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Bioavailability Study of Metformin

Bioavailability Study of Metformin

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Bioequivalence Study of Metformin Hydrochloride Tablets

A Dissertation Submitted to the Vinayaka Missions University in Partial Fulfillment of the Requirement for the Degree of M.S. by Research in Pharmaceutical Technology

By K. Kaliloor Rahman [Enrollment No. 172074 / 206081090084(New)] Under the Guidance of
Mr. K. Senthilkumar M.Pharm (Industrial Pharmacy) Department of Pharmaceutical Chemistry, The Sarada Vilas College of Pharmacy Sarada Vilas Campus Krishanamurthypuram, Mysore -570004 (Karnataka), INDIA

Jan - 2011

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DEDICATED TO MY FAMILY AND TEACHERS

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DECLARATION

I, K. Kaliloor Rahman, hereby declare that the project report entitled “Bioequivalence Study of Metformin Hydrochloride Tablets” is submitted to, Vinayaka Missions University, Salem, in JAN– 2011, has been carried out by me, under the guidance of Mr. K. Senthilkumar M.Pharm (Industrial Pharmacy), Professor The Sarada Vilas College of Pharmacy, Sarada Vilas Campus, Krishanamurthypuram, Mysore -570004.This work is an original and independent, which has not been submitted for any other degree of this or any other university.

Place: Chennai Date:

K. Kaliloor Rahman [Enrollment No. 172074 / 206081090084(New)]

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GUIDE CONSENT LETTER
To The Additional Controller of Examinations, Directorate of Distance Education, Vinayaka Missions University, Salem.

Sub: M.S. Pharmaceutical Technology (By Research) project work – External Guide – Consent Regard I am happy to give my consent to guide the project work of - K. Kaliloor Rahman (Enrollment No. 172074 / 206081090084(New)), on “Bioequivalence Study of Metformin Hydrochloride Tablets” in partial fulfillment of the requirements of M.S. Pharmaceutical Technology, Vinayaka Missions University, Directorate of Distance Education, Salem. For the information of the concerned I am a post graduate in Pharmacy with 13 years of experience. A copy of my credentials along with Bio-data is enclosed for reference.

Guide - Signature & date

Mr. K. Senthilkumar M.Pharm (Industrial Pharmacy) Professor The Sarada Vilas College of Pharmacy Sarada Vilas Campus Krishanamurthypuram Mysore -570004

PLACE :

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CERTIFICATE

This is to certify that the dissertation entitled “Bioequivalence Study of Metformin Hydrochloride Tablets” submitted by K. Kaliloor Rahman (Enrollment No. 172074 / 206081090084(New)), in partial fulfillment of the requirements of M.S. by Research in Pharmaceutical Technology from Vinayaka Missions University, Salem, is a bonafide work carried out by him at Azidus laboratories limited (Clinical and Formulation Research Organisation, vandalur, Chennai-600048, under my guidance and supervision. He has satisfactorily completed the work.

Signature of Guide

Mr. K. Senthilkumar M.Pharm (Industrial Pharmacy) Professor The Sarada Vilas College of Pharmacy Sarada Vilas Campus Krishanamurthypuram Mysore -570004

PLACE : DATE : EXAMINERS 1.____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ 2. ____________________________________________ ____________________________________________ ____________________________________________

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ACKNOWLEDGEMENT
• I am highly indebted to Mr. K. Senthil Kumar, Professor, The Sarada Villas College of Pharmacy, Mysore, for his untiring help, constructive criticism, constant guidance and encouragement at every stage of the investigation and compilation of this thesis. • I convey my heartfelt thanks to Mr. Olaganathan .A MD, of Azidus Laboratories Limited, Clinical & Formulation Research Organisation. 23, School Road, Rathnamangalam, Vandalur, Chennai – 600 048. •

I wish to express my humble thanks to Mr. Bhanu Prasad, MD of Bhan Pharmaceutical Laboratories Limited,.

I thank my wife Dr.Shamsunnisa, B.U.M.S., for her support and constant encouragement during my entire work.

I gratefully acknowledge all my friends and colleague for their valuable help, encouragement and suggestions during the conduct of the project work.

It is a humble pleasure to thank all of those who have indulged in this project to make it a grand success.

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1
1 2 3 4

Table of Contents
Table of Contents ..................................................................................................................... 7 List of Abbreviations ............................................................................................................. 10 Abstract .................................................................................................................................. 13 3.1 Bioequivalence study of Metformin Hydrochloride ...................................................... 13 Introduction ............................................................................................................................ 14 4.1 Drug therapy .................................................................................................................. 14 4.2 Metformin ...................................................................................................................... 14 4.3 DRUG PROFILE ........................................................................................................... 15 4.3.1 Physical and chemical properties ............................................................................... 15 4.3.2 Pharmacodynamic ...................................................................................................... 15 4.3.3 Pharmacokinetics ....................................................................................................... 15 4.3.4 Adverse events ........................................................................................................... 17 DEFINITIONS ....................................................................................................................... 19 5.1 Bioavailability: ............................................................................................................... 19 5.2 Bioequivalence:.............................................................................................................. 19 5.3 Pharmaceutical equivalents:........................................................................................... 19 5.4 Pharmaceutical alternatives: .......................................................................................... 19 5.5 Pharmacodynamic evaluation: ....................................................................................... 19 5.6 Pharmacokinetics: .......................................................................................................... 19 5.7 Reference product: ......................................................................................................... 19 5.8 Therapeutic equivalents: ................................................................................................ 19 5.9 Internal standard: ........................................................................................................... 19 5.10 Pharmacokinetic Terms ................................................................................................. 20 5.10.1 C max ..................................................................................................................... 20 5.10.2 C min...................................................................................................................... 20 5.10.3 T max ..................................................................................................................... 20 5.10.4 AUC 0-t .................................................................................................................. 20 5.10.5 AUC 0-∞ ................................................................................................................ 20 5.10.6 K el ......................................................................................................................... 20 5.10.7 T ½ ......................................................................................................................... 20 REVIEW OF LITERATURE ................................................................................................ 21 6.1 Bioavailability ................................................................................................................ 21 6.2 Measurement of Bioavailability..................................................................................... 21 6.3 Factors Affecting Bioavilability .................................................................................... 21 6.4 Bioequivalence ............................................................................................................... 21 6.5 Molecule Tuning ............................................................................................................ 22 6.6 Method Development..................................................................................................... 22 Study Objectives: Aim of the Present Study.......................................................................... 23 Investigational Plan ................................................................................................................ 24 8.1 Overall Study Design and Plan ...................................................................................... 24 8.2 Discussion of Study Design, Including the Choice of Control Group ........................... 24 8.2.1 Number of Subjects.................................................................................................... 24 8.2.2 Duration of study ....................................................................................................... 24 8.2.3 Washout Period .......................................................................................................... 24 8.2.4 Randomization ........................................................................................................... 24 8.3 Selection of Study Population ........................................................................................ 25 8.3.1 Inclusion criteria ........................................................................................................ 25 8.3.2 Exclusion criteria ....................................................................................................... 26
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5

6

7 8

8.3.3 Laboratory evaluation performed during volunteer screening................................... 27 8.3.4 Subject withdrawal criteria ........................................................................................ 28 8.4 Treatments...................................................................................................................... 28 8.4.1 Treatments administered ............................................................................................ 28 8.4.2 Identity of the Investigational Products ..................................................................... 28 8.4.3 Method of Assigning Subjects to Treatment Groups................................................. 30 8.4.4 Selection of Doses...................................................................................................... 30 8.4.5 Selection and Timing of Dose for Each Subjects ...................................................... 30 8.4.6 Blinding...................................................................................................................... 31 8.4.7 Prior and Concomitant Medication Procedure ........................................................... 31 8.4.8 Treatment Compliance ............................................................................................... 31 8.5 Pharmacokinetic Parameters and Safety Variables........................................................ 31 8.5.1 Pharmacokinetic and Safety Measurements Assessed and Flow Chart ..................... 31 8.5.2 Appropriateness of Measurements ............................................................................. 35 8.5.3 Pharmacokinetic Variables ........................................................................................ 35 8.5.4 Drug Concentration Measurements ........................................................................... 35 9 Study Subjects ........................................................................................................................ 37 9.1 Disposition of Subjects .................................................................................................. 37 9.2 Protocol Deviations ........................................................................................................ 37 9.2.1 Phlebotomy Deviations .............................................................................................. 37 9.2.2 Other deviations ......................................................................................................... 37 10 Analytical Method ................................................................................................................. 38 10.1 Techniques ..................................................................................................................... 38 10.2 Method Validation ......................................................................................................... 38 10.3 Validation of Bioanalytical Method............................................................................... 39 11 Work Flow ............................................................................................................................. 41 11.1 Clinical Operations ........................................................................................................ 41 11.2 Bioanalytical and statistical Operations ......................................................................... 42 12 Data Quality Assurance ......................................................................................................... 43 12.1 Statistical Methods Planned in the Protocol and Determination of Sample Size .......... 43 12.1.1 Statistical and Analytical Plans .............................................................................. 43 12.1.2 Descriptive statistics .............................................................................................. 43 12.1.3 Determination of Sample Size ............................................................................... 43 12.2 Changes in Conduct of the Study or Planned Analyses ................................................. 43 13 Pharmacokinetic Evaluation .................................................................................................. 44 13.1 Data Sets Analyzed ........................................................................................................ 44 13.2 Demographic and other Baseline Characteristics .......................................................... 44 13.3 Measurements of Treatment Compliance ...................................................................... 44 13.4 Pharmacokinetic Results ................................................................................................ 44 13.4.1 Pharmacokinetic Analysis ...................................................................................... 44 13.4.2 Statistical Analysis ................................................................................................. 46 13.4.3 Analysis Charts ...................................................................................................... 48 Chart No:1 .............................................................................................................................. 48 13.4.4 Drug-Drug and Drug-Disease Interactions ............................................................ 50 13.4.5 Pharmacokinetic Conclusions of Metformin ......................................................... 50 14 Safety Evaluation ................................................................................................................... 51 14.1 Extent of Drug Exposure ............................................................................................... 51 14.2 Adverse Events .............................................................................................................. 51 14.3 Clinical Laboratory Evaluation ...................................................................................... 51 14.4 Vital Signs, Physical Findings and Other Observations Related to Safety.................... 51 14.4.1 Recording of Vital Signs and Clinical Examination .............................................. 51
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14.4.2 Post study Safety Evaluation ................................................................................. 51 14.5 Safety Conclusions......................................................................................................... 51 15 Discussion and Overall Conclusions ..................................................................................... 52 16 References .............................................................................................................................. 53

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2

List of Abbreviations
% ± µg 0 C AE AM ANOVA AUC0-∞ AUC0-t BA BE BMI BP CDER CDSCO CI Cmax COA CRF CV CVs Dr Dt ECG EDTA ESR GCP Hb HBsAg HIV HPLC Hr ICF ICH ICMR IEC : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : Percentage Plus or minus Microgram Degree Celsius Adverse Event Ante meridian Analysis Of Variance The area under the plasma concentration versus time curve, from zero to infinity. The area under the plasma concentration versus time curve from time zero to the last measurable concentration. Bioavailability Bioequivalence Body Mass Index Blood Pressure Centre for Drug Evaluation and Research Central Drugs Standard Control Organization Confidence Interval Maximum measured plasma concentration following each ‘treatment’. Certificate of Analysis Case Report Form Coefficient of Variation Curriculum Vitae Doctor Dated Electrocardiogram Ethylene Diamine Tetra- Acetic Acid Erythrocyte Sedimentation Rate Good Clinical Practice Hemoglobin Hepatitis B Surface Antigen Human Immunodeficiency Virus High Performance Liquid Chromatography Hour Informed Consent Form International Conference on Harmonization Indian Council of Medical Research Independent Ethics Committee
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IP IRB IU IUD IV Kel Kg LDH Log Ltd m MD Mg ml mmHg NAV No. OTC P PA PCV pH PK PM Pvt QA R RBC Rh RPM SD SGOT SGPT SOP ST T t1/2

: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :

Investigational Product Institutional Review Board International Unit Intrauterine Device Intra Venous Terminal elimination rate constant Kilogram Lactate Dehydrogenase Logarithm Limited Meters Doctor of Medicine Milligram Milliliter Millimeters of Mercury Not Available Number Over the Counter Protocol Postero Anterior Packed Cell Volume Potential of Hydrogen Pharmacokinetic Post Meridian Private Quality Assurance Reference Red Blood Cell Rhesus Rotations Per Minute Standard Deviation Serum Glutamic Oxaloacetic Transaminase Serum Glutamic Pyruvic Transaminase Standard Operating Procedure Study Test Time required for the plasma drug concentration to decrease by one 1/2.
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THC Tmax US USFDA VDRL WBC WMA Yr

: : : : : : : :

Tetra Hydro Canabinoid Time at which Cmax occurs United States United States Food and Drug Administration Venereal Disease Research Laboratory White Blood Cell World Medical Association Year

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3

Abstract
3.1

Bioequivalence study of Metformin Hydrochloride

Bioequivalence study of Metformin Hydrochloride of two different manufacturers was conducted on 12 healthy volunteers. Metformin is an oral antidiabetic drug belonging to biguanide class of drugs. It lowers both basal and postprandial plasma glucose. It does not stimulate insulin secretion and therefore does not produce hypoglycaemia. Metformin may act via 3 mechanisms: Reduction of hepatic glucose production by inhibiting gluconeogenesis and glycogenolysis In muscle, by increasing insulin sensitivity, improving peripheral glucose uptake and utilisation Delay of intestinal glucose absorption Metformin stimulates intracellular glycogen synthesis by acting on glycogen synthase. It increases the transport capacity of all types of membrane glucose transporters. The primary objective was to investigate the Bioequivalence of Metformin Hydrochloride 500 mg tablets of two different manufacturers, India, in healthy, adult, human subjects under fed conditions. The secondary objective was to assess the safety and tolerability of the test product as compared to the reference product. On analysis the 90 % CI of both the drug were found to be 96.82 to 109.42 and the power (%) of the test drug when compared to the reference was found to be 99.86. Hence the statistical analysis and evaluation of primary pharmacokinetic variables (Cmax, AUC0-t, AUC0-∞) for both test and reference products show that the two formulation were bioequivalent. Company A, India has developed generic version of metformin 500 mg tablets for the purpose of marketing in India. As per the regulatory requirements, this formulation has to be bioequivalent to the already existing formulation containing the same drug in the same quantity. This study has been designed and conducted to determine the bioequivalence of Metformin Hydrochloride 500 mg tablet manufactured two different manufacturers one is a generic formulation and other which is already existing in Indian market a well known brand, under fed conditions. The study was conducted as per Guidance for Industry-Bioavailability and Bioequivalence Studies for Orally Administered Drug Products - General Considerations USFDA, CDER- March 2003 ICH Guidelines for Good Clinical Practices (E6) 1996 Schedule Y of Drugs and Cosmetics Act, 20th January 2005 ICMR Ethical Guidelines for Biomedical Research on Human Subjects 2006 CDSCO Guidelines for BA/BE Studies, March 2005

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4

Introduction
4.1

Drug therapy

Drug therapy is a kinetic and dynamic process, the usual aim of drug therapy is to achieve and maintain effective concentration of drug at the receptor site. However the body is constantly trying to eliminate the drug and therefore it is necessary to balance absorption against elimination so as to maintain desired concentration. The study of this property of maintaining the desired concentration is the major goal of Biopharmaceutics. Biopharmaceutics majorly deals with bioavailability, bioequivalence, pharmacokinetics and pharmacodynamics of drugs. The bioavailability and bioequivalence play an important role in the drug research and development, especially in the generic drug industry as reviewed in design and analysis bioavailabity and bioequivalence studies. The U.S food and drug administration (FDA) was authorized to approve generic drug products under the drug price competition and patent term restoration Act, which was passed in U.S congress in 1984, when a brand name drug product is going off patent, the sponsors may file an abbreviated new drug application (ANDA) to the regulatory agencies for generic approval. The approval of generic drug product does not go through as lengthy and costly, a clinical development as that of new drug product. As the result, generic drug products are relatively cheap, compared to brand name drugs, however, as more generic drug products become available in the marketplace, it has become a public concern whether a generic drug product work as well as brand name drugs in terms of quality and therapeutic effect. A comparative bioavailability study refers to the comparison of bioavailability of different formulations of the same drug or different drug products. When two formulations of the same drug or two drug Products claimed bioequivalence, it is assumed that they will provide the same therapeutic effect or that they are therapeutically equivalent. Two drug products are considered pharmaceutical equivalents, if they contain identical amounts of the same active ingredient; two drugs are identified as pharmaceutical alternatives to each other if both contain an identical therapeutic moiety, but not necessarily in the same amount or dosage form or as the same salt or ester. Two drug products are said to be bioequivalent, if they are pharmaceutical equivalent (i.e. similar dosage forms made, perhaps by different manufacturers) or pharmaceutical alternatives (i.e. different dosage forms) and if there rates and extents of absorption do not show a significant difference when administered at the same molar dose of the therapeutic moiety under similar experimental conditions 4.2

Metformin

Metformin is an oral antidiabetic drug belonging to biguanide class of drugs. It lowers both basal and postprandial plasma glucose. It does not stimulate insulin secretion and therefore does not produce hypoglycaemia. Metformin stimulates intracellular glycogen synthesis by acting on glycogen synthase. It increases the transport capacity of all types of membrane glucose transporters.

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4.3

DRUG PROFILE

Metformin is an oral antidiabetic drug belonging to biguanide class of drugs. It was first introduced in the 1950s in Europe, and subsequently approved in the USA. 4.3.1 Physical and chemical properties Chemical Name: N, N-Dimethylimidodicarbonimidic diamide Hydrochloride Molecular formula: C4H1lN5•HCL Molecular structure:

Molecular weight: 165.62 Solubility in alcohol: 1 in 100 Solubility in water: 1 in 2 Metformin is a white, crystalline powder that is almost odorless. It has a bitter taste and is hygroscopic. It is prepared by chemical synthesis. It is freely soluble in water, slightly soluble in alcohol; practically insoluble in acetone and dichloromethane. 4.3.2 Pharmacodynamic Metformin is a biguanide with antihyperglycaemic effects, lowering both basal and postprandial plasma glucose. It does not stimulate insulin secretion and therefore does not produce hypoglycaemia. a) Metformin may act via 3 mechanisms: Reduction of hepatic glucose production by inhibiting gluconeogenesis and glycogenolysis b) In muscle, by increasing insulin sensitivity improving peripheral glucose uptake and utilization c) Delay of intestinal glucose absorption Metformin stimulates intracellular glycogen synthesis by acting on glycogen synthase. It increases the transport capacity of all types of membrane glucose transporters. 4.3.3 Pharmacokinetics Absorption 4.3.3.1

Gas-liquid chromatography, high pressure liquid chromatography and mass fragmentography have been employed for the determination of Metformin concentrations in plasma and urine. These methods are highly specific and sensitive. The limit of detection is 20µg.
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Metformin is incompletely absorbed, fecal recovery being about 30% of an oral dose. The absorption is slower than the elimination. Peak plasma concentrations of about 2 mg.I-1 are reached after 2 h or later. Oral bioavailability of usual doses is 50-60%. The difference between absorbed and available drug may reflect minor presystemic clearance of the drug or binding to the intestinal wall. Higher doses are proportionally less available, probably because of decreased absorption. Concomitant food intake may slightly impair Metformin absorption; absorption is completed within 6 h. Although there is absorption over the whole range of the intestine, the main part of the drug appears to be absorbed at a confined area in the upper part of the intestine. 4.3.3.2 Distribution The distribution of Metformin is rapid, but low transfer to a deep compartment seems to occur. Mean values for the apparent volume of distribution range from 63 to 276 L in different pharmacokinetic studies. Metformin accumulates in the walls of esophagus, stomach, duodenum, in salivary glands and kidneys. Metformin is not bound to plasma protein. There is a slowly increasing binding to blood cells indicated by an increasing blood: plasma drug concentration ratio with time. Metformin is found in saliva, but in a lower concentration than in plasma. 4.3.3.3 Metabolism and Elimination The mean plasma elimination half-life ranges from 1.5 to 4.5 h. It is prolonged in patients with renal impairment and is correlated to creatinine clearance. Therefore, there may be some prolongation of the half-life in the elderly because of deteriorating renal function. urinary excretion data have revealed a quantitative minor terminal elimination phase with a longer mean half-life, ranging from 8.9 to 19 h. This suggests a small deep compartment with a slow elimination. Metformin is rapidly eliminated by renal excretion. Metformin is excreted unmetabolized in animal studies. But in human studies, it was completely excreted unchanged in one study, but only 80% of an intravenous dose was recovered as unchanged drug in the urine in two studies. 4.3.3.4 Kinetics Of Sustained Release Formulation Of Metformin After an oral dose of the prolonged release tablet Metformin absorption is significantly delayed compared to the immediate release tablet with a Tmax at 7 hours. At steady state, similar to the immediate release formulation, Cmax and AUC are not proportionally increased to the administered dose. The AUC after a single oral administration of 2000mg of Metformin prolonged release tablets is similar to that observed after administration of 1000mg of Metformin immediate release tablets b.i.d. Metformin absorption from the prolonged release formulation is not altered by meal composition. No accumulation is observed after repeated administration of up to 2000mg of Metformin as prolonged release tablets. Renal clearance of Metformin is > 400 ml/min, indicating that Metformin is eliminated by glomerular filtration and tubular secretion. Following an oral dose, the apparent terminal elimination half-life is approximately 6.5 hours. 4.3.3.5 Therapeutic Indications Metformin is indicated for treatment of type 2 diabetes mellitus in adults, particularly in overweight patients, when dietary management and exercise alone does not result in adequate glycemic control. a) Type 2 diabetes (non-insulin-dependent diabetes, NIDDM)
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b) Type I diabetes (insulin-dependent diabetes, IDDM) as adjuvant therapy in combination with insulin 1. Obesity and insulin resistance 2. Hyperlipoproteinemia. 4.3.3.6 Dosage Metformin is available from several manufacturers for oral administration. It is available as film-coated tablets which contain 500 mg or 850 mg Metformin. The tablets should be taken with or after food twice or thrice daily. The tablets should be stored between 15°C and 30°C, in dry conditions and shelf-life of 5 year. It is also available in combination with chlorpropamide, glyburide, and butamide. Metformin hydrochloride is also available as sustained release tablets of 850 and 1000 mg which are ingested orally once or twice daily. 4.3.4 Adverse events Potentially life threatening effect 4.3.4.1

Metformin has no direct toxic effects on bone marrow, liver, or other organs, and tumor-inducing effects have not been encountered in humans. 4.3.4.2 Acute over dosage In a recent review, four cases of overdose have been reported. These patients had no associated renal disease. The first patient, who also took a barbiturate and an antidepressive agent, died, but the other three survived. Peritoneal dialysis was used in the first case and hemodialysis in the fourth, while the second and third patients recovered without dialysis . 4.3.4.3 Severe or irreversible adverse effects Metformin occasionally resulted in lactic acidosis especially in patients with impaired renal function. After a prolonged intake, it may cause vitamin BI2 and folate malabsorption and related symptoms. 4.3.4.4 Symptomatic adverse effect Gastrointestinal adverse effects may occur in 5-30 % of Metformin-treated patients, and seem to be dose related, although this has not been confirmed in a controlled trial. In one study, the overall incidence of adverse events did not increase with dose, although the incidences of digestive disturbances and diarrhea were greater at dosages above 500 mg daily (P < 0.05). Discontinuation of treatment because of gastrointestinal effects may be necessary in about 34%. The symptoms are often seen initially and can probably be minimized or avoided by gradual dose increase. The symptoms which are metallic taste, abdominal distension, nausea, vomiting, diarrhea. and anorexia, may be attributed to the accumulation of the drug in the gastrointestinal mucosa. Another contributing factor may be the proposed histamine H2 agonist capacity of Metformin. 4.3.4.5 Drug interactions Metformin is extensively used in combination with a sulfonylurea. but it is not known whether there is an additive or potentiating effect between these drug. A synergistic effect has been proposed, based on results of one of the US pivotal studies and results of another controlled trial. A combination of Metformin and Clofibrate has been used and might have some advantages.

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Single-dose interaction studies with Furosemide and Nifedipine in healthy volunteers suggest a possible interaction by increasing plasma Metformin levels. However. no such changes were found with Propranolol and Ibuprofen. The absorption of Metformin may be reduced by acarbose and guar gum. Alcohol potentiates the antihyperglycemic and hyperlactatemic effect of Metformin by inhibiting gluconeogenesis. Hence Populations treated with Metformin should preferably avoid alcohol and alcoholism is a definite contraindication.

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5

DEFINITIONS
5.1

Bioavailability:

The bioavailability of drug is defined as the rate and extent to which the active drug ingredient or therapeutic moiety is absorbed and becomes available at the site of drug action. 5.2

Bioequivalence:

Two medicinal products are bioequivalent if they are pharmaceutical equivalents or alternatives and if their bioavailabilities (rate and extent) after administration in the same molar dose are similar to such degree that their effects, with respect to both efficacy and safety, will be essential the same. 5.3

Pharmaceutical equivalents:

Pharmaceutical equivalents are drug products that contain identical amounts of the identical active drug ingredient, i.e., the same salt or ester of the same therapeutic moiety, in identical dosage forms, but not necessarily containing the same inactive ingredients. 5.4

Pharmaceutical alternatives:

Pharmaceutical alternatives are drug products that contain the identical therapeutic moiety, or its precursor, but not necessarily in the same amount or dosage form or as the same salt or ester. 5.5

Pharmacodynamic evaluation:

Pharmacodynamic evaluation is measurement of the effect on a patho-physiological process as a function of time, after administration of two different products to serve as a basis for bioequivalence assessment. 5.6

Pharmacokinetics:

Pharmacokinetics deals with the changes of drug concentration in the drug product and changes of concentration of a drug and/or its metabolite(s) in the human or animal body following administration of the drug product i.e., the changes of drug concentration in the different body fluids and tissues in the dynamic system of liberation, distribution, body storage, binding metabolism and excretion. 5.7

Reference product:

The reference product is a pharmaceutical product which is identified by the Licensing Authority as “Designated Reference Product” and contains the same active ingredient(s) as the new drug. The Designated Reference Product will normally be the global innovator’s product. An applicant seeking approval to market a generic equivalent must refer to the Designated Reference Product to which all generic versions must be shown to be bioequivalent. For subsequent new drug applications in India the Licensing Authority may, however, approve another Indian product as Designated Reference Product 5.8

Therapeutic equivalents:

Therapeutic equivalents are drug products that contain the same active substance or therapeutic moiety and, clinically show the same efficacy and safety. 5.9

Internal standard:

Internal standard is a test compound, which is structurally similar to the analyte added to both calibration standards and samples at known and concentration. Signal from analyte is compared with signal from the internal standard to determine analyte concentration.

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5.10 Pharmacokinetic Terms 5.10.1 C max This is the maximum drug concentration achieved in systemic circulation following drug administration. 5.10.2 C min This is the minimum drug concentration in systemic circulation following multiple dosing at steady state. 5.10.3 T max It is the time required to achieve maximum drug concentration in systematic circulation. 5.10.4 AUC 0-t Area under the plasma concentration – time curve from 0 h to the last quantifiable concentration to be calculated using the trapezoidal rule. 5.10.5 AUC 0-∞ Area under the plasma concentration – time curve, from zero to infinity to be calculated as the sum of AUC 0- plus the ration of the last measurable concentration to the elimination rate constant. 5.10.6 K el Apparent first-order terminal elimination rate constant calculated from semi-log plot of the plasma concentration versus time curve. 5.10.7 T ½ Elimination half life of a drug is the time necessary to reduce the drug concentration in the blood, plasma, or serum to one-half after equilibrium is reached.

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6

REVIEW OF LITERATURE
6.1

Bioavailability

Bioavailability describes the fraction of the dose of a drug that enters into the systemic circulation. Bioavailability means the rate and extent to which the active substance or therapeutic moiety is absorbed from a pharmaceutical form and becomes available at the site of action. All the drug products except intravenous injections undergo transportation mechanism and subsequent absorption in to systemic circulation. Where as in intravenous injection, the drug is injected directly in to systemic circulation so the bioavailability of the drug is 100% and the bioavailability of any drug product such as solid (Tablets, capsules), semi solid (ointments, creams) and liquid (Solutions, suspensions, emulsions) is compared with that of i.v injection. Any alteration in the drug’s bioavailability is reflected in its pharmacological effect. Other processes that play a role in the therapeutic activity of a drug are distribution and elimination. The movement of drug between one compartment and the other (generally blood and the extra vascular tissues) is referred to as drug distribution. Elimination is defined as the process that tends to remove the drug from the body and terminate its action. 6.2

Measurement of Bioavailability

The two major pharmacokinetic methods used to measure bioavailability are: 1) Plasma level-time studies 2) Urinary excretion studies The 3 parameters of plasma level-time studies which are considered important for determining bioavailability are 1) The peak plasma concentration (Cmax) that gives an indication whether the drug is sufficiently absorbed systemically to provide a therapeutic response. 2) The peak time (tmax) that gives an indication of the rate of absorption. 3) The area under the plasma level-time curve (AUC) that gives a measure of the extent of absorption or the amount of drug that reaches the systemic circulation. The 3 major parameters examined in urinary excretion data obtained with single dose study are 1) The maximum urinary excretion rate 2) The time for maximum excretion rate and 3) Cumulative amount of drug excreted in the urine. 6.3

Factors Affecting Bioavilability

The absolute bioavailability of a drug, when administered by an extravascular route, is usually less than one (i.e. F<1). Various factors reduce the availability of drugs prior to their entry into the systemic circulation such as A. Physicochemical factors of the drug B. Physiological factors of the individual C. Formulation factors 6.4

Bioequivalence

Medicinal products are pharmaceutical equivalents if they contain the same amount of the same active substance in the same dosage form that meet the same or comparable standard.
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Bioequivalence studies are needed in the following three situations: a) When the proposed marketed dosage form is different than that used in the pivotal clinical trials. b) When significant changes are made in the manufacturer of the marketed formulation. c) When a new generic formulation is tested versus innovator marketed product. In testimony before the Edward’s Committee recommending changes in the Food and Drug Administration (FDA) in November 1990, Dr.Benet had suggested that approval of the majority of new orally administered drug products was based on the same bioequivalency criteria that are used for the approval of generic drugs. Upon Dr.Benet’s request the FDA reviewed the new molecular entities approved during the past decade. Only thirty-eight percent of approved new molecular entities for oral dosage form administration utilized the final marketed formulation in the pivotal clinical trials. Fifty-nine percent used a different formulation in the clinical trial and therefore were required to prove bioequivalence of the final marketed formulation to that used in the clinical trial. Thus, for the majority of new drugs administered orally during the past decade, clinical studies were not carried out in the marketed formulation, just as is the case with the approval of generic drugs. 6.5

Molecule Tuning

Tuning is the adjustment of the resolution offsets to enable us to get the best response (peak widths) from a specific sample or compound. Tuning can be performed automatically (Resolution Optimization) or manually (Manual Tuning). 6.6

Method Development

Method development usually requires selecting the method requirements and deciding on what type of instrumentation to utilize. In the development stage, decision regarding choice of column, mobile phase, detector(s), and the method of quantitation must be addressed. In this development the stability of analyte in the spiked samples should be determined. The method development and establishment for a Bioanalytical method include determination of d) Selectivity e) Precision and accuracy f) Recovery g) Calibration curves h) Stability of analyte in spiked samples

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7

Study Objectives: Aim of the Present Study

The primary objective was to investigate the Bioequivalence study of Metformin Hydrochloride of two different manufacturers in healthy, adult, human subjects under fed conditions. The secondary objective was to assess the safety and tolerability of the test product as compared to the reference product.

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8

Investigational Plan
8.1

Overall Study Design and Plan

This study was designed to be an open label, balanced, randomized, two treatment, two sequence, two period, single dose, cross over, bioequivalence study under fed condition. 8.2

Discussion of Study Design, Including the Choice of Control Group

The study was designed with 12 + 02 (standby) subjects as a single-dose cross over study. In this study Bioequivalence study of Metformin Hydrochloride of two different manufacturers were compared. The Pharmacokinetic data obtained from the subjects after administering Reference tablets was considered as the data of active control group with which the data of Test drug was compared. 8.2.1 Number of Subjects A total number of 14 [12+ 02 (stand by)] subjects were enrolled in the study as per the Institutional Ethics Committee approved protocol. All the subjects completed the study and as per the protocol, the first 12 subjects were selected for the pharmacokinetic and statistical analysis. 8.2.2 Duration of study The complete duration of the study was 06 days including the 3 days wash out period between the two periods. The subjects who participated in the study underwent a screening procedure within 28 days of start of the study. Upon entering into the study, in each period, the subjects reported to the clinical facility atleast 12 hours before dosing and then they were housed till 24 hours post-dose. 8.2.3 Washout Period A wash out period of 03 days was kept between the two periods which was more than 5 half lives of the investigational product. 8.2.4 Randomization The order of receiving the test and reference products for each subject during the study was determined as per the randomization schedule. The randomization was balanced and the randomization schedule was generated using the randomization table. The randomization table and the dispensing records were kept under controlled access. The study personnel involved in dispensing (the dispenser) and the Principal Investigator were accountable for ensuring compliance to randomization schedule.

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Table-1: Randomization Schedule

Subject sequence Period I Number 1 RT R
2 3 4 5 6 7 8 9 10 11 12 RT TR RT TR TR RT TR RT RT TR TR

Period II T T R T R R T R T T R R T R

13 14

RT TR

R T R T T R T R R T T R T

Key: T = TEST PRODUCT

R = REFERENCE PRODUCT

8.3

Selection of Study Population

All subjects underwent a screening procedure which included demography, medical, personal & family history, general examination, vital parameters measurement, system examination, and site specific examination. The laboratory investigations such as ECG, hematological, biochemical, serological & urinary analysis were performed less than 28 days prior to the commencement of study. Chest X ray was taken for all the subjects except for Subject no. 06 who had taken chest X ray in the past 6 months of the study which was normal and the same X ray has been utilized for this study. The subjects were selected on the basis of the inclusion and exclusion criteria mentioned in the protocol. 8.3.1 Inclusion criteria Healthy male and female volunteers of 18 to 45 yrs. Female volunteers practicing an acceptable method of birth control as judged by the investigator(s), such as condom with spermicide, diaphragm with spermicide, intrauterine device (IUD), or abstinence throughout the duration of the study; or of postmenopausal (no menses) status of at least 1 year; or surgically sterile (bilateral tubal ligation, bilateral oophorectomy, or hysterectomy). Willing to give informed written consent and comply with the study requirements. Body Mass Index (BMI) between 18.50 – 24.99 Kg/m2 and body weight not less than 45 kg. Healthy individuals as evaluated by demography, personal history, medical history and general clinical examination. Vital parameters - BP should be within the range 100 – 139 mmHg systolic and 60 – 89 mmHg diastolic. Pulse rate within the range of 60 – 100 / min.
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Normal biochemical, hematological and urinary parameters. Normal Chest X - ray PA view & ECG in 12 leads. Negative serum and urine pregnancy test (females only) (in addition, urine pregnancy test to be performed on the day of check in during each period). Negative HIV 1 & 2 antibodies, Hepatitis B surface antigen, hepatitis C antibody and VDRL tests. Negative urine test for drugs of abuse for morphine, barbiturates, benzodiazepines, amphetamine, THC & cocaine (to be performed on the day of check in during each period). Negative alcohol breath analysis (to be performed on the day of check in during each period). 8.3.2 Exclusion criteria Subjects incapable of understanding the informed consent. History of any major surgical procedure in the past 3 months. History of diabetes mellitus, tuberculosis and systemic hypertension. History suggestive of cardiac, gastrointestinal, respiratory, hepatic, renal, endocrine, neurological, metabolic, psychiatric or hematological systems, judged to be clinically significant. History of dysphagia. History of any medical disorder that is of significance in the investigator’s opinion. Chronic alcohol intake of more than 2 units per day for the past 6 months or consumption of alcohol in the past 48 hrs of study. History of smoking 9 or more cigarettes or beedies per day and / or inability to withhold smoking or consumption of tobacco containing products during the study. History of any drug abuse in the past 12 months. History of Metformin intake for any reason in the past 7 days. History of hypersensitivity to Metformin and related drugs or other excipients in the formulation. History of allergy to vegetables and / or food substances and / or any other manifestations suggestive of hypersensitivity reactions. Present or past history of intake of drugs which potentially modify kinetics / dynamics of Metformin or any other medication judged to be clinically significant by the investigator. Consumption of grapefruit / its products within 10 days prior to the start of study. Intake of any prescription drug or over-the counter (OTC) drugs within 7 days prior to study and / or intake of any drug in the past that could affect the kinetics or dynamics of Metformin in view of investigator. Subjects with clinically significant abnormal values of laboratory parameters. Subjects who had participated in any other clinical study during the last 3 months.

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Subjects who had bled more than 350 ml in the past 3 months from the date of start of study either for blood donation or for any other reason. Female subjects who are currently pregnant or breastfeeding. History of habituation to coffee, tea or other xanthine containing products and inability to withhold the intake during the in-house stay. 8.3.3 Laboratory evaluation performed during volunteer screening The following laboratory tests (Table 2-a, 2-b, 2-c & 2-d) were performed during screening of the volunteers apart from chest X ray and 12 lead ECG . Chest X-ray was taken to all the subjects expect subject No. 06, because, the Subject No.06 was taken Chest X-ray within 6 months which was normal. And the same X-ray was considered for this study also. Hematology (Table 1-a) Blood Group and Rh Type Total WBC count Differential Leucocytes count Erythrocyte Sedimentation Rate (ESR) Bleeding time Hemoglobin Packed Cell Volume (PCV) Red Blood Cell count Platelet count Clotting time Serology (Table 1-b) Hepatitis B & Hepatitis C HIV 1 & 2 Biochemistry (Table-1c) Blood sugar-Random Creatinine Bilirubin total, direct & indirect Total protein Globulin Alkaline phosphatase LDH Urea Electrolytes (sodium, potassium, bicarbonate, chloride) SGOT & SGPT Albumin A/G ratio Gamma glutamyl transferase Total cholesterol Urinary parameters (Table 1-d) Appearance Colour pH Specific Gravity Albumin Bile Pigment Bile Salt Urobilinogen Acetone RBC
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VDRL

Sugar

Microscopic (pus cells & epithelial cells)

8.3.4

Subject withdrawal criteria

The following criteria were followed as withdrawal criteria in this study. The subject withdraws consent. Development of intolerable adverse event due to study participation as determined by the investigator and / or subject. Development of an intercurrent illness or condition for which the subject requires concomitant medications which may interfere with the kinetics of the study medication. Discovery that the subject entered the study in violation of the protocol or occurrence of a significant protocol violation during the study. The investigator feels that in the best interest of the subject’s health, the subject is to be withdrawn from the trial. Data not known before starting the trial become available and raise concern about the safety of the study drug so that continuation would pose potential risk to any particular subject. When the study medication is administered, if the subject vomits the medication within 4 hrs, then the subject will be withdrawn from the study. If the subject is non cooperative and / or indisciplined. 8.4

Treatments
8.4.1 Treatments administered

All the subjects fasted overnight for atleast 10 hours prior to the scheduled time of high fat high calorie breakfast (approximately 1000 kcal). Dosing was done exactly 30 minutes after the start of the breakfast. As per the randomization schedule, either one tablet of Metformin Hydrochloride 500 mg Test (Generic), or Reference (Branded) containing Metformin Hydrochloride 500 mg was administered to each subject with 240 ml of water by trained study personnel, in such a way that each subject has consumed one unit of test and one unit of reference product at the end of the study. Subject no. 06, in period I, did not consume all the 240 ml of water provided for ingestion of investigational product. This subject has left around 06 ml of water. The study personnel insisted that he should consume all the water, but the subject said that his stomach was very full and he was unable to take the remaining, little quantity of water. 8.4.2 Identity of the Investigational Products The investigational products were stored in the pharmacy inside the humidity chamber at 21°C ± 4°C with the relative humidity of 55% ± 5%, under restricted access. The details of the Investigational products (IPs) including the inventory are given in Table 3 and Table 4.

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Table-3: The details of the investigational products administered to the subjects during the study.
Product Treatment ID Product Name Manufacture Date Expiry Date Strength Dosage Form Bio-batch Size Production Batch Size Potency Content Uniformity (Mean, %CV) Dose Administered Route of Administration Test Reference

T Metformin Hydrochloride Generic April 2008 March 2011 500 mg Tablets NAV NAV 97.97% NAV 500 mg
Oral

R Metformin Hydrochloride Branded April 2008 March 2011 500 mg Tablets NAV NAV 97.47% NAV 500 mg
Oral

Table-4: Inventory and retention of Investigational products. Number of units dispensed but unused 01 01

Product type

Number of units received 80 80

Number of units dispensed 15 15

Number of units used 14 14

Number of units remaining – for retention 66 66

Test (T) Reference (R)

The IPs 80 units of both test and reference products were supplied. There were no obviously damaged IPs found. One unit of test and reference products was taken for physical verification. The dispensing for period I & II was carried out for 14 subjects, 14 units of test and 14 units of reference IPs were dispensed in the appropriately labeled dosing containers. One unit of test and reference IPs were dispensed extra as stand by IPs. The stand by IPs was not used and were kept in the humidity chamber along with the remaining IPs. At the end of the study, 66 units of both test and reference IPs were remaining. The remaining IPs included the IPs taken for physical verification and the IPs that were remaining at the end of the study. The sponsor of the study requested to send the remaining IPs back to their premises and they assured that the IPs will be stored for a minimum period of 5 years.
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8.4.3

Method of Assigning Subjects to Treatment Groups

The order of receiving the test and reference products for each subject during the study was determined randomly. Equal allocation of treatments or balanced randomization was ensured. The randomization schedule as well as the dispensing record were kept under controlled access. The study personnel involved in dispensing and the Principal Investigator were accountable for ensuring compliance to randomization schedule. 8.4.4 Selection of Doses 500 mg of metformin is one of the recommended doses for treating type II diabetes mellitus and can be subjected to bioequivalence estimation for generic marketing in India. 8.4.5 Selection and Timing of Dose for Each Subjects All subjects fasted overnight for at least 10 hours prior to the scheduled time of high fat high calorie breakfast. Drinking water was not allowed for one hour before and one hour after study drug administration (except the 240 ml of water used for dosing). Apart from this, drinking water was allowed at all times. In period I & II, 14 subjects were dosed, with either of the investigational products (Test or reference) in a sequential order from 08.00 AM to 08.26 AM, as per randomization schedule. Single dosing station was used for dosing purpose. The subjects who consumed test drug in period I were dosed with reference drug in period II and vice versa. In both the periods, dosing interval between two subjects was 2 minutes. The details of dosing schedule are given below in Table 5. Table-5: Details of Dosing Schedules in the Study (Dosing was carried out for each Subject with 2 minutes interval).
Period I Subject Number 01 02 03 04 05 06 07 08 09 10 11 Treatment R Time of dosing 08.00 AM Period II Treatment T T R T R R T R T T R Time of dosing 08.00 AM

R T R T T R T R R T

08.02 AM 08.04 AM 08.06 AM 08.08 AM 08.10 AM 08.12 AM 08.14 AM 08.16 AM 08.18 AM 08.20 AM

08.02 AM 08.04 AM 08.06 AM 08.08 AM 08.10 AM 08.12 AM 08.14 AM 08.16 AM 08.18 AM 08.20 AM

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12 13 14

T R T

08.22 AM 08.24 AM 08.26 AM

R T R

08.22 AM 08.24 AM 08.26 AM

8.4.6

Blinding

The study was an open-label study in terms of the drug and dose, but the allocation of the test and reference products were not freely available. The randomization schedule as well as the dispensing records was kept under controlled access. 8.4.7 Prior and Concomitant Medication Procedure The subjects who did not consume any prescribed or over the counter medication in the last 7 days of start of the study, were only included in the study. They were also instructed not to take any medication during the course of the study, including washout period. If drug therapy other than that specified in the protocol was required prior to or during the study, decision would have been taken by the Principal Investigator / Medical Expert whether to continue or discontinue the subject on the basis of the following: The likelihood of pharmacokinetic interactions with other non–study medications given during the course of the study Depending on the time and duration of administration of non-study medications 8.4.8 Treatment Compliance A thorough check of the oral cavity of the subject was carried out immediately after dosing. The duplicate label of the dosing pouch was pasted on the dosing record of the respective subject to ensure the correct allocation of the investigational product as per the randomization schedule. 8.5

Pharmacokinetic Parameters and Safety Variables
8.5.1 Pharmacokinetic and Safety Measurements Assessed and Flow Chart

Pharmacokinetic parameters The following pharmacokinetic parameters were calculated for both test and reference products. AUC0-t : The area under the plasma concentration versus time curve from time 0 to the last time point with measurable concentration, calculated by the linear trapezoidal method. The area under the plasma concentration versus time curve, from time 0 to time infinity. It is calculated as the sum of AUC0-t plus the ratio of the last measurable plasma concentration to the elimination rate constant Maximum measured plasma concentration over the time span specified Time to achieve maximum plasma concentration. If the maximum value occurs at more than one time point, Tmax is defined as the first time point with this value Apparent first order terminal elimination half-life, calculated as 0.693/kel
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AUC0-∞

:

Cmax Tmax

: :

T1/2

:

Kel

:

Apparent first order terminal elimination rate constant calculated from a semi – log plot of the plasma concentration Vs. time curve. The parameter will be calculated by linear least squares regression analysis using the maximum number of points in the terminal log linear phase (e.g. minimum three non-zero plasma concentrations).

The derived PK parameters Kel, t1/2 & AUC0-∞ were reported for all the subjects who completed the study. Safety Measurements In each period, vital parameters such as pulse rate and blood pressure and subject well being assessment were recorded during check in, at 00.00 hour (pre dose), 02.00, 04.00, 06.00, 12.00 and 24.00 hours post dose and during check-out. Body temperature was recorded during check-in and check-out. The post dose vital parameters and subject well being assessment were done within ± 30 minutes of scheduled time and the predose assessment was done within one hour prior to dosing. There were no abnormal and clinically significant values of vital parameters observed in this study. Capillary blood sugar estimation was done at 02.00 hrs post dose and during check-out in each period. No subject has suffered from hypoglycemia and the capillary blood sugar values of all the subjects were more than 50 mg%. The 02.00 hrs post dose blood sugar measurement was done within ± 30 minutes of scheduled time. Time table of events The time table of the events that happened during period I and Period II are given below as Table 6-a and 6-b respectively. All the allowed window period for activities like vital signs & wellbeing assessment, meals and blood sample collection timings etc were considered as per the protocol. Table -6-a: Timetable of Events in Period I
Day Scheduled time Actual Time Activities Reporting at clinical facility. Pre check-in assessment, Urine drug screen, alcohol breath test, clinical examination, vital signs measurement, well being assessment, Obtaining informed Consent and check-in. Dinner Bed time Wake-up call Bathing and morning ablutions IV Cannulation Drinking water restriction starts Pre-dose (00.00 hour) blood sample collection Pre-dose vital examination and well being assessment Breakfast
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0

–12.00 or before

08.00 PM or before

0 0 1 1 1 1 1 1 1

–11.50 to –10.50

08.30 to 08.46 PM

After Dinner –03.00 –03.00 to –02.00 – 02.00 to –01.00 –01.00 –01.00 to 00.00 –01.00 to 00.00 –00.50 05.00 AM 05.00 to 06.00 AM 06.08 to 06.46 AM 07.00 AM 07.01 to 07.28 AM 07.02 to 07.29 AM 07.30 – 08.07 AM

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2

00.00 00.00 00.50 01.00 01.00 01.50 02.00 02.00 02.50 03.00 03.50 04.00 04.00 04.00 04.00 05.00 06.00 06.00 08.00 08.00 12.00 12.00 12.00 16.00 24.00 24.00 24.00 After 24.00

08.00 - 08.26 AM 08.00 AM 08.30 - 08.56 AM 09.00 -09.26 AM 09.34 AM 09.30 -09.56 AM 10.00 -10.26 AM 09.30– 10.18 AM 10.30-10.56 AM 11.00-11.26 AM 11.30 –11.56 AM 12.00-12.26 PM 11.38AM-12.04 PM 12.30 PM 12.01-12.44 PM 01.00-01.26 PM 02.00-02.26 PM 01.46 -02.12 PM 04.00 -04.26PM 04.03-04.33 PM 08.00-08.26 PM 07.30-07.56 PM 08.05-08.43 PM 12.00-12.26 AM 08.00 – 08.26AM 07.30-07.56 AM 08.02-08.35 AM After 08.00 AM

Dosing Posture restriction starts Blood sample collection Blood sample collection End of drinking water restriction Blood sample collection Blood sample collection vital signs & wellbeing assessment & capillary blood glucose measurement Blood sample collection Blood sample collection Blood sample collection Blood sample collection vital signs & wellbeing assessment End of posture restriction lunch Blood sample collection Blood sample collection vital signs and well being assessment Blood sample collection snacks Blood sample collection vital signs & well being assessment dinner Blood sample collection Blood sample collection vital signs & wellbeing assessment breakfast Vital signs & wellbeing assessment, capillary blood sugar measurement and check-out

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Table -6-b: Timetable of Events in Period II
Day 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 –03.00 –03.00 to – 02.00 – 02.00 to –01.00 –01.00 –01.00 to 00.00 –01.00 to 00.00 –00.50 00.00 00.00 00.50 01.00 01.00 01.50 02.00 02.00 02.50 03.00 03.50 04.00 04.00 04.00 04.00 05.00 06.00 Scheduled time –12.00 or before –11.50 to – 10.50 Actual Time 08.00 PM or before 08.30 to 08.44 PM Activities Reporting at clinical facility. Pre check-in assessment, Urine drug screen, alcohol breath test, clinical examination, vital signs measurement, well being assessment and check-in. Dinner Bed time Wake-up call Bathing and morning ablutions IV Cannulation Drinking water restriction starts Pre-dose (00.00 hour) blood sample collection Pre-dose vital examination and well being assessment Breakfast Dosing Posture restriction starts Blood sample collection Blood sample collection End of drinking water restriction Blood sample collection Blood sample collection vital signs & wellbeing assessment and capillary blood glucose measurement Blood sample collection Blood sample collection Blood sample collection Blood sample collection vital signs & wellbeing assessment End of posture restriction lunch Blood sample collection Blood sample collection
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After Dinner 05.00 AM 05.00 to 06.00 AM 06.15 to 06.50 AM 07.00 AM 07.04 to 07.26 AM 07.22 to 07.48 AM 07.30 – 08.05 AM 08.00 - 08.26 AM 08.00 AM 08.30 - 08.56 AM 09.00 -09.26 AM 09.28 AM 09.30 -09.56 AM 10.00 -10.26 AM 09.34– 10.13 AM 10.30-10.56 AM 11.00-11.26 AM 11.30 –11.56 AM 12.00-12.36 PM 11.35AM-12.01 PM 12.46 PM 12.18-12.45 PM 01.00-01.36 PM 02.00-02.26 PM

1 1 1 1 1 1 2 2 2 2 2

06.00 08.00 08.00 12.00 12.00 12.00 16.00 24.00 24.00 24.00 After 24.00

01.35 -02.01 PM 04.00 -04.26PM 04.03-04.32 PM 08.00-08.26 PM 07.30-07.56 PM 08.04-08.43 PM 12.00-12.26 AM 08.00 – 08.26AM 07.30-07.56 AM 08.03-08.37 AM After 08.00 AM

vital signs and well being assessment Blood sample collection snacks Blood sample collection vital signs & well being assessment dinner Blood sample collection Blood sample collection vital signs & wellbeing assessment breakfast Vital signs & wellbeing assessment, capillary blood sugar measurement and check-out

8.5.2

Appropriateness of Measurements

The choice of blood sampling time points was planned to provide an adequate estimation of Cmax, the terminal half-life and at least 80% of AUC0-∞. For determining the bioavailability characteristics of the drug and bioequivalence of the test with reference products, Cmax, AUC 0-t, AUC 0-∞ were compared. The pre-dose sample was collected within one hour prior to drug dosing and collection was completed before the start of breakfast. The post-dose samples were collected within 2 minutes of the scheduled time where the end time of collection to the nearest minute was recorded and hence there was no deviation in the scheduled blood sample collection. The choice of timings to assess the well being, clinical examination and vital signs measurement was planned based on the Tmax of the drug and stages of the study. Clinical Examination and vital signs measurement (blood pressure, oral temperature and pulse rate) were performed and recorded during check in and check out. Apart from this, blood pressure and pulse rate were measured at 00.00 (pre dose) and at 02.00, 04.00, 06.00, 12.00 and 24.00 hrs post dose. Capillary blood sugar measurement was done at 2.00 hrs post dose and during check out. The activities were carried out in both periods similarly. 8.5.3 Pharmacokinetic Variables The pharmacokinetic variables such as AUC0-t, AUC0-∞, AUC0-t / AUC0-∞, Cmax, Tmax, T1/2 and Kel were assessed in this bioequivalence study. The acceptance range of bioequivalence is 80.00% to 125.00% for the 90% confidence intervals for the ratio of least square means of log transformed pharmacokinetic parameters Cmax, AUC0-t and AUC0-∞ of test and reference formulations. 8.5.4 Drug Concentration Measurements Sampling Schedule In each period, a total of 16 blood samples were collected from each subject at the following time points.
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00.00 hour (pre-dose), 00.50, 01.00, 01.50, 02.00, 02.50, 03.00, 03.50, 04.00, 05.00, 06.00, 08.00, 10.00, 12.00, 16.00 and 24.00 hours post-dose. (total of 16 samples – predose sample 8 ml & the 15 remaining samples 5 ml each). Blood samples were collected in a single sampling station. Sample Collection Blood samples were collected through an indwelling cannula placed in forearm veins. The predose sample was collected within one hour prior to drug dosing. The post-dose samples were collected within 2 minutes of the scheduled time where the end time of collection to the nearest minute was recorded. If there was difficulty in obtaining blood sample through intravenous cannula or if the interval between two sampling points was equal to / more than 4 hrs, then blood samples were obtained by direct venous puncture. When intravenous cannula was used to collect blood sample, after insertion of the cannula and after every sample was drawn, 0.5 ml heparinised saline (10 IU/ml) was injected to prevent cannula blockade. When blood samples were collected from intravenous cannula in which heparinised saline was injected previously, initial 0.5 ml of blood was drawn and discarded. Then the required volume of blood was collected. Blood samples were collected using pre-labeled vacutainers containing K2EDTA. Sample Processing After collection, blood samples were centrifuged at 4000 rpm for 10 minutes at 4°C ± 2°C to separate plasma. The centrifuge procedure was started within 15 minutes of the collection of final set of samples. In period I, post dose samples collected at 03.50 and 04.00 hrs were centrifuged a little later than the 15 minutes of collection of last set of samples. After centrifugation, plasma samples were separated into single aliquot and were stored at about -20°C or colder till analysis. After completion of both periods, all the plasma samples were transported to in-house Bioanalytical laboratory. While transferring the samples, adequate measures were taken for maintaining the temperature and integrity of samples. Relationship of Posture, Meal and Water Consumption to Dosing and Sampling The study subjects were not permitted to drink water from one hour prior to dosing and one hour post dosing, except the 240 ml of water given along with drug administration. At all other times, drinking water was made available. The subjects were dosed in their sitting posture and they remained in erect posture for atleast four hours. i.e., the subjects were either sitting or ambulatory but they have not lied down during the first four hours after dosing. All the subjects were provided pre-study dinner, study day breakfast (high calorie high fat), lunch, evening snacks, dinner and post study day breakfast.

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9

Study Subjects
9.1

Disposition of Subjects

Among the volunteers who reported to the facility for the possible inclusion into the study on the day of check-in, 14 eligible volunteers were selected. During selection, preference was given to the volunteers who fulfilled the inclusion and exclusion criteria to the maximum and who reported earlier to the facility. After obtaining informed consent from the 14 volunteers, they were enrolled for the study and all 14 subjects completed both the periods of the study. 9.2

Protocol Deviations
9.2.1 9.2.2 Phlebotomy Deviations Other deviations

All the blood samples were collected at the scheduled time points without any deviation. All the subjects consumed 240 ml of water completely given during administration of the investigational products in both period I & II. But subject no. 06 did not consume all 240 ml and left 6 ml of water in period I. Impact on the study: No significant impact in the study outcome, since the quantity of water left was very little. As per protocol, the blood samples collected have to be centrifuged within 15 minutes of collection of last set of samples for about 10 minutes. But due to power failure, the samples collected at 03.50 hrs and 04.00 hrs post dose in period I were not centrifuged within 15 minutes of collection of last set of samples. Because of this power interruption, the 03.50 hrs samples were centrifuged for 5 minutes extra in addition to 10 minutes and the 04.00 hrs samples were centrifuged for 3 minutes extra. Impact on the study out put: The impact was not significant. The normal reference values of serum protein and chloride were 6.0 – 8.0 g/dl and 95 – 103 mmol/l respectively, as per protocol. But the laboratory which performed the diagnostic tests has changed these reference values and hence the laboratory reports were having the reference values such as 6.0 – 8.3 g/dl and 97 – 107 mmol/l for protein and chloride respectively. For the evaluation of screening laboratory parameters and post study parameters, the reference ranges given in the laboratory report were only considered and not in the protocol. Impact of deviation: not significant. As per protocol the samples have to be stored at or less than -20°C. But the temperature of the deep freezer got increased to -12°C on 19/06/08 between 12.00 PM and 01.00 PM and it reached -20°C at 1.00 PM. The temperature increased to - 18°C on 21/06/08 between 07.00 PM and 08.00 PM and reached -20°C by 08.00 P.M. Impact on the study out put: No significant impact in study outcome. As per protocol, statistical analysis has to be outsourced to Statkon, a statistical organization. But it was performed in-house. The impact of this deviation is not significant.

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10 Analytical Method
10.1 Techniques PROTEIN PRECIPITATION: In protein precipitation, acids or water-miscible organic solvents are used to remove the protein by denaturation and precipitation. The proteins, which are in their cationic form at low pH, form insoluble salts with the acids. Organic solvents, such as methanol, acetonitrile, acetone and ethanol, although having relatively low efficiency in removing plasma proteins, have been widely used in bioanalysis because of their compatibility with high-performance liquid chromatography (HPLC) mobile phases. These organic solvents which lower the solubility of proteins and precipitate them from solutions have an effectiveness which is inversely related to their polarity. A volume of sample matrix is diluted with a volume of organic solvent or other precipitating agent, followed by vortex mixing and then centrifugation or filtration to isolate or remove the precipitated protein mass. Protein precipitation dilutes the sample. When a concentration step is required, the supernatant can be isolated, evaporated to dryness and then reconstituted before analysis. However, matrix components are efficiently removed and will be contained in the isolated supernatant or filtrate. In MS/MS detection systems, matrix contaminants have been shown to reduce the efficiency of ionization process. LIQUID-LIQUID EXTRACTION: Liquid-liquid extraction is the direct extraction of the biological material with a water immiscible solvent. The analyte is isolated by partitioning between the organic phase and the aqueous phase. The analyte should be preferentially distributed in the organic phase under the chosen conditions. For effective LLE some considerations should be taken such as 1) The analyte must be soluble in the extracting solvent. 2) A low viscosity of solvent to facilitate mixing with the sample matrix. 3) The use of pH control allows the fractionation of the sample into acid, neutral and basic components. 4) A large surface area is important to ensure rapid equilibrium. This is achieved by thoroughly mixing using either manual shaking or vortexing. SOLID –PHASE EXTRACTION: The stationary phase comes in the form of a packed syringeshaped catridge, a 24 well plate, which can be mounted on a commercially available extraction manifold. The manifold allows many samples to be processed simultaneously. A typical catridge solid phase extraction manifold can accommodate six disks. Most solid phase extraction manifolds are equipped with a vaccum port. Application of vaccum speeds up the extraction process by pulling the liquid sample through the stationary phase. The analytes are collected in sample tubes inside or below the manifold after they pass through the stationary phase. Solid phase extraction catridges and disks are available with a variety of stationary phases, each of which can separate analytes according to different chemical properties. Most stationary phases are based on silica that has been bonded to a specific functional group. Some of these functional groups include hydrocarbon chains of variable length for reversed phase solid phase extraction, quaternary ammonium or amino groups for anion exchange, and sulfonic acid or carboxyl groups for cation exchange. 10.2 Method Validation Selective and sensitive analytical methods for the quantitative evaluation of drugs and their metabolites (analyte) are critical for the successful conduct of preclinical and clinical pharmacology studies.

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Bioanalytical method validation includes all of the procedures that demonstrate that a particular method used for quantitative measurement of analytes in a given biological matrix, such as plasma, serum or urine, is reliable and reproducible for the intended use. The fundamental parameters for the validation include a) Accuracy b) Precision c) Selectivity d) Sensitivity e) Reproducibility and f) Stability. 10.3 Validation of Bioanalytical Method Plasma samples were analyzed to estimate the concentration of Metformin using a validated Bioanalytical method involving HPLC. The method was validated for sensitivity, specificity, linearity, accuracy, ruggedness and precision (repeatability & reproducibility), percent recovery and stability of samples (interim stability, freeze – thaw stability, bench top stability, auto sampler stability, dry extract, short term & long term stability of stock solution and internal standard). The linearity range for the analytical method was 80 ng/ml to 4000 ng/ml and the limit of quantification was 80 ng/ml. During the subject sample analysis, standard and quality control samples were distributed throughout each batch of subject samples analyzed. The analytical scientists did not have access to randomization and dispensing records. Table-7: Metformin Concentrations (ng/mL) in Subject Plasma Samples from Period I
SUBJECT NO. TIME POINTS (HR) 00.00 00.50 01.00 01.50 02.00 02.50 03.00 03.50 04.00 05.00 06.00 08.00 10.00 12.00 16.00 24.00 BLQ 502.13 985.72 920.64 830.82 799.43 776.22 715.29 681.79 582.47 477.26 282.32 174.58 130.09 BLQ BLQ BLQ 151.69 353.75 404.31 458.31 448.27 489.80 499.13 522.38 463.04 360.34 190.81 175.16 112.16 80.85 BLQ BLQ 425.30 591.80 861.44 950.83 945.49 945.58 921.30 840.26 644.67 518.02 299.58 196.10 142.20 87.98 BLQ BLQ 201.90 656.53 785.93 777.43 831.72 753.89 697.57 634.77 566.95 463.13 252.67 192.68 148.09 96.66 BLQ BLQ 431.59 597.62 807.08 824.08 892.98 934.24 952.23 966.30 820.79 599.84 383.21 269.73 197.32 104.65 BLQ BLQ 209.72 614.80 813.44 817.73 914.57 930.20 948.49 986.20 890.73 736.57 NR 222.46 153.67 BLQ BLQ BLQ 236.05 703.91 793.50 776.03 811.76 797.31 790.60 768.36 682.05 500.12 336.56 201.97 146.16 BLQ BLQ BLQ 160.00 209.28 518.61 547.27 526.84 590.84 572.97 623.99 704.98 627.02 434.77 246.24 178.41 96.11 BLQ BLQ 443.64 632.22 714.39 742.22 735.22 794.77 752.75 704.10 562.88 419.46 278.03 177.97 120.18 BLQ BLQ BLQ 383.48 1055.76 1114.11 1257.97 1236.46 1199.80 1169.18 1133.11 941.82 694.60 469.28 295.67 204.95 109.15 BLQ BLQ 579.16 756.56 822.24 843.89 780.14 673.53 779.06 724.19 505.46 387.34 216.76 148.01 95.27 BLQ BLQ BLQ 391.69 567.53 699.07 783.28 806.71 826.74 847.38 845.00 735.28 521.18 355.19 212.21 150.46 87.90 BLQ 1 2 3 4 5 6 7 8 9 10 11 12

Table-8: Metformin Concentrations (ng/mL) in Subject Plasma samples from Period II

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SUBJECT NO. TIME POINTS (HR) 00.00 00.50 01.00 01.50 02.00 02.50 03.00 03.50 04.00 05.00 06.00 08.00 10.00 12.00 16.00 24.00

1

2

3

4

5

6

7

8

9

10

11

12

BLQ 837.75 803.89 817.91 791.61 796.30 742.20 694.35 688.64 516.24 444.05 289.99 192.22 134.79 BLQ BLQ

BLQ 379.80 646.56 623.29 627.25 737.48 705.75 716.82 708.43 646.08 554.25 413.76 235.73 164.61 84.84 BLQ

BLQ 239.61 435.94 783.32 946.28 1005.83 1113.40 1065.85 944.30 839.39 664.33 420.73 236.85 173.22 87.36 BLQ

BLQ 298.30 554.87 574.83 689.20 708.88 716.36 744.55 706.30 668.72 529.94 358.96 209.91 137.73 BLQ BLQ

BLQ 277.40 695.21 772.19 749.59 822.80 841.39 870.99 721.92 750.05 563.34 364.14 257.69 193.78 102.48 BLQ

BLQ 164.63 474.02 866.57 995.34 1009.38 1033.09 1026.87 1001.55 700.79 692.05 435.50 265.88 186.59 98.17 BLQ

BLQ 145.40 305.11 619.39 796.73 805.50 806.17 849.36 836.32 739.44 533.15 327.45 201.13 164.48 81.98 BLQ

BLQ BLQ 427.15 598.33 552.98 613.10 686.04 736.06 746.92 685.17 544.09 321.26 195.00 123.94 BLQ BLQ

BLQ 621.42 859.81 1028.95 869.15 792.98 760.28 694.36 637.97 432.78 548.22 279.30 186.60 139.81 82.22 BLQ

BLQ 578.65 952.89 1028.01 1290.28 1160.85 1276.35 1278.57 1174.54 1010.91 757.92 514.87 323.79 236.68 116.32 BLQ

BLQ 533.81 911.91 876.21 842.95 903.76 883.89 837.26 751.02 561.84 376.89 237.79 137.25 96.67 BLQ BLQ

BLQ 300.93 683.72 785.42 869.00 840.73 956.76 970.07 931.37 769.91 582.92 355.53 239.94 167.79 102.36 BLQ

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11 Work Flow
11.1 Clinical Operations
Selection of Subjects and ICF

Study Initiation

Drug Dispensing

Medical Examination / Safety Assessment

Check-in of Subjects

Overnight Stay

Safety Assessment and pre-dose sampling

Dosing

Sample Collection / Adverse Event

Medical Examination / Safety Assessment

Check-out of Subjects

Ambulatory (If need) Compilation of Raw QA Audit Data, Medical records, preparation of clinical report Is this last ambulatory of last period?

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11.2 Bioanalytical and statistical Operations
Study Plan Analysis of Clinical Samples

Nomination of Project Leader

Compilation and Review of Raw Data & Chromatograms

Literature Search and Method Development

Pre-method validation & method validation protocol

Pharmacokinetic Analysis using WinNonLin

Method Validation Statistical Analysis using SAS Method Validation Report

Generation & Release of Preliminary QA Audit

Method SOP- Preparation (Drug Specific)

QA Audit Raw data &

Integration, Generation and Issuance of Final Study Report after QA Audit Clinical Report & Medical Records from Clinical Facility Sponsor Archives

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12 Data Quality Assurance
The study underwent quality assurance inspections at various stages (study initiation phase, study in-process phase and reporting phase) for conformance to the study protocol and all the governing SOPs. QA auditors at the end of the study checked all completed CRFs. A statement for adherence to Standard Operating Procedures as well as the pertinent requirements of the ICH ‘Guidance on Good Clinical Practice and Good Laboratory Practice’ for quality assurance is duly signed by the Manger-Quality Assurance is attached as appendix 16.1.7. 12.1 Statistical Methods Planned in the Protocol and Determination of Sample

Size
12.1.1 Statistical and Analytical Plans The log transformed pharmacokinetic parameters (Cmax, AUC0-t, AUC0-∞ ) were analyzed using a Mixed Effects ANOVA Model with the main effects of treatment, period and sequence as fixed factors and subject nested within sequence as random effect factor. A separate ANOVA model was used to analyze each parameter. The sequence effect was tested using the subjects nested within sequence mean square from the ANOVA as the error term. All other main effects were tested against the residual error (mean square error or MSE) from the ANOVA as the error term. The level of significance was 0.05 for period & treatment effect and 0.1 for sequence effect. Each analysis of variance includes calculation of least square means, the difference between the adjusted formulation means and the standard error associated with the difference. The above analysis was done using mixed procedure in SAS version 9.0. Based on the statistical analysis, the test product was concluded bioequivalent to the reference product if the 90% confidence interval for the difference (ratio in the original scale) of least squares means of log transformed pharmacokinetic parameters Cmax, AUC0-t and AUC0-∞ of test and reference formulations were within the bioequivalence range of 80.00-125.00%. 12.1.2 Descriptive statistics Summary statistics (mean, SD, minimum, maximum, Median, Percent of coefficient of variation and geometric mean) was provided by formulation for all metformin pharmacokinetic parameters. Descriptive statistics was also performed for the demographic data of the study subjects. 12.1.3 Determination of Sample Size This study has been conducted as a pilot study in order to get the preliminary pharmacokinetic data of the test and reference products and the sample size of 12+02 is justified at the end of the study by the values of intra subject CV of less than 11.05% and power of more than 99.48%. 12.2 Changes in Conduct of the Study or Planned Analyses There were no changes in either conduct of the study or planned analysis.

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13 Pharmacokinetic Evaluation
13.1 Data Sets Analyzed Fourteen normal healthy, adult male subjects were enrolled into the study and all of them completed both the periods of the study. The samples of the first 12 subjects were analyzed and the data were included for pharmacokinetic and statistical analysis. 13.2 Demographic and other Baseline Characteristics Among the 14 subjects who completed the study, the demographic details such as age, height, weight, BMI, dietary habits of the first 12 subjects were taken for descriptive analysis. Their mean age, height, weight and BMI were 30.33 yr, 1.677m, 66.60 Kg and 23.66 Kg/m2 respectively. All the 14 subjects enrolled into the study were belonging to Asian race and were Non-vegetarians. 13.3 Measurements of Treatment Compliance A thorough check of the oral cavity of the subject was carried out immediately after dosing. The duplicate label of the dosing sachet was pasted on the respective dosing record of the respective subject to ensure the correct allocation of the investigational product as per the randomization schedule. 13.4 Pharmacokinetic Results 13.4.1 Pharmacokinetic Analysis Fourteen normal healthy, adult male subjects were enrolled into the study and all of them completed both the periods of the study. The samples of the first 12 subjects were analyzed and the data were included for pharmacokinetic and statistical analysis. Pharmacokinetic parameters of both Test and Reference formulations were calculated using WinNonlin version 5.2.1 and are given below Table –9a: Summary statistics of pharmacokinetic parameters for test (T) formulation
Pk parameters (Units) Kel (hr-1) T1/2 (hr) T max (hr) C max ng/ml AUC 0-t ng.hr/ml AUC 0-∞ ng.hr/ml AUC 0-t / 0-∞ (%) N 12 12 12 12 12 12 12 Mean 0.18 4.02 2.83 899.00 6379.08 6969.99 91.42 SD 0.04 0.82 1.29 160.77 1143.52 1182.92 1.36 Min 0.13 2.64 0.5 704.98 5071.71 5535.27 88.97 Median 0.17 4.06 3 848.37 6126.24 6678.45 91.47 Max 0.26 5.27 5 1290.28 9431.50 10109.27 93.57 CV% 21.82 20.34 45.36 17.88 17.93 16.97 1.48 Geometric mean 0.18 3.94 2.47 886.93 6297.38 6889.24 91.41

N = Total number of subjects, SD = Standard deviation, CV = Coefficient of Variation

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Table –9b: Individual PK parameter values – Test formulation
Sub. No. 1 2 3 4 5 6 7 8 9 10 11 12 Sequence RT RT TR RT TR TR RT TR RT RT TR TR Period 2 2 1 2 1 1 2 1 2 2 1 1 N Mean SD Min Median Max CV% Geometric Mean Kel (/hr) 0.20 0.17 0.13 0.23 0.16 0.26 0.20 0.16 0.14 0.17 0.21 0.15 12 0.18 0.04 0.13 0.17 0.26 21.80 0.18 Thalf (hr) 3.53 4.09 5.27 3.05 4.39 2.64 3.51 4.43 5.10 4.04 3.37 4.78 12 4.02 0.82 2.64 4.06 5.27 20.30 3.94 Tmax (hr) 0.50 2.50 2.00 3.50 4.00 4.00 3.50 5.00 1.50 2.00 2.00 3.50 12 2.83 1.29 0.50 3.00 5.00 45.40 2.47 Cmax (ng/ml) 837.75 737.48 950.83 744.55 966.30 986.20 849.36 704.98 1028.95 1290.28 843.89 847.38 12 899.00 160.77 704.98 848.37 1290.28 17.90 886.93 AUC0-t (ng.hr/ml) 5540.01 6189.74 6466.69 5412.32 7272.33 6917.33 6044.80 5765.88 6062.73 9431.50 5071.71 6373.98 12 6379.08 1143.52 5071.71 6126.24 9431.50 17.90 6297.38 AUC0-inf (ng.hr/ml) 6227.12 6689.80 7134.97 6018.40 7935.06 7503.22 6459.96 6380.02 6667.11 10109.27 5535.27 6979.74 12 6970.00 1182.92 5535.27 6678.45 10109.27 17.00 6889.24 Ratio (AUC0-t /AUC0-inf) (%) 88.97 92.53 90.63 89.93 91.65 92.19 93.57 90.37 90.93 93.30 91.63 91.32 12 91.42 1.36 88.97 91.47 93.57 1.50 91.41

Table -10a: Summary statistics of pharmacokinetic parameters for reference (R) formulation
Pk parameters (Units) Kel (hr-1) T1/2 (hr) T max (hr) C max ng/ml AUC 0-t ng.hr/ml AUC 0-∞ ng.hr/ml AUC 0-t / 0-∞ (%) N 12 12 12 12 12 12 12 Mean 0.19 3.95 2.75 904.23 6165.77 6768.10 90.92 SD 0.04 0.98 1.01 189.25 1328.08 1371.18 2.00 Min 0.11 2.67 1 522.38 4028.09 4522.95 86.87 Median 0.17 4.17 3 891.45 5652.51 6414.86 90.76 Max 0.26 6.09 4 1257.97 8904.98 9567.05 93.59 CV% 23.97 24.87 36.77 20.93 21.54 20.26 2.20 Geometric mean 0.18 3.84 2.52 884.76 6037.92 6642.71 90.90

N = Total number of subjects, SD = Standard deviation, CV = Coefficient of Variation

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Table 10b: Individual PK parameter values – Reference formulation
Sub. No. 1 2 3 4 5 6 7 8 9 10 11 12 Sequence Period Kel (/hr) Thalf (hr) 3.17 4.24 4.15 6.09 4.41 4.19 3.20 2.81 3.30 4.20 2.67 4.97 12 3.952 0.983 2.67 4.17 6.09 24.9 3.845 Tmax (hr) 1.00 4.00 3.00 2.50 3.50 3.00 2.50 4.00 3.00 2.00 1.00 3.50 12 2.750 1.011 1.00 3.00 4.00 36.8 2.522 Cmax (ng/ml) 985.72 522.38 1113.40 831.72 870.99 1033.09 811.76 746.92 794.77 1257.97 911.91 970.07 12 904.225 189.251 522.38 891.45 1257.97 20.9 884.757 AUC0-t (ng.hr/ml) 5618.72 4028.09 7348.77 5618.50 6681.25 7433.85 5686.30 5024.79 5159.93 8904.98 5432.09 6945.16 12 6156.867 1335.978 4028.09 5652.51 8904.98 21.7 6027.347 AUC0-inf (ng.hr/ml) 6214.46 4522.95 7872.09 6468.00 7333.14 8027.84 6361.71 5526.62 5732.75 9567.05 5804.10 7679.72 12 6759.203 1379.533 4522.95 6414.86 9567.05 20.4 6632.120 Ratio (AUC0-t /AUC0inf) (%) 90.41 89.06 93.35 86.87 91.11 92.60 89.38 90.92 90.01 93.08 93.59 90.44 12 90.901 2.003 86.87 90.68 93.59 2.2 90.881

RT RT TR RT TR TR RT TR RT RT TR TR

1 1 2 1 2 2 1 2 1 1 2 2 N Mean SD Min Median Max CV% Geometric Mean

0.22 0.16 0.17 0.11 0.16 0.17 0.22 0.25 0.21 0.16 0.26 0.14 12 0.185 0.044 0.11 0.17 0.26 24.0 0.180

13.4.2 Statistical Analysis The Summary statistics of untransformed primary pharmacokinetic parameters Cmax, AUC0-t, and AUC0-∞ of test and reference formulations such as geometric mean, least square mean, T/R ratio, Intra subject CV and the level of significance values are given below in Table – 11.

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Table -11: Summary statistics of untransformed primary pharmacokinetic parameters Cmax, AUC0-t, and AUC0-∞ of test and reference formulations
Product / Statistics Least Square Mean -Test Least Square Mean -Reference T/R (%) Ratio 90% CI (T/R) Power (%) Intra-subject CV (%) P-value (ANOVA) Sequence Period Formulation Cmax (ng/ml) 899.00 904.23 99.42 92.13-106.71 99.49 9.88 0.83 0.18 0.89 AUC 0-t (ng.hr/ml) 6379.09 6165.77 103.46 97.02-109.90 99.83 8.55 0.72 0.10 0.35 AUC 0-∞ (ng.hr/ml) 6970.00 6768.10 102.98 96.79-109.18 99.88 8.25 0.76 0.16 0.40

The Summary statistics of log transformed primary pharmacokinetic parameters Cmax, AUC0-t, and AUC0-∞ of test and reference formulations are given below in Table – 12. Table 12: Summary statistics of log transformed primary pharmacokinetic parameters Cmax, AUC0-t, and AUC0-∞ of test and reference formulations
Product / Statistics Least Square Mean -Test Least Square Mean -Reference T/R (%) Ratio 90% CI (T/R) Power (%) Intra-subject CV (%) P-value (ANOVA) Sequence Period Formulation 0.70 0.18 0.96 0.59 0.13 0.32 0.65 0.19 0.37 Cmax (ng/ml) 886.93 884.76 100.25 92.40-108.76 99.48 11.05 AUC 0-t (ng.hr/ml) 6297.38 6037.92 104.30 96.90-112.26 99.79 9.96 AUC 0-∞ (ng.hr/ml) 6889.24 6642.71 103.71 96.67-111.27 99.86 9.53

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13.4.3 Analysis Charts Chart No:1
1400

Plot of Conc VS Time of all Subjects Formulation=R

1200

1000

1 2 3

800

4 5 6

600

7 8

400

9 10 11

200

12

0 0 2 4 6 8 time (hr) 10 12 14 16

Chart No:2
Plot of Conc VS Time of all Subjects Formulation=T

1400

1200

1000

1 2 3

800

4 5 6

600

7 8

400

9 10 11

200

12

0 0 2 4 6 8 time (hr) 10 12 14 16

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Chart No:3
Study No : AZ/BE/ST/001 Plot of mean Conc Vs Time

Plot of Mean Conc VS Time
900 800 700 600 500 400 300 200 100 0 0 2 4 6 8 Time_point 10 12 14 16
R T

Chart No. 4
1000

Plot of Log Scale Mean Conc VS Time
Study No : AZ/BE/ST/001 Plot of log scale mean Conc Vs Time

100
R T

10 0 2 4 6 8 Time_point 10 12 14 16

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13.4.4 Drug-Drug and Drug-Disease Interactions In this study, all the subjects were subjected to a screening procedure to ensure that only healthy adult human volunteers who were apparently not suffering from any disease are enrolled. And during the entire period of the study including wash out period the subjects were not found to be suffering from any significant disease or disorder. Hence there is no study drug (Metformin) – disease interaction in this study. It was ensured that the study subjects have not taken any drug within 7 days of enrollment during the check-in activities of period I. The subjects were dosed with either test or reference (containing Metformin 500 mg) in period I. 24 hours after dosing, the subjects were discharged. During discharge, the subjects were instructed not to consume any drug either prescription or over the counter. During check-in for period II, the fact that they have not consumed any drug in the wash out period was ensured before they were admitted. In period II, the subjects were dosed with alternate treatment containing Metformin 500 mg. No concomitant medication was administered to any subject during their in-house stay. Hence there were no study drug and other drug interaction during the entire period of study. 13.4.5 Pharmacokinetic Conclusions of Metformin The pharmacokinetic variables of both test and reference products were calculated using software Winnonlin version 5.2 and the results were analyzed using software SAS version 9.0. Based on the Pharmacokinetic and statistical evaluations, both the test and reference products containing 500 mg of Metformin hydrochloride are bioequivalent, since the ratio of log transformed primary PK parameters such as Cmax, AUC0-t, AUC0-∞ were well within the bioequivalence range of 80.00 to 125.00%.

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14 Safety Evaluation
14.1 Extent of Drug Exposure In period I fourteen subjects were dosed with a single dose of Test/Reference product containing Metformin 500 mg from 8.00 AM onwards. As per randomization, seven subjects received the Test formulation and the remaining received the Reference formulation. Followed by a washout period of 3 days, in period II, they were again dosed with a single dose of Metformin 500 mg formulation from 8.00 AM onwards. In period II, those who did not receive the Test formulation in period I were dosed with test formulation and vice-versa. 14.2 Adverse Events There were no adverse events reported during the study. 14.3 Clinical Laboratory Evaluation Clinical laboratory evaluations were carried out at the time of screening and also at the end of the study. Capillary blood sugar was tested in all the subjects at 02.00 hrs post dose and at the end of the study both in period I and period II to ensure that no subject is suffering from any drop in blood sugar. The sugar value was found to be well above the hypoglycemic level. 14.4 Vital Signs, Physical Findings and Other Observations Related to Safety 14.4.1 Recording of Vital Signs and Clinical Examination Pulse rate and blood pressure were recorded during check in, check-out, and at 00.00 hour (pre dose) and at 02.00, 04.00, 06.00, 12.00 and 24.00 hours post dose. Body temperature was recorded during check-in and check-out. 00.00 hour vitals were recorded within one hour prior to drug administration. All other vital parameters were recorded with the time flexibility of ± 30 minutes. The vital signs for all the subjects at the above mentioned time points were within normal range. All the subjects were found to be normal at the time of clinical examination conducted during check in and also at check out. 14.4.2 Post study Safety Evaluation Physical examination, vital parameter assessment, capillary blood sugar measurement and wellbeing assessment were performed to all study subjects during subject check-out, in each period, to ensure safety of the study subjects. At the end of period II, the biochemical and haematological parameters of the study subjects were evaluated. 14.5 Safety Conclusions In this study, Metformin tablets both test and reference were well tolerated upon single dose administration to normal healthy adult human subjects under fed conditions. There were no adverse events reported in this study. Vital parameters and well being assessment performed at the scheduled time intervals were normal and within the acceptable range for all the subjects before and after drug administration, in both the periods. Capillary blood sugar measurement performed at 02.00 hrs post dose and during check out in each period showed that no subject suffered from hypoglycemia. Post study safety evaluation to assess the haematological and biochemical parameters was performed at the end of period II. The results showed that there are no significant changes in the respective laboratory parameters.
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15 Discussion and Overall Conclusions
In this study, single oral dose of Metformin 500 mg tablets containing Generic Metformin Hydrochloride 500 mg was compared with single oral-dose of Branded tablets containing Metformin Hydrochloride 500 mg in healthy, adult, human subjects under fasting conditions. The statistical analysis and evaluation of primary pharmacokinetic variables (Cmax, AUC0-t, AUC0-∞) for both test and reference products show that the two formulation were bioequivalent. Hence the test formulation i.e., Generic Metformin 500 mg tablets containing Metformin Hydrochloride 500 mg is bioequivalent to the reference formulation i.e., Branded tablets containing Metformin Hydrochloride 500 mg in healthy, adult, human subjects under fed conditions

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16 References
a) Indian Council of Medical Research ( ICMR): Ethical guidelines for Medical research on human subjects 2006 b) Schedule Y of Drugs and Cosmetics Act, 20th January 2005 c) ICH Guidelines for Good Clinical Practices (E6) 1996 d) Additional Guidance for Organizations performing in Vivo Bioequivalence studies. Geneva: WHO; 2005.p.3-5 e) Guideline for Industry; Structure and Content of Clinical Study Reports, ICH E3, July 1996. f) Leon Shargel, Susanna WU, Andrew BC.(2005) Applied Biopharmaceutics and pharmacokinetics. Fifth edition. Boston: Mc Graw Hill.p.453-99 g) Joao Antonio SF, Jose PJ (2001). Manual for Good Bioavailability and Bioequivalence Practices. Brazil. Vol 1. Module Clinical step. h) Guidelines for Bioavailability and Bioequivalence Studies. Central Drugs Standard Control Organization, Directorate general of health sciences, New Delhi; March 2005. i) ICMR Ethical Guidelines for Biomedical Research on Human Subjects 2006 j) CDSCO Guidelines for BA/BE Studies, March 2005 k) Declaration of Helsinki (WMA General Assembly, Tokyo 2004) l) Guidance for Industry: Bioavailability and Bioequivalence studies for orally Administered Drug Products. U.S. Department of Health and Human Services, FDA: 2002 m) Rasma Chereson, Umesh banakar (1999). Basic Pharmacokinetic: Bioavailability, Bioequivalence and Drug selection. n) Dr. Cory Hawkins, Dr. Melissa Kiser and Dr. James Merdinck. Development and o) Validation of an LC-MS/MS Method. p) Dadger D. and Burnett PE (1995). Issues in Evaluation of Bioanalytical Method selectivity and drug stability. Journal of Pharmaceutical and Biomedical analysis; 14: p.23-31. q) Bressolle F, Bromet-pitit M and Audran M (1996). Validation of Liquid Chromatography and Gas Chromatographic Methods Application to Pharmacokinetics. Journal of Chromatography B; 686: 3-10. r) Green MJ (1996). A Practical Guide to Analytical Method Validation. Analytical Chemistry; 68: p.305A-309A s) Shah VP, Midha KK, Dighe S, Mc Gilveray JI (1992). Analytical Method validationBioavailability, Bioequivalence and Pharmacokinetics Studies, Journal of Pharmaceutical Sciences; 81:p. 309-312 t) Dighe, S.V. and Adams, W.P (1991), Bioequivalence: A United States Regulatory Perspective, in Pharmaceutical Bioequivalence, Welling, P.G., Tse, F.L.S., and Dighe, S.V., eds., Marcel Dekker, Inc., New York, p. 347-380. u) Meyer, M.C (1991), Scientific Basis of Bioavailability and Bioequivalence Testing, Am. Pharm., NS31 (8):47-52. v) Journal of Clinical Pharmacology vol 40.p.no, 1311
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