumonia (03), Pseudomonas aeruginosa (02), Importance of study: This is a growing concern that if a health care provider, providing the substitute of the generic is also the Bioequivalent. For the health care providers to use these brands interchangeable, the bioequivalence of these brands have to be ascertained, this mean that there should be continued post marketing monitoring of the medicines. EXPERIMENT: TESTING PROCEDURE: Test # 1: Cephalexin Monohydrate (As base): Procedure: Perform HPLC assay of Cephalexin as describe below based on HPLC assay method AM1172. Test # 2: Identification: Procedure: Compare manually retention time in HPLC chromatograms of sample with chromatograms of reference standards. Test # 3: Moisture Content: Procedure: Three times separately the moisture content in the powder of the product with the help of suitable Karl Fischer Titrator will be determine and then calculate their mean. Test # 4: Appearance: Procedure: From the 20 capsules take 10 capsules and observe physically before and after opening of the shells. Test # 5: Weight Variation: From 20 capsules take 10 random capsules from composite sample and note individual weight of filled capsules. Calculate their average, minimum, maximum and COV%. Test # 6: Disintegration: Procedure: Prepare a 0.6% v/v solution of HCl (Add carefully 16.2ml of 37% HCl into 500ml of distilled water and then make the solution up to 1000ml with distilled water) and fill the vessel of a suitable disintegration test apparatus with this solution up to recommended height. Maintain the temperature between 35  °C and 39  °C. Place 1 capsule in each of the 6 tubes of the basket and run the equipment. Start the stopwatch and note the time when all capsules are disintegrated. If 1 or 2 capsules fail to disintegrate, repeat the test on 12 additional capsules. The requirements of the test are met if not less than 16 of the 18 capsules tested have disintegrated. Test # 7: Dissolution: Procedure: According to USP 2007 page # 1691. HPLC ASSAY OF CEPHALEXIN BASED ON HPLC ASSAY AM1172 (1.01) FOR CEPHALEXIN CAPSULES.500 MG / CAPSULES. I.PRINCIPLE: The sample is dissolved in water and the insoluble component removed by filtration. An aliquot of the filtrate is diluted with water and examined by reverse phase HPLC. II.APPARATUS 1. A suitable high performance liquid chromatographic system equipped with a variable Wavelength UV Detector. 2. A suitable analytical balance with tolerance of +/- 0.1 mg. 3. A suitable Ultrasonic bath. 4. Suitable Syringes such as BDà ¯Ã†â€™Ã‚ ¤, 10ml. 5. Analytical Column: 150 Ãâ€" 4.6 mm. i.d stainless steel column packed with S5 – ODS2 or equivalent column. 6. Pre-Column: 50 Ãâ€" 4.6 mm .i.d stainless steel column packed with 80 µm Porasil or equivalent is fitted between the pump and injector. 7 Detector Condition: 254nm range 0.5 aufs 8. Pump Condition: Flow rate 1.0 ml / minute. 9. Injection volume: 20 µl 10. pH meter. 11. Mobile phase: See reagents. B) REAGENTS Cephalexin analytical reference standard Methanol HPLC grade Acetonitrile HPLC grade 1- Hydroxybenzotriazole, Reagent grade. Triethylamine ( T.E.A ) analytical grade Orthophosphoric acid, analytical grade 1- Pentanesulfonic acid, sodium salt, Reagent grade (8) Mobile Phase: Dissolve 1g Pentanesulfonic acid sodium salt (Monohydrate) and 15ml of Triethylamine in 850 ml of distilled water, adjust the pH to 3.0 with Orthophosphoric acid. Add 50 ml of Methanol and 100 ml of Acetonitrile, Mix well and degas before use. Note: Caution should be taken when degassing the mobile phase to avoid evaporating the mixture. Internal standard solution: Dissolve 1 gm of 1- Hydroxybenzotriazole in 1000ml of mobile phase, mix well. C) PROCEDURE: This procedure contains the minimum recommended sample and standard Preparation for assays performed manually. Alternate weights and volumes may be substituted if necessary, as long as they are documented and provide nominally equivalent concentration of the analyte. PREPARATION OF REF. STANDARD SOLUTION Accurately weigh approximately 32, 40 and 48 mg of Cephalexin reference Standard in to into three separate 200ml volumetric flasks respectively, pipette 30 ml of the internal standard solution in to each flask and swirl to dissolve, Dilute to volume with distilled water and mix well. SAMPLE PREPARATION Take 20 random capsules from composite sample and note individual weight content of each capsule and determine average content weight of 20 capsules. Accurately weigh about 500 mg sample from composite sample of weight content of 20 capsules in 500ml volumetric flask and record the weight, add water to dissolve and make up the volume with water up to 500 ml mix well and filter. Take 20ml of above dilution in 100ml volumetric flask, add 30ml Internal Standard and then add water upto 100ml mix well and filter through 0.45 µm filter paper. d) CHROMATOGRAPHY Make 20 à ¯Ã‚ Ã‚ ­l injection of a standard to ensure the chromatography system complies with the system suitability criteria. The peak tailing should not be greater than 1.5, peak efficiency should be greater than 4000 plates. The resolution between the two peaks should be greater than 2.5. A typical chromatogram is attached. LIMIT OF CEPHALEXIN MONOHYDRATE CONTENT. CEPHALEXIN THEORY LABEL 500MG / CAPSULE 500MG / CAPSULE (As Cephalexin anhydrous) ( As Cephalexin anhydrous) Acceptance Limit: 475 – 525mg per capsule (95 – 105 %). Regulatory Limit: 462.5 – 550 mg per capsule (92.5 – 110 %) (British Pharmacopeia) REFERENCES: (Augsburger LL,Shangraw RF,Giannini RP, Shah VP, Prasad VK and Brown D (1983) Thiazides VIII: Dissolution Survey of marketed Hydrochlorothiazides tablets. J.Pharma Sci.,72(8):876-881) Anne H. Dantzig, Linda B. Tabas, Linda Bergin , Cefaclor uptake by the proton-dependent dipeptide transport carrier of human intestinal Caco-2 cells and comparison to cephalexin uptake Biochimica et Biophysica Acta (BBA) Biomembranes, Volume 1112, Issue 2, 9 December 1992, Pages 167-173. Chalermporn Thongpoon, Boonsom Liawruangrath, Saisunee Liawruangrath, R. Alan Wheatley, Alan Townshend , Flow injection chemiluminescence determination of cephalosporins in pharmaceutical preparations using tris (2,2†²-bipyridyl) ruthenium (II)-potassium permanganate system , Analytica Chimica Acta, Volume 553, Issues 1-2, 30 November 2005, Pages 123-133. (Farar WE (1985) Antibiotic resistance in developing countries. The Journal of Infectious Diseases,152(6):1103-1106 ) (Glass RI, Huq I, Alim ARM and yunus M (1980) Emergence of multiply antibiotic-resistant vibro cholerae in Bangladesh . 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