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Bioequivalence study of Metformin Hydrochloride (SR) 500 mg tablet

formulations in healthy human subjects
Abstract. Introduction: India has huge potential for generic drugs. Thats why, BABE studies
have become an important part of the clinical research in India. Bioequivalence studies are
performed to prove that, different formulations of a drug product are similar to each other in
terms of safety and efficacy.

Objective: The present study was carried out to assess the safety and efficacy of two Metformin
Hydrochloride (SR) 500 mg tablet formulations. The test formulation was Metformin
Hydrochloride (SR) 500 mg tablet of Vapi Care Pvt. Ltd., India, and the reference standard was
Dibeta SR tablet [containing Metformin Hydrochloride (SR) 500 mg] of Torrent Pharmaceuticals
Ltd., India.

Methodology: The present study was an open label, balanced, randomized, two treatment, two
period, two sequence, single dose, cross over bioequivalence study under fasting condition;
conducted at Auriga Research Ltd., New Delhi, India. Metformin Hydrochloride (SR) 500 mg
tablet of Vapi Care Pvt. Ltd., India, was the Test formulation and Dibeta SR tablet [containing
Metformin Hydrochloride (SR) 500 mg] of Torrent Pharmaceuticals Ltd., India, was the
Reference standard. Hippocrates Independent Ethics Committee (HIEC), New Delhi, India was
the ethical committee for this study. There was no dropout/withdrawal of subjects from this
study. Subjects were randomly assigned to receive a single oral dose of the test and the reference
formulation under fasting condition, with a washout period of seven days. Blood samples were
drawn as per the approved study protocol. Drug concentration in the plasma samples were
measured by using validated LC/MS/MS method. Win Nonlin Version 5.2 software was used for
statistical calculations.

Results and Discussion: The 90% confidence interval for ln-transformed pharmacokinetic
parameters Cmax, AUC0-t and AUC0- of the test formulation was 89.13%, 87.46% and 88.29%,
respectively. The 90% confidence interval for ln-transformed pharmacokinetic parameters Cmax,
AUC0-t and AUC0- of the reference standard was 112.44%, 123.85% and 123.87%, respectively.
The mean test/reference ratio for ln-transformed Cmax, AUC0-t and AUC0- were 100.110%,
104.080% and 104.580%, respectively.

Conclusion: Ln-transformed pharmacokinetic parameters Cmax, AUC0-t and AUC0- of the test
formulation and reference standard, are within the acceptable range of 80125% (to prove
bioequivalence between two drugs). So, the present study concludes that the test formulation
Metformin Hydrochloride (SR) 500 mg of Vapi Care Pvt. Ltd., India is bioequivalent to the
reference standard Dibeta SR tablet [containing Metformin Hydrochloride (SR) 500 mg] of
Torrent Pharmaceuticals Ltd., India.

LIST OF ABBREVIATIONS

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ADR Adverse Drug Reaction

AE Adverse Event

ANDA Abbreviated New Drug Application

ANOVA Analysis of Variance

API Active pharmaceutical ingredient

ARL Auriga Research Ltd.

AUC Area Under Curve

BA Bioavailability

BE Bioequivalence

BP Blood Pressure

BMI Body Mass Index

BUN Blood Urea Nitrogen

CDSCO Central Drugs Standard Control Organization

CI Confidence Interval

cm/s Centimeter/s

Cmax Maximum Plasma Concentration

COA Certificate of Analysis

cGMP current Good Manufacturing Practice

CNS Central Nervous System

CPU Clinical Pharmacological Unit

CRF Case Record Form

CRO Contract Research Organisation

cu.mm Cubic millimetre

CV Coefficient of variation

CYP Cytochrome P

dl Decilitre

ECG Electrocardiogram

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EMEA European Medicine Agency

ESR Erythrocyte Sedimentation Rate

FBS Fasting Blood Sugar

FDA Food and Drug Administration

Gamma GT Gamma Glutamyl Transferase

GCP Good Clinical Practice

GeoLSM Geometric least squares mean

GLP Good Laboratory Practice

gm Gram

GMP Good Manufacturing Practice

h / hr/ Hr Hour

HCT Haematocrit

HCV Hepatitis C Virus

HDL High Density Lipoproteins

HIV Human Immuno-deficiency Virus

Ht Height

HPFB Health Product and Food Brand

ICD Informed Consent document

ICF Informed Consent Form

ICH International Conference on Harmonization

ICMR Indian Council of Medical Research

ICU Intensive care unit

IEC Independent Ethics Committee

IgE Immunoglobulin E

IND Investigation New Drug application

IP Investigational Product

IRB Institutional Review Board

IU International Units

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IV Interavenous

Kel Elimination rate constant

Kg Kilogram

K2EDTA Dipotassium Ethylene Diamine Tetra acetic acid

L Litre

LDL Low Density Lipoproteins

ln/Log Logarithmic/ Logarithmic value to the base e

LSM Least Square Mean

m Metre

Max Maximum value

MCH Mean Corpuscular Haemoglobin

MCHC Mean Corpuscular Haemoglobin Concentration

MCV Mean Corpuscular Volume

MPV Mean Platelet Volume

NDA New Drug Approval

g Microgram

l/ul Microlitre

mEq Milliequivalent

mg Milligram

ml Millilitre

min Minute/Minimum

n/N Number of subjects

NA Not Applicable

NDAs New Drug Application(s)

ng Nanogram

No. Number

P Probability

pg Picogram

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PK Pharmacokinetic

QC Quality Control

R Reference

RBC Red Blood Cell

RDW Red Cell Distribution Width

rpm Revolutions Per Minute

SAE Serious Adverse Event

SD Standard Deviation

SDC Serum drug concentration

SE Standard Error

SGOT Serum Glutamate Oxaloacetate Transaminase

SGPT Serum Glutamate Pyruvate Transaminase

SOP Standard Operating Procedure

Sub No. Subject Number

T Test

THC Tetrahydrocannabinoids

Tmax/tmax Maximum time point

t1/2 Terminal half-life

U Unit

UK United Kingdom

USFDA United State Food and Drug Administration

VLDL Very Low Density Lipoproteins

WBC White Blood Cell

WHO World Health Organization

WMA World Medical Association

Wt Weight

C Degree Celsius

% Percentage

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% RSD Percentage relative standard deviation

List of Tables

Table No. TITLE PAGE No.

24
1 2x2 crossover design

2 Four-sequence and two-period design 25

3 Two-sequence and four-period design 25

4 Four-sequence and four-period design 25

5&6 Two-sequence and three-period design 25

7 Crossover design for three medications 26

8 Crossover design for four medications 27

9 List of Regulatory Authorities 38

10 Clinical Classification of diabetes mellitus 40

11 Categorization of Glucose Status 41

12 Classification of Oral Hypoglcaemic Agents (OHAs) 43

13 Types of Insulin 46

14 Description of Test and Reference Product 63

15 Investigational Product Accountability 65

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16 Administration Time of Study Drugs 67

17 Randomization Schedule 72

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18 Phlebotomy Schedule 73

19 Acceptance Criteria for Bioequivalence 81

20 Subjects Demographic Profile 86

21 Summary of Demographics of the study volunteers 87

22 Clinical Laboratory Evaluation 88-89

89-90
Urine Analysis
23

24 91
Blood Sample Collection

25 Pharmacokinetic Parameters of Test Product and 96

Reference Standard of the Study Drug

26 Primary Pharmacokinetic Parameters of Test and 97

Reference product of the Study Drug

27 Secondary Pharmacokinetic Parameters of Test and 97

Reference product of the Study Drug

28 Pharmacokinetic Bioequivalence Parameters of Test 98

and Reference product of the Study Drug

29 Summary of ANOVA Results obtained from Test and 99

Reference product of the Study Drug

30 100
Deviations in Vital Signs Measurements

31 101
Blood glucose monitoring Period I

32 102
Blood glucose monitoring Period II

33 103
Incidence of Adverse Events

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List of Figures

Figure No. TITLE PAGE No.

1 The stages of development of a typical new drug 6

2 A typical plasma drug concentration-time profile 9

3 Regular graph of first-order kinetics 15

4 Algorithm for the management of Type 2 DM 48

5 Volunteers included in the study 70

6 Flowchart of Bioequivalence Study 84-85

Mean plasma concentration vs. time curve of Test

7 and Reference Metformin Hydrochloride (SR) 92

500mg tablet formulations

Mean of Log plasma concentration of Test and

8 Reference Metformin Hydrochloride (SR) 500mg 93

tablet formulations

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Contents

SECTION TITLE PAGE No.

1 Introduction 1-4

2 Objectives 5

3 Review of Literature 6-61

4 Methodology 62-85

5 Results and Discussion 86-106

6 Conclusion 107

7 References 108-110

8 Annexures 111-123

1. Introduction

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Bioavailability era was started as early as 1945, with the first publication of the concept
of biological availability. The development of analytical techniques, during the 1960s,
made possible the development of methods sensitive enough to allow quantification of
drugs or metabolites, initially in the urine and afterwards in the plasma, making
possible the evaluation and the comparison of bioavailability of different formulations,
as well as demonstration that significant differences among them could occur.

Bioavailability is defined as the rate and extent to which the active ingredient or active
moiety is absorbed from a drug product and becomes available at the site of action. For
drug products that are not intended to be absorbed into the bloodstream, bioavailability
may be assessed by measurements intended to reflect the rate and extent to which the
active ingredient or active moiety becomes available at the site of action. (FDA,2003).

Bioequivalence is defined as the absence of a significant difference in the rate and

extent to which the active ingredient or active moiety in pharmaceutical equivalents or
pharmaceutical alternatives becomes available at the site of drug action when
administered at the same molar dose under similar conditions in an appropriately
designed study. (FDA, 2003)

The approaches to document BE in order of preference are (i) pharmacokinetic (PK)

measurements based on measurement of an active drug and/or metabolite in blood,
plasma, and/or urine; (ii) pharmacodynamic (PD) measurements; (iii) comparative
clinical trials; and (iv) in vitro studies.

The FDA usually considers that the plasma concentration of a drug is a surrogate for
the concentration at the site of action for a systemically acting drug. Proving
equivalence requires integration of several studies, such as PK, PD, controlled-clinical,
in vitro studies, and any other specific model or study that may prove useful in proving
equivalence.

The Drug Price Competition and Patent Term Restoration Act of 1984 (the Hatch
Waxman Amendments), established the current Abbreviated New Drug Application
approval process. The showing that must be made for an Abbreviated New Drug
Application to be approved is quite different from what is required in an New Drug
Application. An New Drug Application applicant must prove that the drug product is
safe and effective. An Abbreviated New Drug Application does not have to prove the

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safety and effectiveness of the drug product, because an Abbreviated New Drug
Application relies on the finding the FDA has made that the reference listed drug is safe
and effective. Instead, an Abbreviated New Drug Application applicant must
demonstrate, among other things,that its drug product is bioequivalent to the reference
listed drug. The scientific premise underlying the HatchWaxman amendments is that
in most circumstances, bioequivalent drug products may be substituted for each other.

A generic drug is bioequivalent to the listed drug if the rate and extent of the
absorption of the drug do not show a significant difference from the rate and extent of
absorption of the listed drug when administered at the same molar dose of the
therapeutic ingredient under similar experimental conditions in either a single dose or
multiple doses.

Bioequivalence studies are performed to demonstrate that different formulations or

regimens of drug product are similar to each other in terms of their therapeutic benefit
(efficacy) and non therapeutic side effects (safety). [1]

Selected reasons for conducting bioequivalence studies (Bert et al; 1991)

New formulation of a medicine is chosen to replace an old formulation (e.g.

new excipients or new salt of the medicine are used).
New dosage form of a medicine replaces an older one (e.g. tablets replace
capsules) or extends the product line.
New physical characteristics of a medicine product exist (e.g. increased density
of a tablet due to higher compression used in its manufacture).
An older supply of the medicine is to be compared with newer one in order to
determine the effect of age and natural changes in the overall composition.
Two lots of an active medicine are compared in which differences are shown to
exist.
A new manufacturing method is used to prepare the medicine or a new
manufacturing site is preparing the medicine and question of bioequivalence is
raised.
A new manufacturer (may be a generic manufacturer or a new supplier of a
brand name product) of a medicine desires to compare the new product with a
standard. [2]

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Need for the study

Globally diabetes affects 246 million people and is expected to affect 366 million by
2030. At least 7 million new cases are reported annually. India accounts for largest
diabetic population with 41 million patients, mounting to 6 percent of the adult
population. [3]

Diabetes mellitus is a heterogenous group of disorders characterized by hyperglycemia;

that results from defects in insulin secretion, insulin action (sensitivity), or both; is
associated with abnormalities in carbohydrate, fat and protein metabolism; and results
in chronic complications including microvascular, macrovascular, and neuropathic
disorders. Among various types of diabetes mellitus, Type 2 diabetes mellitus is the
commonest.

Various classes of oral hypoglycaemics are available for the treatment of Type 2
diabetes mellitus in addition to insulin formulations. These are Sulfonylureas,
Biguanides, Alpha-Glucosidase Inhibitor, Thiazolidinediones (TZD), Meglitinides.
Amylin Analogues, Incretin Mimetics and Dipeptidyl Peptidase-4 (DPP-4) Inhibitors
are the new treatment regimens available for the Type 2 diabetes mellitus. As per the
Global Guideline for Type 2 Diabetes (developed by International Diabetes
Federation, 2005), Metformin is considered as a first line drug.

Metformin is a biguanide hypoglycemic agent used in the treatment of non-insulin-

dependent diabetes mellitus not responding to dietary modification and exercise.
Metformin decreases hepatic glucose production (both gluconeogenesis and
glycogenolysis), decreases intestinal absorption of glucose, and improves insulin
sensitivity of both hepatic and peripheral (muscle) tissues causing an increased uptake
of glucose into these insulin-sensitive tissues.

Metformin is the only biguanide available in the market. Metformin has been used
clinically for 45 years, and has been approved since 1995. Metformin has become the
first-line therapy for glycemic control when oral agents are indicated in overweight
patients. The action of metformin does not involve stimulation of pancreatic insulin
secretion and therefore it is still a beneficial agent when -cell function has declined.
Another advantage of metformin over insulin secretagogues, and sulfonylureas in
particular, is that it does not usually cause hypoglycemia and weight gain. Metformin

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has a short duration of action, with a half-life of between 1.3 and 4.5 hours, and does
not bind to plasma proteins. It is not metabolised and is totally renally eliminated.

If there are no contraindications, Metformin can be used in conjunction with diet as

first-line therapy in patients not adequately controlled on diet alone. As it has a
different mode of action to the sulfonylureas, meglitinide analogues, thiazolidinediones
and -glucosidase inhibitors, it can be valuable when prescribed in combination. [4]

After the patent expires, several companies may manufacture and market different
formulations of a same active substance, with similar qualities and performances, so as
the interchangeability among different formulations, when given in equivalent doses,
presents the same safety and efficacy. Generic substitution has thus provided a means
of supplying the market with inexpensive, efficacious, and safe drug products after
patent expiration, without repeating the entire drug development process. [5]

Thats why Sponsor tried to bring a new generic version of Metformin Hydrochloride
(SR) 500 mg tablet into the market. As per the regulation, bioequivalence study should
be done before marketing. So, a bioequivalence study was conducted at Auriga
Research Ltd, comparing the Test formulation Metformin Hydrochloride (SR) 500 mg
tablet of Sponsor with the Reference standard Dibeta SR tablet [containing Metformin
Hydrochloride (SR) 500 mg] of Torrent Pharmaceuticals Ltd., India.

This was a DCGI submission study, so the CDSCO BA/BE Guidelines, 2005 were
followed in conjunction with Schedule Y to the Drugs and Cosmetic Rules, Indian GCP
Guidelines issued by CDSCO, GLP and the Ethical Guidelines for Biomedical
research on human subjects issued by Indian Council of Medical Research (ICMR
Guidelines). ICH-GCP Guidelines and Declaration of Helsinki were also referred as
and when required.

2. Objectives

2.1 GENERAL OBJECTIVE:

TO ASSESS THE BIOEQUIVALENCE OF THE TEST FORMULATION METFORMIN

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HYDROCHLORIDE (SR) 500 MG TABLET OF SPONSOR WITH THE REFERENCE

STANDARD DIBETA SR TABLET [CONTAINING METFORMIN HYDROCHLORIDE (SR)

500 MG] OF TORRENT PHARMACEUTICALS LTD., INDIA IN HEALTHY HUMAN

ADULT MALE SUBJECTS UNDER FASTING CONDITION.

2.2 SECONDARY OBJECTIVES:

TO STUDY AND EVALUATE THE BIOAVAILABILITY OF THE TEST FORMULATION

METFORMIN HYDROCHLORIDE (SR) 500 MG TABLET OF SPONSOR WITH THE
REFERENCE STANDARD DIBETA SR TABLET [CONTAINING METFORMIN
HYDROCHLORIDE (SR) 500 MG] OF TORRENT PHARMACEUTICALS LTD., INDIA.

TO COMPARE THE BIOAVAILABILITY OF THE TEST FORMULATION WITH THE

REFERENCE STANDARD.

TO ASSESS THE SAFETY OF THE TEST FORMULATION AND THE REFERENCE

STANDARD, ON THE BASIS OF CLINICAL AND LABORATORY EXAMINATION

3. Review of Literature

3.1 Drug Discovery and Development

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Figure 1: The stages of development of a typical new drug

This figure shows in an idealized way the stages of a typical project, aimed at
producing a marketable drug that meets a particular medical need (e.g. to retard the
progression of Parkinsons disease or cardiac failure, or to prevent migraine attacks).

Broadly, the process can be divided into three main components, namely:

Drug Discovery, during which candidate molecules are chosen on the basis of
their pharmacological properties.
Preclinical Development, during which a wide range of non-human studies
(e.g. toxicity testing, pharmacokinetic analysis and formulation) are performed.
Clinical Development, during which the selected compound is tested for
efficacy, side effects and potential dangers in volunteers and patients.

These phases do not necessarily follow in strict succession as indicated in the above
figure, but generally overlap.

3.2 Clinical Development (Phases of Clinical Trials)

Clinical development proceeds through four distinct phases (Friedman et al., 1996)

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3.2.1 Phase I

Phase I trials are performed on a small group (normally 20-80) of normal healthy
volunteers, and their aim is to check for safety (does the drug produce any potentially
dangerous effects, for example on cardiovascular, respiratory, hepatic or renal
function?), tolerability (does the drug produce any unpleasant symptoms, for example
headache, nausea, drowsiness?) and pharmacokinetic properties (is the drug well
absorbed? What is the time course of the plasma concentration? Is there any evidence
of cumulation or non-linear kinetics?). Phase I studies may also test for
pharmacodynamic effects in volunteers (e.g. does a novel analgesic compound block
experimentally induced pain in humans? How does the effect vary with dose?).

3.2.2 Phase II

Phase II studies are performed on groups of patients (normally 100-300) and are
designed to test for efficacy in the clinical situation, and if this is confirmed, to
establish the dose to be used in the definitive Phase III study. Often, such studies will
cover several distinct clinical disorders (e.g. depression, anxiety states and phobias) to
identify the possible therapeutic indications for the new compound and the dose
required. When new drug targets are being studied, it is not until these Phase II trials
are completed that the team finds out whether or not its initial hypothesis was correct,
and lack of the expected efficacy is a common reason for failure.

3.2.3 Phase III

Phase III studies are the definitive double-blind randomized trials, commonly
performed as multicentre trials on 1000-3000 patients, aimed at comparing the new
drug with commonly used alternatives. These are extremely costly, difficult to
organize, and often take years to complete, particularly if the treatment is designed to
retard the progression of a chronic disease. It is not uncommon for a drug that seemed
highly effective in the limited patient groups tested in Phase II to look much less
impressive under the more rigorous conditions of Phase III trials.

Increasingly, phase III trials are being required to include a pharmacoeconomic

analysis, such that not only clinical but also economic benefits of the new treatments
are assessed. The whole process has to comply with an elaborate code known as Good

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Clinical Practice, covering every detail of the patient group, data collection methods,
recording of information, statistical analysis and documentation.

At the end of phase III, the drug will be submitted to the relevant regulatory authority
for licensing. The dossier for this is a massive and detailed compilation of preclinical
and clinical data. Evaluation by the regulatory authority normally takes a year or more,
and further delays often arise when aspects of the submission have to be clarified or
more data are required. Eventually, about two-thirds of submissions gain marketing
approval.

3.2.4 Phase IV

Phase IV studies comprise the obligatory post marketing surveillance designed to detect
any rare or long-term adverse effects resulting from the use of the drug in a clinical
setting in many thousands of patients. Such events may necessitate limiting the use of
the drug to particular patient groups, or even withdrawal of the drug. [6]

3.3 Pharmacokinetics: Basic Considerations

Pharmacokinetics is defined as the kinetics of drug absorption, distribution, metabolism

and excretion (KADME) and their relationship with the pharmacologic, therapeutic or
toxicologic response in man and animals. The applications of pharmacokinetic
principles in the safe and effective management of individual patient is called as
clinical pharmacokinetics.

The process of movement of drug from its site of administration to the systemic
circulation is called as absorption. Bioavailability is defined as the rate and extent
(amount) of drug absorption. The movement of drug between one compartment and the
other (generally blood and the extravascular 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. Elimination occurs by two processes-biotransformation
(metabolism), which usually inactivates the drug, and excretion which is responsible for
the exit drug/metabolites from the body.

3.3.1 Plasma Drug ConcentrationTime Profile

A direct relationship exists between the concentration of drug at the biophase (site of
action) and the concentration of drug in plasma. A typical plasma drug concentration-

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time curve obtained after a single oral dose of a drug and showing various
pharmacokinetic and pharmacodynamic parameters is depicted in Fig.2

Such a profile can be obtained by measuring the concentration of drug in plasma

samples taken at various intervals of time after administration of a dosage form and
plotting the concentration of drug in plasma (Y-axis) versus the corresponding time at
which the plasma sample was collected (X-axis).

Fig.2: A typical plasma drug concentration-time profile showing pharmacokinetic

and pharmacodynamic parameters, obtained after oral administration of single
dose of a drug.

(A) Pharmacokinetic Parameters

The three important pharmacokinetic parameters that describe the plasma level-time
curve and useful in assessing the bioavailability of a drug from its formulation are:

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i) Peak Plasma Concentration (Cmax)

The point of maximum concentration of drug in plasma is called as the peak and the
concentration of drug at peak is known as peak plasma concentration. It is also called
as peak height concentration and maximum drug concentration. Cmax is expressed in
mcg/mL.

The peak represents the point of time when absorption rate equals elimination rate of
drug. The portion of curve to the left of peak represents absorption phase i.e. when the
rate of absorption is greater than the rate of elimination. The section of curve to the
right of peak generally represents elimination phase i.e. when the rate of elimination
exceeds rate of absorption.

ii) Time of Peak Concentration (tmax)

The time for drug to reach peak concentration in plasma (after extravascular
administration) is called as the time of peak concentration. It is expressed in hour (hr)
and is useful in estimating the rate of absorption. Onset time and onset of action are
dependent upon tmax.

iii) Area Under the Curve (AUC)

It represents the total integrated area under the plasma level-time profile and expresses
the total amount of drug that comes into the systemic circulation after its
administration. AUC is expressed in mcg hr/mL. It is the most important parameter in
evaluating the bioavailability of a drug from its dosage form as it represents the extent
of absorption.

(B) Pharmacodynamic Parameters

The various pharmacodynamic parameters are:

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i) Minimum Effective Concentration (MEC)

It is defined as the minimum concentration of drug in plasma required to produce the

therapeutic effect. It reflects the minimum concentration of drug at the receptor site to
elicit the desired pharmacologic response. The concentration of drug below MEC is
said to be in the subtherapeutic level.

In case of antibiotics, the term Minimum Inhibitory Concentration (MIC) is used. It

describes the minimum concentration of antibiotic in plasma required to kill or inhibit
the growth of microorganisms.

ii) Maximum Safe Concentration (MSC)

Also called as Minimum Toxic Concentration (MTC), it is the concentration of drug in

plasma above which adverse or unwanted effects are precipitated. Concentration of
drug above MSC is said to be in the toxic level.

iii) Onset of Action

The beginning of pharmacologic response is called as onset of action. It occurs when

the plasma drug concentration just exceeds the required MEC.

iv) Onset Time

It is the time required for the drug to start producing pharmacologic response. It
corresponds to the time for the plasma concentration to reach MEC after administration
of drug.

v) Duration of Action

The time period for which the plasma concentration of drug remains above the MEC
level is called as duration of drug action.

vi) Intensity of Action

It is the maximum pharmacologic response produced by the peak plasma concentration

of drug. It is also called as peak response.

vii) Therapeutic Range

The drug concentration between MEC and MSC represents the therapeutic range. [7]

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3.3.2 Determination of Main pharmacokinetic measures

Pharmacokinetic measures assessed by bioequivalence derive directly from drug

concentration curve along time, which is characterized by the quantification of a given
number of biological samples related to previously established collection times.

The first and most important measure assessed is the area under the curve of the drug
plasma concentration versus time, often used to measure absorption length or the total
amount of drug absorbed by the body after a single-dose administration of a
medication. The determination of bioequivalence between two medications results from
the comparison of AUCs obtained from the experiment. By its mathematical
representation

AUC= FD/ Ke.Vd

This measure is noticed to be directly proportionate to the amount of drug effectively

absorbed and available to be distributed through the body (FD). The term Ke.Vd,
expresses the drug total clearance, i.e., its elimination speed from the distribution
volume, where Ke corresponds to the drug total elimination speed constant in the body.

Among several methods for the determination of AUC from zero time to last collection
time (tk) the most used one is that of trapezoids (Chow & Liu, 2006). This method
comprises the sum of trapezium areas as determined by collection times and respective
concentrations.

Let us assume that C0, C1, C2, Ck, are the concentrations obtained in an experiment for
collection times 0, t1, t2, tk, respectively. AUC from zero to tk, denoted by AUCtk, is
obtained as follows:

The area under the curve of concentration versus time (AUC) may also be extrapolated
and calculated from zero time up to the time related to the complete elimination of the
drug. This measure is referred to in literature as the area under the curve from zero time
to infinite. The additional portion is expressed by a relationship between the last
concentration measured Ck and the drug elimination speed constant Ke. The

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elimination constant is calculated for each volunteer as the slope coefficient of

regression line adjusted for the 4 to 6 last values converted to log10, multiplied by
2.303. The area under the curve from zero to infinite is obtained as follows: [8]

3.3.3 Determination of Other pharmacokinetic measures

Other pharmacokinetic measures that shall also be presented in the bioequivalence

studies, even though they do not require comparative statistical handling, are: Apparent
Volume of Distribution (Vd), Clearance (Cl) and Elimination half-life (t1/2).

i) Apparent Volume of Distribution (Vd)

Apparent Volume of Distribution (Vd) is defined as the hypothetical volume of body

fluid into which a drug is dissolved or distributed. It is called as apparent volume
because all parts of the body equilibrated with the drug do not have equal
concentration. It is expressed in liters.

Apparent Volume of Distribution = Amount of the drug in the body / Plasma drug
concentration

i.e. Vd = X / C

Drugs that are highly lipid soluble, such as digoxin, have a very high volume of
distribution (500 litres). Drugs, which are lipid insoluble, such as neuromuscular
blockers, remain in the blood, and have a low Vd.

ii) Clearance (Cl)

Clearance (Cl) is defined as the theoretical volume of body fluid containing drug (i.e.
that fraction of apparent volume of distribution) from which the drug is completely
removed in a given period of time. It is expressed in ml/min or liters/hour.

Clearance = Rate of elimination / Plasma drug concentration

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i.e. Cl = dX/dt/C

iii) Elimination half-life (t1/2)

Elimination half-life (t1/2) is defined as the time taken for the amount of drug in the
body as well as plasma concentration to decline by one-half or 50% its initial value. It
is expressed in hours or minutes. It is also called as biological half-life. After four half-
lives, elimination is 93.75% complete.

Half-life is related to elimination rate constant (KE) by the following equation:

t1/2= 0.693/ KE

Elimination half-life is a secondary parameter that depends upon the primary

parameters clearance and apparent volume of distribution according to following
equation:

t1/2= 0.693 Vd/ Cl

If the Apparent Volume of Distribution (Vd) is increased, then elimination rate constant
(KE) will decrease, the Elimination half-life (t1/2) will increase, but the clearance (Cl)
will not change. [9]

3.3.4 First Order Kinetics (Linear Kinetics)

First order process is one whose rate is directly proportional to the concentration of
drug undergoing reaction i.e. greater the concentration, faster the reaction. It is because
of such a proportionality between rate of reaction and the concentration of drug that a
first-order process is said to follow linear kinetics.

A constant fraction of the drug in the body is eliminated per unit time. The rate of
elimination is proportional to the amount of drug in the body. The majority of drugs are
eliminated by this way. Most of the pharmacokinetic processes like absorption,
distribution follow first order kinetics. [10]

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Fig.3: Regular graph of first-order kinetics

3.4 Bioavailability and Bioequivalence

During its existence, a new drug, is considered to have 3 life periods. The first of them
is before its approval for public use, when pre-clinical and clinical studies are carried
out, to evaluate efficacy and safety. The second comprises the life span of its patent, in
which a marketing exclusivity is still valid. The last period begins just after the patent
expires and other companies may market the product, as a similar or as a generic drug.
Thus, several companies may manufacture and market different formulations of a same
active substance, with similar qualities and performances, so as the interchangeability
among different formulations, when given in equivalent doses, presents the same safety
and efficacy.

Bioavailability era was started as early as 1945, with the first publication of the concept
of biological availability. The development of analytical techniques, during the 1960s,
made possible the development of methods sensitive enough to allow quantification of

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drugs or metabolites, initially in the urine and afterwards in the plasma, making
possible the evaluation and the comparison of bioavailability of different formulations,
as well as demonstration that significant differences among them could occur.

Bioavailability is defined as the rate and extent to which the active ingredient or active
moiety is absorbed from a drug product and becomes available at the site of action. For
drug products that are not intended to be absorbed into the bloodstream, bioavailability
may be assessed by measurements intended to reflect the rate and extent to which the
active ingredient or active moiety becomes available at the site of action. (FDA,
2003).

Bioequivalence is defined as the absence of a significant difference in the rate and

extent to which the active ingredient or active moiety in pharmaceutical equivalents or
pharmaceutical alternatives becomes available at the site of drug action when
administered at the same molar dose under similar conditions in an appropriately
designed study. (FDA, 2003)

The approaches to document Bioequivalence in order of preference are

pharmacokinetic (PK) measurements based on measurement of an active

drug and/or metabolite in blood, plasma, and/or urine;
pharmacodynamic (PD) measurements;
comparative clinical trials; and
in vitro studies.

The FDA usually considers that the plasma concentration of a drug is a surrogate for
the concentration at the site of action for a systemically acting drug. Proving
equivalence requires integration of several studies, such as PK, PD, controlled-clinical,
in vitro studies, and any other specific model or study that may prove useful in proving
equivalence.

The concept of Bioequivalence and the required proof by the regulatory agencies has
evolved over the past several decades:

In the United States, the 1902 federal law for biologics, particularly vaccines,
required evaluation for safety, purity, and potency.
The 1906 Food and Drugs Act added drugs other than biologics.

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The 1938 FDC Act created FDA and evaluation of new drugs based on data in a
filed NDA.
The 1962 law added effectiveness requirements for the approval of NDA. 1902
Federal law required that biologics (vaccines) be evaluated for safety, purity,
and potency.
The 1906 Food and Drugs Act added drugs other than biologics.
The 1938 FDC Act created FDA and required safety evaluation on new drugs
before marketing based on data in an NDA.
The 1962 law added effectiveness requirement for approval of an NDA; in the
1960s, the FDA permitted marketing of similars, while corresponding pioneer
products underwent
Drug Efficacy Study Implementation (DESI) reviews. Similars came into the
market between 1938 and 1962.
In 1970 the FDA terminates marketing of similars unless DESI pioneer
showed safety and efficacy.
Similar manufacturer submits ANDA with formulation and manufacture
information;
(The Supreme Court in the United States vs. Generix Drug Corporation
supported FDA requirement for ANDA).
The 1984 generic law in the United States (WaxmanHatch) created a generic
approval system for all new drugs, including those approved after 1962. The
FDA finalized the bioequivalence (BA/BE) regulations, wherein the pioneer
shows BA in NDA; similars to DESI-effective pioneers show BE leading to
first United States first generics. Several revisions to the bioequivalence
(BA/BE) regulations, were made including the most recent one in April 2006.
The Drug Price Competition and Patent Term Restoration Act of 1984 (the
HatchWaxman Amendments), established the current Abbreviated New Drug
Application approval process. The showing that must be made for an
Abbreviated New Drug Application to be approved is quite different from what
is required in an New Drug Application. An New Drug Application applicant
must prove that the drug product is safe and effective. An Abbreviated New
Drug Application does not have to prove the safety and effectiveness of the
drug product, because an Abbreviated New Drug Application relies on the
finding the FDA has made that the reference listed drug is safe and effective.

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Instead, an Abbreviated New Drug Application applicant must demonstrate,

among other things, that its drug product is bioequivalent to the reference listed
drug. The scientific premise underlying the HatchWaxman amendments is that
in most circumstances, bioequivalent drug products may be substituted for each
other.

A generic drug is bioequivalent to the listed drug if the rate and extent of the
absorption of the drug do not show a significant difference from the rate and extent of
absorption of the listed drug when administered at the same molar dose of the
therapeutic ingredient under similar experimental conditions in either a single dose or
multiple doses.

Bioequivalence studies are performed to demonstrate that different formulations or

regimens of drug product are similar to each other in terms of their therapeutic benefit
(efficacy) and non therapeutic side effects (safety). [11]

3.5 Different Steps in Bioavailability/Bioequivalence (BA/BE) Studies

3.5.1Clinical Step

The clinical phase is comprehended from volunteer selection up to hospital discharge

and the last follow-up return.

When a bioavailability/bioequivalence center is assembled to carry out clinical step, it

should be reminded that the participants of such studies are not entitled to direct
therapeutic benefits from their participation. Therefore, all possible measures should be
taken in order to minimize risks related to drug administration or hospitalization, in
addition to providing the most comfortable situation as possible to the subjects.

When organizing a clinical research unit to carry out bioavailability/bioequivalence

studies, it should be taken into consideration that such center is not only comprised of a
physical facilitiy with a specific legislation, but mainly by the interaction of a staff that
is, by definition, multidisciplinary. Considering the multidisciplinarity of the
professional staff required for the performance of the clinical step of the studies, as well
as the importance of this phase for the following steps (analytical and statistical) and
final outcome of the study, it is mandatory that all professionals involved have a
general knowledge about the analytical and statistical steps, since their participation

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may influence the final outcome of the study.

3.5.2 Bioanalytical Step

The performance of a literature research is the first step for the search of the
bioanalytical method. Once the method exists, it should be tested for its reproducibility.
In the event there is no bioanalytical method for a given drug, the analytical center
should develop a method that responds satisfactorily to the study aimed.

The previous performance of the steps required for the development of the analytical
method for bioequivalence studies assures to the analytical center and its contractor that
the services hired will be carried out at the deadline foreseen and with the required
reliability of the results, which will be evaluated for registration purposes of the study
drug. In this context, we may state that contracted party and contractor will not waste
their time and additional financial resources in the event the studies performed are
rejected in the end due to the inappropriateness of the method used and undetermined
storage conditions.

3.5.3 Statistical Step

Concentration levels of a drug in the body depend partially on the administration route,
which may be classified as intravascular or extravascular. Intravascular administration
is carried out directly into blood stream by intravenous or intra-arterial route.
Extravascular includes oral, intramuscular, subcutaneous, transdermal and other
administration routes. When a drug is administered to the body, it generally passes
through the absorption, distribution, metabolism, and finally, elimination stages. Thus,
bioavailability is generally determined by pharmacokinetic measures, i.e., those relating
to the drug absorbed amount and the absorption process speed. These measures may be
obtained from the drug quantification results in biological fluids such as blood or urine,
after single-dose extravascular administration.

A bioequivalence study basically refers to the comparison of the main pharmacokinetic

measures as observed in the experiment, related to the products to be tested. Two
medications are considered as bioequivalent if their absorption amounts and speeds do
not present statistically significant differences when administered at the same molar
dose of the active ingredient, under the same experimental conditions.

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The choice of designs and statistical methods for data analysis are two important
aspects in a bioequivalence study. These two aspects are much related to each other,
once the analysis method depends on the used design. The most used experimental
planning in bioavailability / bioequivalence assays is the crossover one.

Elimination period (washout) is defined as a sufficiently large time interval between

two treatment periods for the carryover effect of a formulation administered within a
treatment period to be eliminated from the experimental units for next period.

Drug effect is the one observed during the period in which it is administered, while
carryover effect is the drug effect that persists after the end of the dosage period. The
existence of carryover effect means that there are different carryover effects in the
sequences of treatment; The non-existence of carryover effect does not necessarily
imply such effects are null, but that if existing, they have the same intensity in both
sequences of treatment.

3.6 Study Designs in Bioequivalence Studies

3.6.1 General Study Designs in Bioequivalence Studies

Usually a complete crossover design is used. With this design, each subject receives all
products with a washout period between each dose administration.

(A). Open-Label

A term used to describe the situation when both the researcher and the participant in a
research study know the treatment the participant is receiving. Open-label is the
opposite of double blind when neither the researcher nor the participant knows what
treatment the participant is receiving.

(B). Blinding (synonym: masking)

Keeping group assignment (e.g. to treatment or control) secret from the study
participants or investigators. Blinding is used to protect against the possibility that
knowledge of assignment/drug may affect patient response to treatment, provider
behaviors (performance bias) or outcome assessment (detection bias). Blinding is not
always practical (e.g. when comparing surgery to drug treatment). The importance of

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blinding depends on how objective the outcome measure is; blinding is more important
for less objective outcome measures such as pain or quality of life.

(C). Double Blind (synonym: double masked)

Neither the participants in a trial nor the investigators (outcome assessors) are aware of
which intervention the participants are given. The purpose of blinding the participants
(recipients and providers of care) is to prevent performance bias. The purpose of
blinding the investigators (outcome assessors, who might also be the care providers) is
to protect against detection bias.

(D). Triple Blind (synonym: triple masked)

An expression that is sometimes used to indicate that knowledge of which study

participants are in which comparison group is kept secret from the statistician dosing
the analysis as well as from the study participants and investigators (outcome
assessors).

(E). Performance Bias

Systematic differences in the care provided apart from the intervention being evaluated.
For example, if patients know they are in the control group, they may be more likely to
use other forms of care, patients who know they are in the experimental (intervention)
group may experience placebo effects, and care providers may treat patients differently
according to what group they are in. Blinding of study participants (both the recipients
and providers of care) is used to protect against performance bias.

(F). Detection Bias (synonym: ascertainment bias)

Systematic differences between comparison groups and how, outcomes are ascertained
diagnosed or verified.

(G). Concealment of allocation

The process used to prevent foreknowledge of group assignment in a randomized

controlled trial, which should be seen as distinct from blinding. The allocation process
should be impervious to any influence by the individual making the allocation by

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having the randomization process administered by someone who is not responsible for
recruiting participants; for example, a hospital pharmacy, or a central office. Using
methods of assignment such as date of birth and case record numbers (see quasi-
random allocation) are open to manipulation. Adequate methods of allocation
concealment include: centralized randomization schemes; randomization schemes
controlled by a pharmacy; numbered or coded containers in which capsules from
identical-looking, numbered bottles are administered sequentially; on-site computer
systems, where allocations are in a locked unreadable file; and sequentially numbered
opaque, sealed envelopes.

(H). Randomized Controlled Trial (RCT)

A study in which patients are allocated at random (by chance alone), to receive one of
several clinical interventions. One of these interventions is the standard of comparison
or control. The control may be a standard practice, a placebo (sugar pill), or no
intervention at all. Someone who takes part in a randomized controlled trial (RCT) is
called a participant or subject. RCTs seek to measure and compare the outcomes after
the participants receive the interventions. Because the outcomes are measured, RCTs
are quantitative studies.

Randomization is the process of assigning clinical trial participants to treatment groups.

Randomization gives each participant a known (usually equal) chance of being
assigned to any of the groups. Successful randomization requires that group assignment
can not be predicted in advance.

(I). Crossover Design

A research design where the subjects will get both the treatments in sequence. Contrast
this with a parallel groups design where some subjects get the first treatment and
different subjects get the second treatment. The crossover design represents a special
situation where there is not a separate comparison group. In effect, each subject serves
as his/her own control. In addition, since the same subject receives both treatments,
there is no possibility of covariate imbalance. Ideally, in a crossover design, a subject is
randomly assigned to a specific treatment order. Some subjects will receive the

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standard therapy first, followed by the new therapy (AB). Others will receive the new
therapy first, followed by the standard therapy (BA).

(J). Parallel Groups Design

A research design, where we apply the treatment and the control (or the two treatments)
simultaneously to two separate groups of subjects. Contrast this with a crossover design
where each subject gets the treatment and then the control (or the control and then the
treatment) in sequence.

(K) Cross Sectional Design

A research design, where subjects are assessed at a single time in their lives. A cross
sectional study is fast and can study a large number of patients at little cost or effort.
Also, we dont have to worry about patients dropping out during the course of the
study. This study is efficient at identifying association, but may have trouble deciding
cause and effect. With data at only one time point, we do not know whether the chicken
or the egg came first.

(L). Cohort Design

An observational design, where patients are selected based on an exposure variable.

Contrast this with a case control design. For example: In Beral et al (1999), general
practitioners in the United Kingdom recruited 23,000 women who were using oral
contraceptives and a similar number of women who were not using oral contraceptives.
The researchers followed both groups of women for 25 years and noted how many
died.

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3.6.2 Commonly used Designs in Bioequivalence Studies (Design Types)

3.6.2.1 Crossover design for two medications (T = Test, R = Reference)

A) Table 1: 2x2 crossover design

This is a conventional not-replicated design with two formulations, two periods, two
sequences that may be represented as follows:

Sequence Period 1 Period 2

1 R T
2 T R

Each individual is randomly assigned to RT or TR sequence in two dosage periods.

That is, individuals assigned to RT (TR) sequence receive formulation R (T) in the first
dosage period and formulation T (R) in the second dosage period. Dosage periods are
separated by enough time for the drug received in the first period to be fully
metabolized and/or eliminated from the body when the second dosage period starts.

Randomization for a 2x2 crossover study may be carried out through tables of random
numbers or randomization procedures implemented by statistical software.

B) Replicated crossover design

This design is recommended for bioequivalence studies of formulations with modified-

release dosage or highly variable products (intra-individual variation coefficient
30%), including the quick-release, and modified-release ones and other oral
administration products.

The same test and reference formulation batches shall be used for this design for
replicated administration. The periods shall be sufficiently spaced (washout) to assure
non-existence of carryover effects. More commonly used replicated crossover designs
to compare two formulations are:

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i) Table 2: Four-sequence and two-period design (Balaams design):

Sequence Period 1 Period 2

1 T T
2 R R
3 R T
4 T R

ii) Table 3: Two-sequence and four-period design

Sequence Period 1 Period 2 Period 3 Period 4

1 T R R T
2 R T T R

iii) Table 4: Four-sequence and four-period design

Sequence Period 1 Period 2 Period 3 Period 4

1 T T R R
2 R R T T
3 T R R T
4 R T T R

iv) Table 5: Two-sequence and three-period design

Sequence Period 1 Period 2 Period 3

1 T R T
2 R T R

Or

Table 6: Two-sequence and three-period design

Sequence Period 1 Period 2 Period 3

1 T R R
2 R T T

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A larger number of volunteers are recommended for the three-period design, as

compared with the four-period design in order to achieve the same statistical power for
bioequivalence.

3.6.2.2 Crossover design for three medications (Williams design)

In order to compare three formulations of a drug, there is a total of three possible

comparison pairs among formulations: formulation 1 versus formulation 2, formulation
1 versus formulation 3, and formulation 2 versus formulation 3. Since the number of
formulations to be compared is large, more sequences and consequently more
individuals are required, which may be unfeasible. A practicular design proposed by
Williams (1949) has balancing properties and requires few sequences and periods. A
design is said to be balanced if it fulfills the following conditions:

Each medication is administered one single time to each volunteer;

The number of volunteers receiving medication has to be equal in each period;
The number of volunteers receiving medication i in any period followed by
medication j next
period is the same for all ij.

Table 7: Williams design is illustrated as follows: (T1 = Test 1, T2 = Test 2, R =

Reference)

Sequence Period 1 Period 2 Period 3

1 R T2 T1
2 T1 R T2
3 T2 T1 R
4 T1 T2 R
5 T2 R T1
6 R T1 T2

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3.6.2.3 Table 8: Crossover design for four medications (Williams design):

Sequence Period 1 Period 2 Period 3 Period 4

1 R T3 T1 T2
2 T1 R T2 T3
3 T2 T1 T3 R
4 T3 T2 R T1

T1 = Test 1, T2 = Test 2, T3= Test 3 R = Reference

3.6.3 Selection of the experimental design

Selecting an appropriate design when planning a bioequivalence study is an important

issue. The answer to this issue depends on several factors, such as:

Number of formulations to be compared;

Drug characteristics and availability
Study objective
Inter- and intra-individual variability;
Study duration and number of periods employed;
Cost of addition of a volunteer related to the addition of one period;
Dropout rate.

Data analysis, result interpretation and bioequivalence determination among

formulations directly depend on the selected design. Thus, all above-mentioned factors
shall be carefully evaluated for a proper design to be chosen. [12]

3.7 Contract Research Organization

In the Code of Federal Regulations (CFR), the U.S. Food and Drug Administration
regulations state that a CRO is "a person [i.e., a legal person, which may be a
corporation] that assumes, as an independent contractor with the sponsor, one or more
of the obligations of a sponsor, e.g., design of a protocol, selection or monitoring of
investigations, evaluation of reports, and preparation of materials to be submitted to the
Food and Drug Administration." [21 CFR 312.3(b)]

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Services offered by CROs include: product development, formulation and

manufacturing; clinical trial management (preclinical through phase IV); clinical,
medical and safety monitoring; preclinical, toxicology, and clinical laboratory services
for processing trial samples; data management, biostatistics and medical writing
services for preparation of an FDA New Drug Application (NDA), Abbreviated New
Drug Application (ANDA), or Biologics License Application (BLA); regulatory affairs
support; and many other complementary services. CROs range from large, international
full service organizations to small, niche specialty groups and can offer their clients the
experience of moving a new drug or device from its conception to FDA marketing
approval without the drug sponsor having to maintain a staff for these services.

3.7.1 Outsourcing in clinical research

The CRO industry developed mostly in the late 1990s when pharmaceutical R&D
efforts became more complex and competition in rapidly-growing therapeutic areas
increased. Particularly over the last few years, this forced the pharmaceutical industry
to utilize downsizing strategies more to concentrate resources on core skills. As
industry margins come under increasing pressure, companies could begin outsourcing
aspects of their development, manufacturing or marketing processes so as to
concentrate on their core specialties.

Outsourcing has been particularly influential in the pharmaceutical industry as the

success of a large pharmaceutical company depends on competence in fields as diverse
as combinatorial chemistry, computer integrated manufacturing and marketing
medicines directly to consumers.

External cost pressures have acted as a major driver for the pharmaceutical outsourcing
market. At bottom, the outsourcing market has developed in response to the downward
and upward cost pressures exerted on pharmaceutical manufacturers profit margins.
Given that such pressures are likely to increase in the future, CROs will become more
and more important strategic partners for pharmaceutical companies. It is, therefore, in
the latters interest to consider probable developments in the CRO market and its major
players.

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3.7.2 Reasons for outsourcing to CROs

Outsourcing offers a number of advantages to drug companies. These include:

Sponsor can convert the fixed costs of maintaining the personnel, expertise and
facilities like data management necessary for clinical trial management into
variable costs
Non-availability of services in-house
Knowledge of regulatory affairs in a particular country of interest
Increased complexity of clinical trials
Necessity for medical and clinical knowledge in specific therapeutic areas or
indications
Increased amount of data required from clinical trials
Multinational and multi-center nature of current clinical trials
Large requirement of patient populations
Regionalized diseases

To effectively evaluate the potential impact of outsourcing, it is important to

understand the CRO selection process and the key factors in CRO selection.

3.7.3 CRO market size and growth

Industry analysers IMS Health and BCC Research estimate that the global
pharmaceutical market will grow at about a 5% rate in 2009 to over $820 billionand be
worth over $1 trillion by 2013. Pharmaceutical and biotechnology companies in the US
spent approximately $59 billion on R&D in 2007, which equates to roughly 18% of
their sales and is a 5% increase from the previous year.

A significant portion of R&D budgets are used for the outsourcing services offered by
the CRO industry, approximately $15 billion in 2007. This figure is expected to grow at
15% over the next seven years and should increase further with the broadening of the
spectrum of services outsourced to cover the entire value chain. As outsourced services
in developing countries such as China and India move up the value chain to cover
phase 1/2 trials, the total contracts value may go up to $20 billion by 2010. Further,
certain therapeutic areas within pharmaceutical development are slated for an even
greater growth curve, namely the oncology class, expected to see continued growth of
upwards of 21% over the next few years due to the large target market, strong unmet

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medical need, and overwhelming number of drugs currently in development (667 for
cancer vs. 252 for CNS disorders, 206 for cadiovascular disorders, and 186 for
infections). [13]

3.8 Role and Responsibilities of a Research Team

(A) Principal Investigator

Investigator is a person responsible for the conduct of the clinical trial at a trial site. If a
trial is conducted by a team of individuals at a trial site, the investigator is the
responsible leader of the team and may be called the principal investigator (PI).

The principal investigator (PI) is charged to conduct objective research that generates
independent, high quality, and reproducible results. The Principal Investigator is
responsible for the management and integrity of the design, conduct, and reporting of
the research project and for managing, monitoring, and ensuring the integrity of any
collaborative relationships. Additionally, the principal investigator is responsible for
the direction and oversight of compliance, financial, personnel, and other related
aspects of the research project to assure research is conducted in accordance with
applicable regulatory requirements and ethics committee. It is a responsibility of the
principal investigator to assure that the rights, safety and well-being of the research
subjects are guaranteed, correctly and promptly.

(B) Clinical Investigator

A clinical investigator is responsible for ensuring that an investigation is conducted

according to the signed investigator statement, the investigational plan, and applicable
regulations; for protecting the rights, safety, and welfare of subjects under the
investigator's care; and for the control of drugs under investigation.

(C) Project Manager

A project manager is the person accountable for accomplishing the stated project
objectives. The project manager's role in a nutshell is the overall responsibility for the
successful planning, execution, monitoring, control and closure of a project. Key
project management responsibilities include creating clear and attainable project
objectives, building the project requirements, and managing the triple constraint for
projects, which are cost, time, and quality (also known as scope).

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(D) Quality Manager

Quality assurance (QA) refers to a program for the systematic monitoring and
evaluation of the various aspects of a project, service, or facility to ensure that
standards of quality are being met.

A quality manager, sometimes called a quality assurance manager, coordinates the

activities required to meet quality standards. Quality managers also monitor and advise
on the performance of the quality management system and produces data and report on
performance, measuring against set conditions.

They liaise with other managers and staff throughout the organisation to ensure that the
QA system is functioning properly. Where appropriate, the quality manager advises on
changes and their implementation and provides training, tools and techniques to enable
others to achieve quality.

(E) Clinical Research Coordinator (CRC)

Clinical Research Coordinator (CRC) is the individual working for the investigator or a
university/academic institution who handles most of the administrative responsibilities
of a clinical trial/clinical study, is the liaison between the investigators site and the
sponsor, and reviews all data and records related to the clinical trial/clinical study.
Clinical Research Coordinator (CRC) is responsible for the entire conduct of the study,
using good clinical practice (GCP) under the auspices of the Principal Investigator (PI).

(F) Clinical Research Associate (CRA)

A Clinical Research Associate (CRA) also called as a Monitor, is an individual

employed by a pharmaceutical or medical device manufacturer, or by a contract
research organization (CRO) usually acting on a sponsor's behalf or by an academic
institute conducting clinical trials.

The main function of a clinical research associate is to monitor clinical trials. A clinical
research associate ensures compliance with the clinical trial protocol, checks clinical
site activities, makes on-site visits, reviews Case Report Forms (CRFs) and
communicates with clinical research investigators.

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(G) Medical Writer

Medical writing is the activity of producing scientific documentation by a specialized

writer. The medical writer typically is not one of the scientists or doctors who
performed the research.

A medical writer, working with doctors, scientists, and other subject matter experts,
creates documents that effectively and clearly describe research results, product use,
and other medical information. The medical writer also makes sure that documents
comply with regulatory requirement, ethics committee, Good Clinical Practice (GCP)
and other applicable guidelines in terms of content, format and structure. Their main
responsibility in a research team is the preparation of study protocol and clinical study
reports.

(H) Pharmacist

Pharmacists are healthcare professionals who practice the science of pharmacy. Their
main responsibility is receipt and accountability of investigational product.

(I) Phlebotomist

A phlebotomist is a qualified technician trained to draw blood samples from the

volunteers. Phlebotomy is the act of drawing blood either for testing or transfusion.
Phlebotomists collect blood primarily by performing venipuncture or cannulation.

(J) Nurse

A nurse is a healthcare professional who, in collaboration with other members of a

health care team, is responsible for treatment, safety, and recovery of acutely or
chronically ill individuals; health promotion and maintenance within families,
communities and populations; and, treatment of life-threatening emergencies in a wide
range of health care settings. Their main responsibility in a research team is Vitals
monitoring e.g. BP (Blood Pressure), RR (Respiratory Rate), PR (Pulse Rate) and oral
temperature.

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(K) Custodian

A custodian is the designated person of a research team, under whose direct

supervision, volunteers reside during the conduct of study. Their main responsibility in
a research team is the supervision of check in and check out of volunteers, supervise
distribution of study meals, supervise the other facilities being provided to the
volunteers.

(L) Volunteer Development Officer

A volunteer development officer (VDO) is the designated person of a research team,

whose main responsibility in a research team is the development and maintenance of
adequate volunteer data base. [13]

3.9 Special Considerations for modified-release drug products (CDSCO BA/BE

Guidelines, March 2005):

Modified release products include:

Delayed release
Sustained release
Mixed immediate and sustained release
Mixed delayed and sustained release
Mixed immediate and delayed release

Generally, these products should:

Act as modified-release formulations and meet the label claim.

Preclude the possibility of any dose dumping effect.
There must be a significant difference between the performance of modified
release product and the conventional release product when used as reference
product.
Provide a therapeutic performance comparable to the reference immediate-
release formulation administered by the same route in multiple doses (of an
equivalent daily amount) or to the reference modified-release formulation.
Provide consistent pharmacokinetic performance between individual dosage
units; and

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Produce plasma levels which lie within the therapeutic range (where
appropriate) for the proposed dosing intervals at steady state.

If all of the above conditions are not met but the applicant considers the formulation to
be acceptable, justification to this effect should be provided.

I. Study Parameters

Bioavailability data should be obtained for all modified release drug products although
the type of studies required and the pharmacokinetic parameters which should be
evaluated may differ depending on the active ingredient involved. Factors to be
considered include whether or not the formulation represents the first market entry of
the drug substance, and the extent of accumulation of the drug after repeated dosing.

If the formulation is the first market entry of the drug substance, the products
pharmacokinetic parameters should be determined. If the formulation is a second or
subsequent market entry then comparative bioavailability studies using an appropriate
reference product should be performed.

II. Study Design

Study design will be single dose or single and multiple dose based on the modified
release products that are likely to accumulate or unlikely to accumulate both in the
fasted and non-fasting state. If the effect of food on the reference product is not known
(or it is known that food affects its absorption), two separate two-way cross-over
studies, one in the fasted state and the other in the fed state, may be carried out. If it is
known with certainty (e.g. from published data) that the reference product is not
affected by food, then a three-way cross-over study may be appropriate with:

a) The reference product in the fasting state

b) The test product in the fasted state, and
c) The test product in the fed state.

III. Requirements for modified release formulations unlikely to accumulate

This section outlines the requirements for modified release formulations which are used
at a dose interval that is not likely to lead to accumulation in the body (AUC 0-T/AUC0-
0.8).

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When the modified release product is the first market entry of that type of dosage form,
the reference product should normally be the innovators immediate-release
formulation. The comparison should be between a single dose of the modified release
formulation and doses of the immediate-release formulation which it is intended to
replace. The latter must be administered according to the established dosing regimen.

When the modified release product is the second or subsequent entry on the market,
comparison should be with the reference modified release product for which
bioequivalence is claimed.

Studies should be performed with single dose administration in the fasting state as well
as following an appropriate meal at a specified time. The following pharmacokinetic
parameters should be calculated from plasma (or relevant biological matrix)
concentrations of the drug and/or major metabolite(s): AUC0-T, AUC0-t, AUC0-, Cmax
(where the comparison is with an existing modified release product), and Kel.

The 90% confidence interval calculated using log-transformed data for the ratios (Test:
Reference) of the geometric mean AUC (for both AUC0-T and AUC0-t) and Cmax (where
the comparison is with an existing modified release product) should generally be within
the range 80 to 125% both in the fasting state and following the administration of an
appropriate meal at a specified time before taking the drug.

The pharmacokinetic parameters should support the claimed dose delivery attributes of
the modified-release dosage form.

IV. Requirements for modified release formulations likely to accumulate

This section outlines the requirements for modified release formulations that are used at
dose intervals that are likely to lead to accumulation (AUC0-T/AUC0- < 0.8).

When a modified release product is the first market entry of the modified release type,
the reference formulation is normally the innovators immediate-release formulation.
Both a single dose and steady state doses of the modified release formulation should be
compared with doses of the immediate-release formulation which it is intended to
replace. The immediate-release product should be administered according to the
conventional dosing regimen.

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Studies should be performed with single dose administration in the fasting state as well
as following an appropriate meal. In addition, studies are required at steady state. The
following pharmacokinetic parameters should be calculated from single dose studies:
AUC0-T, AUC0-t, AUC0-, Cmax (where the comparison is with an existing modified
release product), and Kel. The following parameters should be calculated from steady
state studies: AUC0-T(ss), Cmax, Cmin, Cpd and degree of fluctuation.

When the modified release product is the second or subsequent modified release entry,
single dose and steady state comparisons should normally be made with the reference
modified release product for which bioequivalence is claimed.

The 90% confidence interval for the ratio of geometric means (Test: Reference drug) of
AUC (for both AUC0-T and AUC0-t) and Cmax (where the comparison is with an existing
modified release product) determined using log-transformed data should generally be
within the range 80 to 125% when the products are compared after single dose
administration in both the fasting state and the fed state.

The 90% confidence interval for the ratio of geometric means (Test: Reference drug)
for AUC0-T(ss), Cmax and Cmin determined using log-transformed data should generally
be within the range 80 to 125% when the formulations are compared at steady state.

The pharmacokinetic parameters should support the claimed attributes of the modified-
release dosage form.

Pharmacodynamic data may reinforce or clarify interpretation of differences in the

plasma concentration data.

When these studies do not show bioequivalence, comparative efficacy and safety data
may be required for the new product. [14]

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3.10 Regulatory guidelines for the conduct of bioavailability and bioequivalence

studies

It is the objective of regulatory guidance to define, for products with a systemic effect,
when bioavailability or bioequivalence studies are necessary and to formulate
requirements for their design, conduct, and evaluation. The possibility of using in vitro
instead of invivo studies with pharmacokinetic end points is also envisaged.

Guidance documents are meant to provide assistance to industry and healthcare

professionals on how to comply with the policies and governing statutes and
regulations. They also serve to provide review and compliance guidance to staff,
thereby ensuring that mandates are implemented in a fair, consistent and effective
manner.

Guidance documents are administrative instruments not having force of law and, as
such, allow for flexibility in approach. Alternate approaches to the principles and
practices described in document may be acceptable provided they are supported by
adequate scientific justification. Alternate approaches should be discussed in advance
with the relevant program area to avoid the possible finding that applicable statutory or
regulatory requirements are met.

The requirements for conduct of Bioavailability and Bioequivalence studies vary from
country to country. These changes are reflected in the guidelines issued by the different
regulatory authorities across the world.

Some of the Regulatory Authorities which have issued guidelines for the conduct of
Bioavailability and Bioequivalence studies are as follows:

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Table 9: List of Regulatory Authorities

Nation Regulatory Authority

Australia Therapeutic Goods Administration (TGA)

Brazil National Health Surveillance, Agency

Canada Health Products and Food Branch, Ministry of Health, Canada

China State Drug Administration (SDA)

European Union European Agency for the Evaluation of Medical Products

(EMEA)
India Central Drugs Standard Control Organization (CDSCO)

Japan Ministry of Health and Welfare (MHW)

Malaysia Ministry of Health

Saudi Arabia Saudi Food & Drug Authority

Singapore The Health and Safety Authority

South Africa Medicines control Council (MCC)

South Korea Korea Food and Drug Administration (KFDA)

United States of Food and Drug Administration (FDA)

America
World Health Organization

s [15]

regulatory
authorities own

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3.11 Diabetes Mellitus

Diabetes mellitus is a heterogenous group of disorders characterized by hyperglycemia;

that results from defects in insulin secretion, insulin action (sensitivity), or both; is
associated with abnormalities in carbohydrate, fat and protein metabolism; and results
in chronic complications including microvascular, macrovascular, and neuropathic
disorders.

3.11.1 Epidemology

Globally diabetes affects 246 million people and is expected to affect 366 million by
2030. At least 7 million new cases are reported annually. India accounts for largest
diabetic population with 41 million patients, mounting to 6 percent of the adult
population.

Type 1 diabetes mellitus results from autoimmune destruction of the cells of the
pancreas. Type 1 diabetes mellitus develops in childhood or early adulthood, although
some latent forms do occur. Type 1 DM accounts for up to 10% of all cases of DM and
is likely initiated by the exposure of a genetically susceptible individual to an
environmental agent.

Among various types of diabetes mellitus, type 2 diabetes mellitus is the commonest.
The prevalence of type 2 DM is increasing. Type 2 DM accounts for as much as 90% of
all cases of DM. Type 2 Diabetes Mellitus is characterized by insulin resistance and at
least initially, a relative lack of insulin secretion. Most individuals with type 2 diabetes
exhibit abdominal obesity which itself causes insulin resistance. Multiple risk factors
for the development of type 2 DM have been identified, including family history (i.e.,
parents or siblings with diabetes); obesity (i.e., 20% over ideal body weight, or body
mass index [BMI] 25 kg/m2); habitual physical inactivity; race or ethnicity;
previously identified impaired glucose tolerance or impaired fasting glucose;
hypertension (140/90 mm Hg in adults); high density lipoprotein (HDL) cholesterol
35 mg/dL and/or a triglyceride level 250 mg/dL; history of gestational diabetes
mellitus or delivery of a baby weighing >9 pounds; history of vascular disease; and
polycystic ovary disease. While the prevalence of type 2 DM increases with age, the
disorder is increasingly being recognized in adolescence. Much of the rise in adolescent

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type 2 DM is related to an increase in adiposity and sedentary lifestyle, in addition to an

inheritable predisposition [16]

3.11.2 Classification

The classification adopted by WHO is given in Table 1.

Table10: Clinical Classification of diabetes mellitus

1. Diabetes Mellitus (DM)

a) Insulin-dependent diabetes mellitus (IDDM, Type 1)

b) Non-insulin-dependent diabetes mellitus (IDDM, Type 2)
c) Malnutrition-related diabetes mellitus (MRDM)
d) Other types (secondary to pancreatic, hormonal, drug-induced, genetic and other
abnormalities)

2. Impaired glucose tolerance (IGT)

3. Gestational diabetes mellitus (GDM)

[17]

3.11.3 Diagnostic tests

Random blood sugar (RBS): 200 mg/dL (Normal Value: 60-150 mg/dl)
Fasting blood sugar (FBS): 126 mg/dL (Normal Value: 60-110 mg/dl)
Post Prandial blood sugar (PPBS): 200 mg/dL (Normal Value: 90-140 mg/dl)
Oral glucose tolerance test (OGTT, 75 g glucose): 200 mg/dL
(Normal Value: <140 mg/dl)
Glycosylated hemoglobin (Hb A1C): >7% (Normal Value: <7%)
C-peptides
Urine analysis
Criteria for screening diabetes in asymptomatic, undiagnosed individuals: > 45
years, Younger people (obese, first degree relative with DM, high risk ethnic
population, baby wt> 9 lb with GDM, HTN, HDL35 mg/dl, TG250 mg/dL)

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Table 11: Categorization of Glucose Status

Normal IGT Diabetes mellitus

FPG <100 mg/dl 100-125 mg/dl 126 mg/dL

OGTT <140 mg/dl 140-199 mg/dl 200 mg/dL

IGT: Impaired Glucose Tolerance, FPG: Fasting Plasma Glucose, OGTT: Oral Glucose
Tolerance Test

Impaired Glucose Tolerance (IGT) describes a state intermediate-at risk group-

between diabtets mellitus and normality. It can only be defined by the Oral Glucose
Tolerance Test (OGTT).

A glucose tolerance test in medical practice is the administration of glucose to

determine how quickly it is cleared from the blood. The test is usually used to test for
diabetes, insulin resistance, and sometimes reactive hypoglycemia. The glucose is most
often given orally so the common test is technically an Oral Glucose Tolerance Test
(OGTT).

3.11.4 Management of Type 2 Diabetes Mellitus

Goals of therapy in diabetes mellitus are directed at reducing symptoms of

hyperglycemia, reducing the onset and progression of retinopathy, nephropathy, and
neuropathy complications, intensive therapy for associated cardiovascular risk factors,
and improving quality and quantity of life.

Metformin should be included in the therapy for all type 2 DM patients, if tolerated and
not contraindicated, as it is the only oral antihyperglycemic medication proven to
reduce the risk of total mortality and cardiovascular death, according to the United
Kingdom Prospective Diabetes Study.

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Intensive glycemic control is paramount for reduction of microvascular complications

(neuropathy, retinopathy, and nephropathy) as evidenced by the Diabetes Control and
Complications Trial in type 1 DM and the United Kingdom Prospective Diabetes Study
(UKPDS) in type 2 DM. The UKPDS also reported that control of hypertension in
patients with diabetes will not only reduce the risk of retinopathy and nephropathy, but
also reduce cardiovascular risk.

Knowledge of the patients quantitative and qualitative meal patterns, activity levels,
pharmacokinetics of insulin preparations, and pharmacology of oral antihyperglycemic
agents are essential to individualize the treatment plan and optimize blood glucose
control while minimizing risks for hypoglycemia and other adverse effects of
pharmacologic therapies.

Treatment of type 2 DM often necessitates use of multiple therapeutic agents

(combination therapy), including oral hypoglycemics and insulin to obtain glycemic
goals.

Aggressive management of cardiovascular disease risk factors in type 2 DM is

necessary to reduce the risk for adverse cardiovascular events or death. Smoking
cessation, use of antiplatelet therapy as a primary prevention strategy, aggressive
management of dyslipidemia minimally to goal lowdensity lipoprotein-cholesterol
(LDL-C) (<100 mg/dL) and secondarily to raise high-density lipoprotein-cholesterol
(HDL-C) to 40 mg/dL, and treatment of hypertension (again often requiring multiple
drugs) minimally to <130/80 mm Hg are vital.

Prevention strategies for type 2 DM are established. Lifestyle changes, dietary

restriction of fat, aerobic exercise for 30 minutes 5 times a week, and weight loss, form
the backbone of successful prevention. To date, medications have been less effective
than lifestyle changes to prevent progression to type 2 DM.

Patient education and ability to demonstrate self-care and adherence to therapeutic

lifestyle and pharmacologic interventions are crucial to successful outcomes.
Multidisciplinary teams of health care professionals including physicians (primary care,
endocrinologists, ophthalmologists, and vascular surgeons), podiatrists, dietitians,
nurses, pharmacists, social workers, behavioral health specialists, and certified diabetes
educators are needed to optimize these outcomes in persons with diabetes mellitus.

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Patient education and ability to demonstrate self-care and adherence to therapeutic

lifestyle and pharmacologic interventions are crucial to successful outcomes.
Multidisciplinary teams of health care professionals including physicians (primary care,
endocrinologists, ophthalmologists, and vascular surgeons), podiatrists, dietitians,
nurses, pharmacists, social workers, behavioral health specialists, and certified diabetes
educators are needed to optimize these outcomes in persons with diabetes mellitus. [18]

Table 12: Classification of Oral Hypoglcaemic Agents (OHAs)

1.Sulfonylureas:

i. First generation: Tolbutamide, Chlorpropamide

ii. Second generation: Glibenclamide (Glyburide), Glipizide, Gliclazide,
Glimepiride.

2. Biguanides: Metformin

3. Alpha-glucosidase inhibitor: Acarbose, Miglitol

4. Thiazolidinediones (TZD): Rosiglitazone, Pioglitazone

5.Meglitinides: Repaglinide, Nateglinide

Amylin analogues (Pramlintide), Incretin mimetics (Exenatide) and Dipeptidyl

Peptidase-4 (DPP-4) inhibitors (Saxagliptin, Sitagliptin.) are the new treatment
regimens available for the Type 2 diabetes mellitus. Amylin Analogues and Incretin
mimetics are given through subcutaneous route whereas Dipeptidyl Peptidase-4 (DPP-
4) inhibitors are given through oral route. [19]

3.11.4.1 Sulfonylureas (Insulin Secretagogues):

Stimulates the release of insulin from pancreatic beta cells.

First generation Sulfonylureas
Tolbutamide (Daily dose: 0.5-2 g)
Chlorpropamide (Daily dose: 0.1-0.5 g)
ADRs: Hypoglycemia, Gastric irritation, Blood dyscrasias, Disulfiram like
reactions.
Second generation Sulfonylureas

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Glimepiride (Daily dose: 1-6 mg)

Glipizide (Daily dose: 5-20 mg)
Glyburide (Daily dose: 5-15 mg)
Gliclazide (Daily dose: 40-240 mg)
ADRs: Hypoglycemia and weight gain.
Sulfonylureas are Contraindicated in severe hepatic and renal impairment.
Place in therapy:
Used in patients with Type 2 Diabetes mellitus, in whom administration
improves the early phase of insulin release that is refractory to acute glucose
stimulation.
Because of its short duration of action, which is independent of renal function,
tolbutamide is probably the safest sulfonylurea to use if liver function is normal.
Acetohexamide & Chlorpropamide are now rarely used. Chlorpropamide has a
prolonged biologic effect & severe hypoglycemia can occur especially in the
elderly.
Glyburide, Glipizide, Gliclazide and Glimepiride are 100-200 times more potent
than tolbutamide. These drugs should be used with caution in patients with
cardiovascular disease or in elderly patients, in whom prolonged hypoglycemia
would be especially dangerous.

3.11.4.2 Biguanides

Metformin (Daily dose: 0.5-2.5 g)

Increased insulin receptor binding, stimulation of tissue uptake of glucose,
reduced gastrointestinal absorption of carbohydrates, inhibition of hepatic
gluconeogenesis
ADRs: Gastrointestinal disturbances, Lactic acidosis
Does NOT cause hypoglycemia or weight gain
Place in therapy: useful in obese patient as it does not cause weight gain. Can be
used along with diet as second line therapy in patients not adequately controlled
on diet alone. Valuable when prescribed in combination.

3.11.4.3 Alpha glucosidase inhibitors

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Alpha- glucosidase inhibitors competitively inhibit the glucosidase enzymes in

the gut that digest dietary starch and sucrose.
Acarbose (Daily dose: 75-300 mg)
Miglitol (Daily dose: 75-300 mg)
To be taken with first bite of food
ADRs: flatulence, abdominal pain, diarrhea, elevated LFTs.
Place in therapy: for patients inadequately controlled on diet alone or on diet
and other oral hypoglcaemic agents (OHAs). The GIT adverse effect limits its
use.

3.11.4. Thiazolidinediones

Agonist of PPAR gamma. Increases skeletal muscle cell sensitivity to insulin

and decreases hepatic glucose production
Rosiglitazone (Daily dose: 4-8 mg)
Pioglitazone (Daily dose: 15-45 mg)
ADRs: oedema, headache, abdominal pain, myalgia, increased LFTs, anaemia,
weight gain during trial.
Place in therapy: should be offered to patients in combination with a
sulphonylurea or metformin, if uncontrolled blood sugar with these agents.

3.11.4.5 Meglitinides

Increases insulin secretion by the pancreas

Repaglinide (Daily dose: 1.5-8 mg)
ADRs of Repaglinide: hypoglycemia, abdominal pain.
Natiglinide (Daily dose: 180-480 mg)
Place in therapy: an effective first line therapy in type 2 diabetes mellitus. May
be used in combination with metformin to produce a synergistic effect. Most
beneficial in patients who experience problems with post-prandial glucose
elevation and as single therapy in patients who eat at unpredictable times.

3.11.4.6 Insulin

Insulin is mainstay in the treatment of diabetes mellitus (type 1 and type 2 also). Type 1
treatment necessitates insulin therapy. Currently, the basal-bolus insulin therapy or
pump therapy in motivated individuals often leads to successful glycemic outcomes.

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Basal-bolus therapy often includes a basal insulin for fasting and post-absorptive
control, and rapid acting bolus insulin for mealtime coverage. Therapeutically, use of
basal-bolus therapy in type 2 DM is increasing.

(A) Table 13: Types of Insulin

Type Onset (hrs) Peak (hrs) Duration (hrs) Appearance

Ultra-short 15-30 min 1-2 hrs 3-5 hrs Clear

acting (Aspart,
Lispro,
Glulisine)

Short acting 0.5-1.0 hrs 2-3 hrs 3-6 hrs Clear

(Regular,
Semilente)

Intermediate 2-4 hrs 6-10 hrs 16-24 hrs Cloudy

acting (NPH,
Lente)

Long acting 4-6 hrs 18 hrs 24-36 hrs Cloudy

(Ultralente, (Ultralente),
Glargine, Insulin
Clear
detemir ) (Glargine)

NPH: Neutral Protamine Hagedorn, hrs: hours, min: minute

(B) Insulin Dosing

Single daily injection of intermediate acting insulin.

Multiple injection (basal-bolus): Short acting insulin before meals with
intermediate or Long acting insulin once or twice daily. Two-daily injections of
intermediate acting insulin. [Split mixed regimen: 30:70 (Regular: NPH)].
Three daily injections of short acting (or Ultra-short acting) insulin in
combination with a single injection of long acting insulin.
Continuous subcutaneous insulin infusion.

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(C) Site

Abdomen, thigh, arms, buttocks

(D) Route

Subcutaneous.

(E) Storage & Mixing of Insulin

Regular + NPH (), Regular + Lente (X), NPH + Lente (X)

(F) Adverse Reactions to Insulin

Lipohypertrophy (thickness of s/c tissue)

Hypersensitivity reactions
Hypoglycemic symptoms:

(a) Due to adrenaline release (sweating, palpitations, tremors, tachycardia,

hunger)

(b) Neuroglcopenic cause (restlessness, irritability, mental instability, tingling

sensation)

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3.11.4.7 Figure 4: Algorithm for the management of Type 2 Diabetes Mellitus

[20]

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3.12 Metformin (Study Drug)

3.12.1 Summary

Metformin Hydrochloride Sustained Release tablet is an oral antihyperglycemic drug

used in the management of type 2 diabetes.

Metformin Hydrochloride indicated as an adjunct to diet and exercise to improve

glycemic control in patients with type 2 diabetes. It may be used concomitantly with a
sulfonylurea or insulin to improve glycemic control in adults (17 years of age and
older).

3.12.2 Introduction

Metformin is an oral hypoglycaemic agent used to treat patients with non-insulin-

dependent diabetes mellitus, but its effect on embryonic tissues has not been well
studied. Early-somite mouse embryos were exposed in whole embryo culture to
metformin (02000 g/ml) and assayed for glucose uptake and glycolysis at 6, 12 and
24 hours. Embryos exposed to metformin for 6 hours were also evaluated for glucose
uptake in the presence of 0 or 100 cytochalasin B. Glucose uptake was increased in
embryos exposed to metformin at 2000 g/ml for 6 hours and 1000 g/ml or more for
12 hours. Glycolysis was increased in embryos exposed to metformin at 2000 g/ml for
6 or 24 hours and 1000 g/ml or more for 12 hours, producing lactate concentrations up
to six times higher than controls. Glut-1 was increased in embryos exposed to
1000 g/ml or more for 6 hours, and metformin-stimulated glucose uptake was
significantly decreased by cytochalasin B. Thus, glucose uptake and glycolytic
metabolism are increased in early-somite mouse embryos in response to high
concentrations of metformin in vitro, and the mechanism of increased glucose uptake
appears to involve a cytochalasin B-sensitive glucose transporter.

3.12.3 Physical, chemical and pharmaceutical properties:

Metformin hydrochloride (N,N-dimethylimidodicarbonimidic diamide hydrochloride) is

not chemically or pharmacologically related to any other classes of oral
antihyperglycemic agents. The structural formula is as shown:

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Metformin hydrochloride is a white to off-white crystalline compound with a molecular

formula of C4H11N5 HCl and a molecular weight of 165.63. Metformin hydrochloride
is freely soluble in water and is practically insoluble in acetone, ether, and chloroform.
The pKa of metformin is 12.4. The pH of a 1% aqueous solution of metformin
hydrochloride is 6.68.

3.12.4 Storage

Store at 2025 C (6877 F); excursions permitted to 1530 C (5986 F).

Dispense in light-resistant containers.

3.12.5 Pharmacology (Mechanism of Action)

Metformin is an antihyperglycemic agent which improves glucose tolerance in patients
with type 2 diabetes, lowering both basal and postprandial plasma glucose. Its
pharmacologic mechanisms of action are different from other classes of oral
antihyperglycemic agents. Metformin decreases hepatic glucose production, decreases
intestinal absorption of glucose, and improves insulin sensitivity by increasing
peripheral glucose uptake and utilization. Unlike sulfonylureas, metformin does not
produce hypoglycemia in either patients with type 2 diabetes or normal subjects except
in special circumstances and does not cause hyperinsulinemia. With metformin therapy,
insulin secretion remains unchanged while fasting insulin levels and day-long plasma
insulin response may actually decrease.

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3.12.6 Pharmacokinetics
3.12.6.1 Absorption

The absolute bioavailability of a Metformin hydrochloride 500 mg tablet given under

fasting conditions is approximately 50% to 60%. Studies using single oral doses of
Metformin hydrochloride 500 mg to 1500 mg, and 850 mg to 2550 mg, indicate that
there is a lack of dose proportionality with increasing doses, which is due to decreased
absorption rather than an alteration in elimination. Food decreases the extent of and
slightly delays the absorption of metformin, as shown by approximately a 40% lower
mean peak plasma concentration (Cmax), a 25% lower area under the plasma
concentration versus time curve (AUC), and a 35-minute prolongation of time to peak
plasma concentration (Tmax) following administration of a single 850 mg tablet of
metformin with food, compared to the same tablet strength administered fasting. The
clinical relevance of these decreases is unknown.

Following a single oral dose of Metformin hydrochloride sustained release; Cmax is

achieved with a median value of 7 hours and a range of 4 hours to 8 hours. Peak plasma
levels are approximately 20% lower compared to the same dose of Metformin
hydrochloride, however, the extent of absorption (as measured by AUC) is similar to
Metformin hydrochloride.

At steady state, the AUC and Cmax are less than dose proportional for Metformin
hydrochloride sustained release within the range of 500 mg to 2000 mg administered
once daily. Peak plasma levels are approximately 0.6, 1.1, 1.4, and 1.8 g/mL for 500,
1000, 1500, and 2000 mg once-daily doses, respectively. The extent of metformin
absorption (as measured by AUC) from Metformin hydrochloride sustained release at a
2000 mg once-daily dose is similar to the same total daily dose administered as
Metformin hydrochloride tablets 1000 mg twice daily. After repeated administration of
Metformin hydrochloride sustained release, metformin did not accumulate in plasma.

Within-subject variability in Cmax and AUC of metformin from Metformin

hydrochloride sustained release is comparable to that with Metformin hydrochloride.

Although the extent of metformin absorption (as measured by AUC) from the
Metformin hydrochloride sustained release tablet increased by approximately 50%
when given with food, there was no effect of food on Cmax and Tmax of metformin.

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Both high and low fat meals had the same effect on the pharmacokinetics of Metformin
hydrochloride sustained release.

3.12.6.2 Distribution

The apparent volume of distribution (V/F) of metformin following single oral doses of
Metformin hydrochloride 850 mg averaged 654 358 L. Metformin is negligibly
bound to plasma proteins, in contrast to sulfonylureas, which are more than 90%
protein bound. Metformin partitions into erythrocytes, most likely as a function of time.
At usual clinical doses and dosing schedules of Metformin hydrochloride, steady state
plasma concentrations of metformin are reached within 24 to 48 hours and are
generally <1 g/mL. During controlled clinical trials of Metformin hydrochloride,
maximum metformin plasma levels did not exceed 5 g/mL, even at maximum doses

3.12.6.3 Metabolism

Intravenous single-dose studies in normal subjects demonstrate that metformin is

excreted unchanged in the urine and does not undergo hepatic metabolism (no
metabolites have been identified in humans) nor biliary excretion.

3.12.6.4 Elimination

Renal clearance is approximately 3.5 times greater than creatinine clearance, which
indicates that tubular secretion is the major route of metformin elimination. Following
oral administration, approximately 90% of the absorbed drug is eliminated via the renal
route within the first 24 hours, with a plasma elimination half-life of approximately 6.2
hours. In blood, the elimination half-life is approximately 17.6 hours, suggesting that
the erythrocyte mass may be a compartment of distribution.

3.12.7 Indication & Dosing Information

Metformin hydrochloride sustained-release tablets is indicated as an adjunct to diet and

exercise to improve glycemic control in adults with type 2 diabetes mellitus.

The usual starting dose of metformin SR (metformin hydrochloride extended-release

tablets) is 500 mg once daily with the evening meal. Dosage increases should be made
in increments of 500 mg weekly, up to a maximum of 2000 mg once daily with the
evening meal. If glycemic control is not achieved on metformin SR 2000 mg once

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daily, a trial of metformin SR 1000 mg twice daily should be considered. If higher

doses of metformin are required, metformin should be used at total daily doses up to
2550 mg administered in divided daily doses, as described above.

3.12.8 Side Effects

Less common side effects of Metformin HCL include:

Diarrhea
Nausea
Upset Stomach
Abdominal Bloating
Gas
Loss of Appetite
These side effects generally go away after you take the medicine for a while. Taking
your medicine with meals can help reduce these side effects.

3.12.9 Adverse Events

Adverse reactions reported in greater than 5% of the Metformin patients, and that were
more common are Diarrhea, Nausea/vomiting, Flatulence, Asthenia, Indigestion,
Abdominal discomfort, Headache.

Other Adverse reactions less commonly reported were abnormal stools, hypoglycemia,
myalgia, lightheaded, dyspnea, nail disorder, rash, sweating increased, taste disorder,
chest discomfort, chills, flu syndrome, flushing, palpitation, upper respiratory infection
and taste disturbance.

3.12.10 Drug Interactions

3.12.10.1 GlyburideIn a single-dose interaction study in type 2 diabetes patients,

coadministration of metformin and glyburide did not result in any changes in either
metformin pharmacokinetics or pharmacodynamics. Decreases in glyburide AUC and
Cmax were observed, but were highly variable.

The single-dose nature of this study and the lack of correlation between glyburide
blood levels and pharmacodynamic effects make the clinical significance of this
interaction uncertain.

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3.12.10.2 FurosemideA single-dose, metformin-furosemide drug interaction study in

healthy subjects demonstrated that pharmacokinetic parameters of both compounds
were affected by coadministration. Furosemide increased the metformin plasma and
blood Cmax by 22% and blood AUC by 15%, without any significant change in
metformin renal clearance. When administered with metformin, the Cmax and AUC of
furosemide were 31% and 12% smaller, respectively, than when administered alone,
and the terminal half-life was decreased by 32%, without any significant change in
furosemide renal clearance. No information is available about the interaction of
metformin and furosemide when coadministered chronically.

3.12.10.3 NifedipineA single-dose, metformin-nifedipine drug interaction study in

normal healthy volunteers demonstrated that coadministration of nifedipine increased
plasma metformin Cmax and AUC by 20%and 9%, respectively, and increased the
amount excreted in the urine. Tmax and half-life were unaffected. Nifedipine appears to
enhance the absorption of metformin. Metformin had minimal effects on nifedipine.

3.12.10.4 Cationic drugsCationic drugs (e.g., amiloride, digoxin, morphine,

procainamide, quinidine, quinine, ranitidine, triamterene, trimethoprim, or vancomycin)
that are eliminated by renal tubular secretion theoretically have the potential for
interaction with metformin by competing for common renal tubular transport systems.
Such interaction between metformin and oral cimetidine has been observed in normal
healthy volunteers in both single-and multiple-dose, metformin-cimetidine drug
interaction studies, with a 60% increase in peak metformin plasma and whole blood
concentrations and a 40% increase in plasma and whole blood metformin AUC. There
was no change in elimination half-life in the single-dose study. Metformin had no effect
on cimetidine pharmacokinetics. Although such interactions remain theoretical (except
for cimetidine), careful patient monitoring and dose adjustment of Metformin
hydrochloride or Metformin hydrochloride extended release and/or the interfering drug
is recommended in patients who are taking cationic medications that are excreted via
the proximal renal tubular secretory system.

3.12.10.5 OtherCertain drugs tend to produce hyperglycemia and may lead to loss of
glycemic control. These drugs include the thiazides and other diuretics, corticosteroids,
phenothiazines, thyroid products, estrogens, oral contraceptives, phenytoin, nicotinic
acid, sympathomimetics, calcium channel blocking drugs, and isoniazid. When such

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drugs are administered to a patient receiving Metformin hydrochloride or Metformin

hydrochloride extended release, the patient should be closely observed for loss of blood
glucose control. When such drugs are withdrawn from a patient receiving Metformin
hydrochloride, the patient should be observed closely for hypoglycemia.

In healthy volunteers, the pharmacokinetics of metformin and propranolol, and

metformin and ibuprofen were not affected when coadministered in single-dose
interaction studies.

Metformin is negligibly bound to plasma proteins and is, therefore, less likely to
interact with highly protein-bound drugs such as salicylates, sulfonamides,
chloramphenicol, and probenecid, as compared to the sulfonylureas, which are
extensively bound to serum proteins.

3.12.11 Warning - Lactic Acidosis

Lactic acidosis is a rare, but serious, metabolic complication that can occur due to
metformin accumulation during treatment with metformin or metformin SR; when it
occurs, it is fatal in approximately 50% of cases. Lactic acidosis may also occur in
association with a number of pathophysiologic conditions, including diabetes mellitus,
and whenever there is significant tissue hypoperfusion and hypoxemia.

3.12.12 GENERAL PRECAUTIONS

3.12.12.1 Macrovascular OutcomesThere have been no clinical studies establishing
conclusive evidence of macrovascular risk reduction with metformin or metformin SR
or any other anti-diabetic drug.

3.12.12.2 Monitoring of renal functionMetformin is known to be substantia lly

excreted by the kidney, and the risk of metformin accumulation and lactic acidosis
increases with the degree of impairment of renal function. Thus, patients with serum
creatinine levels above the upper limit of normal for their age should not receive
metformin or metformin SR.

3.12.12.3 Use of concomitant medications that may affect renal function or

metformin disposition Concomitant medication(s) that may affect renal function or
result in significant hemodynamic change or may interfere with the disposition of

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metformin, such as cationic drugs that are eliminated by renal tubular secretion should
be used with caution.

3.12.12.4 Hypoxic states Cardiovascular collapse (shock) from whatever cause,

acute congestive heart failure, acute myocardial infarction and other conditions
characterized by hypoxemia have been associated with lactic acidosis and may also
cause prerenal azotemia. When such events occur in patients on metformin or
metformin SR therapy, the drug should be promptly discontinued.

3.12.12.5 Surgical procedures Metformin or metformin SR therapy should be

temporarily suspended for any surgical procedure (except minor procedures not
associated with restricted intake of food and fluids) and should not be restarted until the
patient's intake has resumed and renal function has been evaluated as normal.

3.12.12.6 Alcohol intakeAlcohol is known to potentiate the effect of metformin on

lactate metabolism. Patients, therefore, should be warned against excessive alcohol
intake, acute or chronic, while receiving metformin or metformin SR.

3.12.12.7 Impaired hepatic function Since impaired hepatic function has been
associated with some cases of lactic acidosis, metformin and metformin SR should
generally be avoided in patients with clinical or laboratory evidence of hepatic disease.

3.12.13 Contraindications

Metformin hydrochloride is contraindicated in patients with:

1. Renal disease or renal dysfunction (e.g., as suggested by serum creatinine levels

1.5 mg/dL [males], 1.4 mg/dL [females] or abnormal creatinine clearance)
which may also result from conditions such as cardiovascular collapse (shock),
acute myocardial infarction, and septicemia.
2. Known hypersensitivity to metformin hydrochloride.
3. Acute or chronic metabolic acidosis, including diabetic ketoacidosis, with or
without coma. Diabetic ketoacidosis should be treated with insulin.

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3.12.14 OVERDOSE AND ITS MANAGEMENT

Overdose of metformin hydrochloride has occurred, including ingestion of amounts
greater than 50 grams. Hypoglycemia was reported in approximately 10% of cases, but
no causal association with metformin hydrochloride has been established. Lactic
acidosis has been reported in approximately 32% of metformin overdose cases.
Metformin is dialyzable with a clearance of up to 170 mL/min under good
hemodynamic conditions. Therefore, hemodialysis may be useful for removal of
accumulated drug from patients in whom metformin overdosage is suspected. [21]

3.12.15 Clinical Studies conducted on Metformin (Study Drug)

Various clinical studies conducted on metformin have proven its Antidiabetic (Oral
hypoglycemic) Activity.

A 24-week, double-blind, placebo-controlled study of Diabeta SR, taken once daily

with the evening meal, was conducted in patients with type 2 diabetes who had failed to
achieve glycemic control with diet and exercise (HbA1c 7.0-10.0%, FPG 126-270
mg/dL). Patients entering the study had a mean baseline HbA1c of 8.0% and a mean
baseline FPG of 176 mg/dL. After 12 weeks treatment, mean HbA1c had increased
from baseline by 0.1% and mean FPG decreased from baseline by 2 mg/dL in the
placebo group, compared with a decrease in mean HbA1c of 0.6% and a decrease in
mean FPG of 23 mg/dL in patients treated with Diabeta SR 1000 mg once daily.
Subsequently, the treatment dose was increased to 1500 mg once daily if HbA1c was
7.0% but <8.0% (patients with HbA1c 8.0% were discontinued from the study). At
the final visit (24-week), mean HbA1c had increased 0.2% from baseline in placebo
patients and decreased 0.6% with Diabeta SR. [22]

A 16-week, double-blind, placebo-controlled, dose-response study of Diabeta SR, taken

once daily with the evening meal or twice daily with meals, was conducted in patients
with type 2 diabetes who had failed to achieve glycemic control with diet and exercise
(HbA1c 7.0-11.0%, FPG 126-280 mg/dL).Compared with placebo, improvement in
glycemic control was seen at all dose levels of Diabeta SR (metformin hydrochloride
extended-release tablets) and treatment was not associated with any significant change
in weight. [23]

A 24-week, double-blind, randomized study of Diabeta SR, taken once daily with the
evening meal, and metformin hydrochloride tablets, taken twice daily (with breakfast

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and evening meal), was conducted in patients with type 2 diabetes who had been
treated with metformin hydrochloride 500 mg tablets twice daily for at least 8 weeks
prior to study entry. The metformin hydrochloride tablets dose had not necessarily been
titrated to achieve a specific level of glycemic control prior to study entry. Patients
qualified for the study if HbA1c was 8.5% and FPG was 200 mg/dL.After 12 weeks
of treatment, there was an increase in mean HbA1c in all groups; in the Diabeta SR
1000 mg group, the increase from baseline of 0.23% was statistically significant. [24]

3.12.16 Nonclinical Studies conducted on Metformin (Study Drug)

Metformin absorption is relatively slow and may extend over about 6 hours. Animal
14
studies with metformin, labelled with C have shown that the drug is neither
concentrated by liver cells nor is it excreted in the bile; it is concentrated in the
intestinal mucosa and salivary glands.

It has been shown that, following a 2 g dose of metformin, the blood level remains
under 10 mcg/mL even at the peak, occurring 2 hours after absorption. During the
experiments, metformin was shown to be devoid of any notable action in the body,
apart from its specific metabolic activity.

In the healthy animal, metformin lowers blood sugar only at a nearly lethal dose.
Different animal species are of unequal sensitivity. On the other hand, the animal with
experimental diabetes is sensitive to a much lower dosage, providing some insulin is
still secreted. The antihyperglycemic action of metformin is probably mediated through
insulin: Metformin improves the K co-efficient of glucose assimulation. Metformin
improves the co-efficient of insulin efficiency. In the obese diabetic with
hyperinsulinemia, metformin is reported to normalize insulin output. This normalizing
effect is concurrent to that of glycemia. Metformin has little effect on liver glycogen of
the healthy animal. In low and average doses, no change occurs. In high doses nearing
lethal levels, liver glycogen decreases. This lowering precedes the fall in blood sugar.
This reaction represents a defense mechanism tending to mobilize body reserves in
order to combat hypoglycemia.

In the diabetic animal with a low liver glycogen reserve, the opposite occurs and
metformin builds up glycogen stores of the liver. In vitro, on muscular tissue isolated in
Warburgs apparatus, metformin increases glucose uptake by the muscle. This action

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follows an aerobic pathway. Even in high concentration, contrary to phenethyl-

biguanide, metformin apparently does not block respiration or change carbohydrate
metabolism via the anaerobic pathway.

Metformin is eliminated in faeces and urine. It is rapidly excreted by the kidneys in an

unchanged form. Renal clearance is 450 mL/minute; this appears to explain the absence
of accumulation.

Metabolites of metformin have not been identified, neither by radio-active nor by

chemical methods. A single Rf spot is always present following radiochromatographic
study of urine and always corresponds to that of pure metformin. Administration during
10 consecutive days has not shown any sign of accumulation. Inhibition of
glyconeogenesis has been observed in animals following its stimulation by fasting,
cortisol, alcohol or other substrates such as alanine lactate or pyruvate. However, such
an effect varies according to the type and dosage of the biguanide used, nutritional state
of the animal species and design of experimental model.

This inhibition of glyconeogenesis is observed only in the presence of insulin and it

does not appear to play an important role in man. Inhibition of intestinal absorption of
sugars, which is not related to a malasorption phenomenon has been observed with
biguanides under certain experimental conditions in animal and in man. In one study, a
20% retardation of galactose absorption was observed in man receiving metformin.
However, such an effect of metformin could not be confirmed in another study in man.

Recent findings appear to indicate that most of the metabolic effects of the biguanides
are exerted through a single mechanism, namely inhibition of fatty acid oxidation and
of acetyl-CoA generation.

However, inhibition of insulin-stimulated lipogenesis which has also been observed

appears to be due to the inhibition of acetyl-CoA carboxylase by the biguanides. Such
an effect may explain, at least partly, the weight-reducing effect exerted by these drugs
in obese diabetic patients.

The effects of metformin treatment on advanced glycation endproduct formation and

peripheral nerve function in streptozotocin-induced diabetic rats were examined.
Streptozotocin-induced diabetic rats were treated with low dose metformin (5065 mg
kg1 daily) or high dose metformin (500650 mg kg1 daily) for 10 weeks. While the

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metformin-untreated diabetic group showed a significant increase of advanced

glycation endproducts (6.1-fold in the lens, 1.6-fold in the sciatic nerve, 2.3-fold in the
renal cortex, and 1.9-fold in plasma; all P<0.01) compared with the healthy control
group, both metformin-treated groups had significantly less advanced glycation
endproduct deposition. The % decrease in the diabetes-induced increase in advanced
glycation endproduct formation by low and high dose metformin treatment was 25%
and 72% in the lens (both P<0.01), 31% and 42% in the sciatic nerve (both P<0.05),
and 16% and 33% in the renal cortex (P<0.05 and P<0.01), respectively. However, the
plasma advanced glycation endproduct level showed no significant difference from that
in the untreated diabetic group, in spite of slight decrease in plasma glucose and
glycated hemoglobin levels in the metformin-treated groups. The diabetes-induced
sciatic nerve conduction velocity deficits were improved by 46% and 42% by low and
high dose metformin treatment, respectively (both P<0.01). These data suggest that
metformin may have a direct antiglycative action, which in turn contributes to
amelioration of peripheral nerve function. Thus, metformin treatment may be effective
in the prevention of diabetic complications through not only lowering plasma glucose,
but also directly inhibiting advanced glycation endproduct formation.

3.12.17 Toxicological Studies conducted on Metformin (Study Drug)

3.12.17.1 Carcinogenesis, Mutagenesis, Impairment of Fertility and Teratogenic
Effects

Long-term carcinogenicity studies have been performed in rats (dosing duration of 104
weeks) and mice (dosing duration of 91 weeks) at doses up to and including 900
mg/kg/day and 1500 mg/kg/day, respectively.

These doses are both approximately four times the maximum recommended human
daily dose of 2000 mg based on body surface area comparisons. No evidence of
carcinogenicity with metformin was found in either male or female mice. Similarly,
there was no tumorigenic potential observed with metformin in male rats. There was,
however, an increased incidence of benign stromal uterine polyps in female rats treated
with 900 mg/kg/day.

There was no evidence of a mutagenic potential of metformin in the following in vitro

tests: Ames test (S. typhimurium), gene mutation test (mouse lymphoma cells), or

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chromosomal aberrations test (human lymphocytes). Results in the in vivo mouse

micronucleus test were also negative.

Fertility of male or female rats was unaffected by metformin when administered at

doses as high as 600 mg/kg/day, which is approximately three times the maximum
recommended human daily dose based on body surface area comparisons.

3.12.17.2 Teratogenic Effects of Metformin (Study Drug)

Recent information strongly suggests that abnormal blood glucose levels during
pregnancy are associated with a higher incidence of congenital abnormalities. Most
experts recommend that insulin be used during pregnancy to maintain blood glucose
levels as close to normal as possible. Because animal reproduction studies are not
always predictive of human response, DIBETA SR should not be used during
pregnancy unless clearly needed.

There are no adequate and well-controlled studies in pregnant women with DIBETA
SR. Metformin were not teratogenic in rats and rabbits at doses up to 600 mg/kg/day.
This represents an exposure of about two and six times the maximum recommended
human daily dose of 2000 mg based on body surface area comparisons for rats and
rabbits, respectively. Determination of fetal concentrations demonstrated a partial
placental barrier to metformin. [25]

4. Methodology

4.1 Study Site

This study was conducted at Auriga Research Ltd (A Delhi based CRO). Auriga
Research Ltd. is the sister organization of Arbro Pharmaceuticals Ltd, New Delhi and
both the organizations are located in Kirti Nagar Industrial Area, New Delhi. The
various facilities offered by Auriga Research Ltd. are as follows:

I. Clinical Facility

30 bed ward area.

2 bed ICU equipped with vital monitors, defibrillator, ECG, emergency
equipments and emergency medicines.

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Areas for volunteer recruitment, screening, ICF presentation and counseling.

Dining and Recreation area for volunteers.
Pharmacy equipped with stability chambers and refrigerated storage.
Phlebotomy area for drawing blood samples of volunteers.
Referigerated Centrifuge (RCF) area for plasma separation.
Deep freezer for plasma storage.
Report writing and documentation cell.
Archival room for the completed study documents.
Collaboration with nearby Kalra Hospital, for referring any medical emergency.

II. Bioanalytical Facility

LC-MS/MS lab.
HPLC and GC lab.
ICP-MS lab.
GC-MS lab.
Cold room and Deep freezer for sample storage (at -800C).
Sample preparation area.
Wet lab equipped with Refrigerated centrifuges, Airshaft free micro and
analytical balance areas, Fume hoods.
Dark lab.

III. Statistical Facility

21 CFR part 11 compliant softwares
PK and Statistical capability
SAS- Statistical Analysis Software
WinNonlin version 5.2 software
Statistical input to protocol design (sample size, experimental design,
randomization code)
Statistical programming, analysis and reporting

4.2 Investigational Drug Products (Study Drug)

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Metformin Hydrochloride (SR) 500 mg tablet was the Test Product and Dibeta SR
tablet [containing Metformin Hydrochloride (SR) 500 mg] was used as the Reference
standard.

4.2.1 Description of Test and Reference Product

Metformin Hydrochloride (SR) 500 mg tablet was the Test Product and Dibeta SR
tablet [containing Metformin Hydrochloride (SR) 500 mg] was the Reference standard.

Table 14: Description of Test and Reference Product

Test (T) Reference (R )

Product Metformin hydrochloride Dibeta SR

SR

Dosage Form Tablet Tablet

Strength 500 mg 500 mg

Generic Name Metformin hydrochloride Metformin hydrochloride

Manufactured By Sponsor Acme Formulation Pvt. Ltd,

India. Marketed by (Torrent
Pharmaceuticals Ltd, India)

Batch/Lot No. VM-001 AF9010

Mfg Date Jun 2009 May 2009

Expiry Date May 2011 Apr 2011

4.2.2 Accountability of Drug Products (Receipt, Storage and Retention)

The sponsor had supplied sufficient quantities of the study formulations for
conduct of the study & for retention purpose.
The drug products along with Certificate of Analysis (COA) was received by
the Pharmacist or Principal Investigator or designee.
The COA had product description/appearance, identification, in vitro
dissolution data and assay as per specifications/pharmacopoeia

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Reference products was supplied in the original manufacturers packing and the
test products was supplied in an appropriate package deemed to maintain the
integrity of the products
Records were made of the receipt and dispensing of clinical supplies to provide
a complete accountability of all supplies
The supplies were stored at controlled temperature or as per label instructions in
Pharmacy accessible only to the Pharmacist or authorized personnel
Batch number, manufacturing date, expiry date and physical description for
both products was included in the final report.
At the conclusion of the study, the unused supplies were retained in accordance
to applicable regulatory requirements regarding retention of reserve sample.

Accountability of drug products is mentioned in the following Table 15.

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Table 15: Investigational Product Accountability

DISPENSING

Period I

Treatment Batch Initial Quantity Quantity Quantity

Code No. Dispensed Remained
Metformin
149+01*
hydrochloride 06+01** 142
VM-001 =150 tablets
SR 500 mg (T )

Dibeta SR 149+01*
AF-9010 06+01** 142
500 mg (R) =150 tablets

Period II

Treatment Batch Initial Quantity Quantity Quantity

Code No. Dispensed Remained
Metformin 141+01*
hydrochloride VM-001 06+01** 134
SR 500 mg (T ) =142 tablets

Dibeta SR 141+01*
AF-9010 06+01** 134
500 mg (R) =142 tablets

RETENTION

Quantity Test (T) Reference (R)

Retained 134 134

Remarks: 01*: 1 tablet taken for identification

01**:Extra tablet dispensed

4.2.3 Assignment to Treatment Sequences

The following information was given on the dosing label prepared for the study:
Study No.
Period No.
Subject No.
Randomization Code
Date of Dosing
Dated initials of Pharmacist

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4.2.4 Labeling
Whether a subject receives test or reference products during the study was
determined according to randomization schedule generated and authorized by
PK Scientist.
The Clinical Investigator and the personnel involved in dispensing of study
drugs were accountable for ensuring compliance to randomization schedule.

4.2.5 Dose and Route of Administration of Investigational Drug Products

Subjects were receiving each treatment according to the randomization schedule.
Dosing was done after an overnight fast of at least 10 hrs and fasting was continue until
4 hrs post dose. Lunch, snacks and dinner were provided at 4, 9 & 13 hrs post dose
respectively.

4.2.6 Assessment of Compliance

Compliance was assessed by conducting a thorough examination of the oral cavity by
trained study personnel after dosing

4.2.7 Timing of Dose for each period

Administration of the study drug was performed between 09:00-09:10 hrs, on the first
day of the study in both the periods; as mentioned in Table 16.

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Table 16: Administration Time of Study Drugs

Period I Period II

Sub. Date Intended Actual Date Intended Actual

No. Time (am) Time (am) Time (am) Time (am)

1 31-10-09 09:00 09:00 07-11-09 09:00 09:00

2 31-10-09 09:02 09:02 07-11-09 09:02 09:02

3 31-10-09 09:04 09:04 07-11-09 09:04 09:04

4 31-10-09 09:06 09:06 07-11-09 09:06 09:06

5 31-10-09 09:08 09:08 07-11-09 09:08 09:08

6 31-10-09 09:10 09:10 07-11-09 09:10 09:10

7 31-10-09 09:00 09:00 07-11-09 09:00 09:00

8 31-10-09 09:02 09:02 07-11-09 09:02 09:02

9 31-10-09 09:04 09:04 07-11-09 09:04 09:04

10 31-10-09 09:06 09:06 07-11-09 09:06 09:06

11 31-10-09 09:08 09:08 07-11-09 09:08 09:08

12 31-10-09 09:10 09:10 07-11-09 09:10 09:10

4.3 Study Design (Annexure-II)

4.3.1 Design
An open label, balanced, randomized, two treatment, two period, two sequence, cross
over bioequivalence study of single dose of Metformin Hydrochloride (SR) 500 mg
tablet of Sponsor and Dibeta SR tablet [Containing Metformin Hydrochloride (SR) 500
mg] of Torrent Pharmaceuticals Ltd., India in healthy human adult male subjects under
fasting condition.

4.3.2 Choice of the Doses

The dosage of one Metformin tablet was chosen as it is recommended by the DCGI.

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4.3.3 Duration of Samples

Sampling till 24 hours was taken into account based on the terminal half life of
Metformin which is 6.2 hours.

4.3.4 Dosing
After a supervised overnight fast, subjects had received a single oral dose of the
assigned formulation, with 240 ml of 20% solution of glucose, according to the
randomization schedule generated and authorized by Biostatistician. Mouth check was
done by the study personnel doing dosing in the presence of other study personnel
responsible for dosing supervision. To prepare 20% of glucose, 48 gram of
commercially available glucose powder was dissolved in 240 mL of water.

4.3.5 Sample Size

Hippocrates Independent Ethics Committee (HIEC), New Delhi has given the approval
to conduct this study and allowed to enroll twelve subjects for this particular study
based on ethical reason.

4.3.6 Criteria Check

4.3.6.1 Inclusion Criteria
The subjects were included based on the following criteria:
1) Subject who are able and ready to provide written informed consent.
2) Subjects must be healthy human beings within 18-45 years of age (both
inclusive).
3) Subjects should be of ideal body weight in relation to height according to Life
Insurance Corporation of India height-weight chart for non-medical cases.
4) Subjects must be of normal health as determined by medical history and
physical examination, ECG and laboratory tests performed within 21 days prior
to the commencement of the study.
5) Subjects whose screening laboratory values are within normal limits or
considered by the physician / Principal Investigator to be of no clinical
significance.

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4.3.6.2 Exclusion Criteria

The subjects were excluded based on the following criteria:
1) Subjects incapable of understanding the informed consent process or not ready
to sign informed consent.
2) Subjects with Hypersensitivity and/or intolerance and/or contraindication to
Metformin or any ingredients of the formulation or related group of
drugs.Subjects with active or a history of peptic ulceration.
3) Subject with resting hypotension (BP <90 /60) or hypertension (BP 140 /90).
4) Subject with Resting Pulse rate below 60 / min. and above 100 /min.
5) Subjects with or prior history of clinically significant, Cardiovascular,
pulmonary, hepatic, renal, hematological, gastrointestinal, endocrine,
immunologic, dermatologic, musculoskeletal, neurological or psychiatric
disease.
6) Subjects with a history of known food allergy.
7) Subjects who have suffered any illness or who have been hospitalized within
the last 4 weeks preceding the start of the study.
8) Subjects who have taken over the counter or prescribed medications, including
any enzyme modifying drugs within the last 14 days prior to the study.
9) Subject who have a History of drug abuse or alcoholism i.e. alcohol
consumption > 2 units / day or 10 units / week (one unit of alcohol = 50 ml
spirit or 200 ml wine or 500ml beer).
10) Subject who have Smoking History of > 10 Cigarettes / day or Tobacco
consumption > 4 packets / day
11) Subjects who participated in any other clinical trial requiring repeated blood
sampling or a blood donation program or blood volume of more than 350 mL,
in the past three months ( approx. 90 days) (This 350 mL includes the total
blood volume that will occur during the study).
12) Subject with clinically significant abnormal lab values / abnormal ECG or chest
X-ray.
13) Subject who has difficulty with donating blood.

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4.3.7 Recruitment of Volunteers

Volunteers who fulfilled all the study requirements including inclusion and exclusion
criteria (as mentioned above) were allowed to participate in the study.

Recruited Volunteers

N=12

Period I Period II

N=12 N=12

Test Reference Test Reference

N=06 N=06 N=06 N=06

Completed Completed Completed Completed

N=06 N=06 N=06 N=06

Withdrawn Withdrawn Withdrawn Withdrawn

N=00 N=00 N=00 N=00

Figure 5: Volunteers included in the study

4.3.8 Restrictions and Prohibitions

4.3.8.1 Restrictions
Subjects were instructed as follows:
1) Not to smoke or consume tobacco containing products and xanthine products
for at least 24 hours before dosing and for the entire duration of blood
collection.
2) To abstain from alcohol for 48 hours before dosing and for the entire duration
of blood collection.

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3) Not to consume any medication (including over-the counter products) for 14

days preceding the study and for the entire duration of the study. If concomitant
medication is required during the time of sample collection the subjects will be
treated accordingly, and a decision to continue or discontinue the subjects will
be made, based on the time the medication was administered, pharmacology
and pharmacokinetics of study and non study medication.
4) Grapefruit or grapefruit juice should not be allowed at least 48 hrs prior to
dosing as grapefruit alters the pharmacokinetics of most of the orally
administered drugs.

4.3.8.2 Prohibitions
The subjects were prohibited from smoking or consuming tobacco containing products,
xanthine containing products and alcohol during their stay in the clinical facility.

4.3.9 Blinding
Auriga Research Limited had make no changes in the appearance of test or reference
product. While dosing, the treatments were labeled as mentioned under section 4.2.4.
The pharmacist or a person designated by the principal investigator had dispensed and
label the product as per randomization code.

4.3.10 Randomization Schedule

A balanced block randomization schedule was generated before the start of the study
utilizing WinNonlin Version 5.2 by the biostatistician. Subjects received the assigned
formulation in each period, according to a randomization schedule with the following
possible sequences, TR or RT [where T = Test Product and R = Reference Standard].

Equal allocation of subjects to each sequence was ensured. Study personnel involved in
the sample analysis were kept blinded from the randomization code during the entire
study. Persons involved in dispensing of study drug and in verifying the dispensing
activity were accountable for ensuring compliance with the Randomization schedule.
The randomization schedule used in this study was as follows:

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Table 17: Randomization Schedule

Sub. Sequence Code Sequence
Period 1 Period 2
No.

1 B RT R T

2 A TR T R

3 A TR T R

4 B RT R T

5 B RT R T

6 A TR T R

7 A TR T R

8 B RT R T

9 A TR T R

10 B RT R T

11 A TR T R

12 B RT R T

4.3.11 Blood Sampling

A total of 28 blood samples were collected during the study. The blood samples were
collected pre-dose (2 x 3 ml) within a period of 1.5 hour before dosing and at (1.0, 2.0,
3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 12.0, 18.0, 24.0) hours post-dosing in K2EDTA
vacutainer. The pre-dose and post-dose samples up to 12 hours were collected via an
indwelling intravenous cannula / scalp vein set placed in a forearm vein of the subjects.
After 12 hours post-dose samples were taken directly by venipuncture. Intravenous
indwelling cannula / scalp vein set was kept in situ as long as possible by injecting 0.5
ml of 5 IU/ml of heparin in normal saline solution to maintain the potency of cannula /
scalp vein set for collection of the post-dose samples.

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The blood samples were collected and transferred into pre-labeled (mentioning Study
number, Subject number, Period and sampling time point) K2EDTA sample collection
tube. If for any reason the indwelling cannula / scalp vein set is blocked or must be
removed for practical reasons, direct venipuncture was done. For each subject the total
number of blood samples drawn during the study was 28 and the total volume of blood
drawn including 10 ml for screening, 11 ml of blood which is discarded (i.e., 0.5 ml of
blood is discarded before each sample is drawn to remove the heparin which is in the
indwelling cannula / scalp vein set), had not exceed 111 ml for the entire study.
The choice of the blood sampling time schedule was intended to cover the expected
time of the peak plasma concentrations of metformin, which was at around 7 (4-8) hour
(Drugs @ FDA, 2008). It was also extended for sufficient time to be able to obtain a
reliable estimate of the extent of absorption. The last sample collection was at 24.00
hours post-dose, which was more than 3 times the maximum reported half-life of
metformin 6.2 hours, and was cover at least 80% of the AUC0-.

4.3.11.1 Table 18: Phlebotomy Schedule

Pre dose 1.0 2.0 3.0 4.0 5.0

Phlebotomy
6.0 7.0 8.0 9.0 10.0 12.0
Schedule (hrs)

18.0 24.0

4.3.12 Handling of Plasma Samples

All the blood samples were centrifuged under refrigeration immediately after its
collection to separate plasma at 2 to 100C, 4000 100 rpm for 10 minutes. The blood
samples was kept in wet ice bath before centrifugation. The separated plasma were
transferred to pre-labeled polypropylene tubes in single aliquot and stored upright in a
box containing dry ice or in a freezer at a temperature -200C or colder for interim
storage. Finally the samples were transferred and stored in a freezer at (80 10)0C or
colder until completion of analysis.
4.3.13 Sample Transportation

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The samples were transported to the Arbro Pharmaceutical Ltd analytical section
within 12 hr of sample separation and stored at (80 10)0C or colder till the time of
analysis.
4.3.14 Criteria for Recording & Reporting Blood Sample Deviation
Blood samples were collected at times specified under phlebotomy schedule. The
actual time of collection of each blood sample was the time at which sample collection
end and the hour and minutes on clock display at that time was recorded on the
appropriate raw data forms. A window period of 2 minutes from the scheduled time
point is allowed for the in-house blood draws. All deviations outside the allowed range
as above were documented as protocol deviations. In all such instances appropriate
time corrections for the actual time of sample collection, was incorporated at the time
of data analysis.
4.3.15 Study Meals
Standardized meals (Annexure-VIII) prepared by dietician was served to subjects. An
overnight fasting of at least 10 hrs was ensured prior to dosing and fasting state was
continued until 4 hrs post dose. Lunch, snacks and dinner were provided at 4, 9 & 13
hrs post dose respectively. Water was restricted from 1 hr Predose to 2 hrs post dose.
Subsequently water was provided ad libitum. 60 mL of 20% glucose solution was
provided at the interval of 15 minutes till 4 hrs of post dose, and whenever required
after blood glucose monitoring or safety reason.
4.3.16 Housing
From at least 12 hours prior to drug administration until after 24 hours. post dose
4.3.17 Washout Period
At least 07 days between treatments.
4.3.18 Activity Levels
Subjects were seated upright for the first 2 hours following drug administration.
However, should medical events occur at any time, subjects may be placed in an
appropriate position; Subjects were prohibited from any strenuous or athletic activity
during housing.
4.4 Clinical Assessment
4.4.1 Subject Screening
Subject Screening was done 21 days prior to the start of the study. Medical histories
and demographic data including name, sex, age, race, body weight (kg), and height
(cm), and smoking habits were recorded. Each subject has to undergo a complete

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general physical examination and laboratory tests as mentioned in the next section
4.4.2. Only medically healthy subjects with clinically acceptable laboratory profiles,
chest X-ray (performed within 6 months prior to study start) and ECG (performed
within 21 days prior to study start) were enrolled into the study.

4.4.2 Clinical / Diagnostic Laboratory Tests

The following tests were performed during screening of volunteers:
Chest X-ray and ECG
Haematology
Haemoglobin
Total and differential leukocyte count
Platelet count
Blood Group & Rh Typing
Routine Biochemical Investigations
Total Cholesterol
FBS
PPBS
Creatinine
Urea
Blood Urea Nitrogen
LFT
Total bilirubin
Total proteins
Alkaline phosphatase
SGOT/AST
SGPT/ALT
Urine analysis
Colour
Transparency
pH
Specific gravity
Protein
Glucose
Microscopic examination
Serology
HIV Test 1 and 2
Test for Hepatitis B

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Test for Hepatitis C

VDRL test

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4.5 Safety Measurement during the Study

4.5.1 Clinical Safety
4.5.1.1 Vitals
A physician, or a nurse under the supervision of a physician, had monitored the vital
signs of the subjects. Sitting blood pressure, radial pulse rate and oral temperature were
measured at admission, prior to dosing and at 2, 6, 12, 18 and 24 hours post-dosing. In
case, vital sign measurements overlap with the blood sample collection or any other
scheduled catering activity, the vital sign measurements were done either after or
before the activity as per the convenience of subject and medical personnel. All
observations were written in the study raw data forms.
4.5.1.2 Medical Examination
Clinical investigator had done the medical examination of the subjects at admission,
prior to dosing, once in every 12 hrs and prior to discharge in each period of the study.
4.5.1.3 Assessment of Well Being
Subjects well being were assessed at admission, Predose, once in every 4 hrs post dose
(except at 16 and 20 hrs), and at discharge in each period. In addition, at any time,
subjects may report spontaneously any side effects, inconvenience to the monitoring
staff. This information was also be recorded in the raw data forms
4.5.1.4 Evaluation of Blood Glucose
Blood glucose was monitored by finger prick method at the interval of 1 hr after drug
administration till 4 hrs. post dose.
4.5.2 Detection and Management of Adverse Events (Annexure IX & X)
The clinical investigator or a medical officer was available within the clinical facility
until 24 hrs post dose during each period. Medically qualified personnel or any
specialist had been also appointed on a call-duty until the completion of the study.
Subjects were monitored throughout the study period for adverse events. Subjects were
informed to bring to the notice of the doctor or nurse or any other staff any adverse
event that may occur during their in house stay.
4.5.3 Documentation and Reporting of Adverse Events
No adverse event was reported in this study.
4.6 Ethical Considerations
4.6.1 Basic Principles
This research was carried out in accordance with the clinical research guidelines
established by the Basic principles defined in the U.S.21 CFR Part 312.120, ICH GCP,

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Indian GCP and the principles enunciated in the Declaration of Helsinki (Ethical
Principles for Medical Research Involving Human Subjects, revised by 59th WMA
General Assembly, Seoul, October 2008).

4.6.2 Independent Ethics Committee (Annexure-I)

1) An informed consent document for general screening and the general screening
procedure approved by the ethics committee was used for this study.
2) Hippocrates Independent Ethics Committee (HIEC), New Delhi had reviewed this
protocol, and given the approval for the conduct of this study.

4.6.3 Informed Consent Document (Annexure VII)

1) Screening of volunteers for participation in this study was done after taking an
informed consent from the volunteers. Screening was performed within 21 days
prior to start of the study.
2) An oral presentation of the Informed Consent Document (ICD) was provided to all
the volunteers in the presence of a medical officer to resolve medical/general
queries of the volunteers.
3) The purpose of the study, the procedures to be carried out and the potential hazards
were described to the subjects, in a language that the subject comprehends. The
subjects were required to read the informed consent document (ICD) which
summarizes the discussion prior to check-in. Ample time was given to the
volunteers to read and understand the contents of the informed consent form. The
informed consent form describes in non-technical terms the study procedures and
potential hazards. The ICD was then signed and dated by the subject, study
personnel obtaining consent and clinical investigator /designee. The subject were
then allowed to check in. A copy of the informed consent document was given to
the subjects.

4.6.4 Termination of the Study

Auriga Research Ltd., India and the Sponsor had the right to terminate the study at any
time. The reason for this termination had to be provided to the subjects. The Principal
Investigator and the IEC reserves the right to discontinue the study for safety reasons at
any time.

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4.6.5 Volunteer Insurance/ Medical treatment for injury

All the subjects were covered by insurance for any study related injuries.

4.6.6 Volunteer Compensation for participation

The subjects were compensated for their time utilized in the study on a prorate basis of
30:70 i.e. after Period I & after Period II as per Ethics Committee recommendations on
compensation.

4.6.7 Withdrawal / Dropout of subject from study

Any subject is free to withdrawal from the study at any time without having to give any
reasons thereof.
The investigator may withdraw a subject from the study for any of the valid reasons
which he thinks is appropriate in view of the safety and well-being of subject, GCP
Principles or objective of the project, in particular for:
1) Any serious side effects including hypersensitivity.
2) Subject develops any medical illness.
3) Administered concomitant medication for any adverse event interacting with study
medication.
4) Due to non-compliance with study protocol.
5) Any abnormal laboratory test considered to be of clinical significance.
6) Willful withholding of information.
7) Any serious protocol violation (The subject fails to comply with the requirements
of the protocol).
8) Requirements of study protocol, if are too onerous.
9) Error in dosing.
10) Inter-current illness requiring treatment.
In any of these cases the compensation to Subjects will be made as per the
compensation structure approved by IEC. The final report will include reasons for
withdrawal. In the present study, no withdrawal or dropout of the subject was noted.

4.7 Analytical Procedures

4.7.1 Method Description
1) A validated bioanalytical method developed as per the regulatory requirements
for the quantification of Metformin in plasma was employed for subjects
sample analysis.

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2) Analysis of the subjects sample was including distribution of quality control

samples throughout each batch of study samples analyzed. Samples from all
subjects who completed the study was analyzed.
3) All concentration values below the limit of quantification (BLQ) was set to be
zero for all pharmacokinetic and statistical calculations. Any missing samples
were reported as M and was not be included for Pharmacokinetic or statistical
analysis.
4) The analyst had no access to randomization schedule during the course of the
analysis.
4.7.2 Validation Protocol
The bioanalytical methods were developed and validated as per the regulatory
requirements
This includes the following:
1) Specificity was checked on six independent sources of the same matrix.
2) Five precision and Accuracy batches were checked.
The between batch CVs for low, medium and high QC concentrations
should be 15% and for LOQ QC should 20%
The between batch mean value should be within 15% of the nominal
value at low, medium and high QC concentrations and should not
deviate by more than 20% at the LOQ QC level.
1) Stability which includes bench top, freeze thaw, in-injector, long term and stock
solution stability.
2) Validation for anti-coagulant effects, if required.
3) Recovery studies of drug and IS from the biological matrix.
4) Sensitivity of the method (LOQ).
5) Dilution integrity.

4.8 Pharmacokinetic and Statistical Analysis

Pharmacokinetic and Statistical analyses will include data from all subjects who
complete the study. If subjects are withdrawn due to adverse events their plasma
concentrations obtained from the bio-analytical laboratory will not be used in
calculation of pharmacokinetic parameters or for statistical analysis. However, these
concentrations will be tabulated in a separate table.

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4.8.1 Pharmacokinetic Parameters & Analysis

Pharmacokinetic parameters for Metformin was calculated using non compartmental
model of WinNonlin Version 5.2 as follows:
4.8.1.1 Primary Parameters:
1) Cmax: Maximum measured plasma concentration over the time span specified
2) AUC0-t : The area under the plasma concentration versus time curve, from time
0 to the last measurable concentration, where t = time of last identifiable
concentration, calculated using linear trapezoidal method.
3) AUC0-: The area under the plasma concentration versus time curve from time
0 to infinity.
4.8.1.2 Secondary Parameters:
1) Tmax: Time of the maximum measured plasma concentration. If the maximum
value occurs at more than one time point, Tmax is defined as the first time point
with this value.
2) Kel: The elimination rate constant.
3) t 1/2: The terminal elimination half-life.
4) AUC0-t / AUC0-: The ratio of AUC0-t to AUC0-
No value of AUC0-, t1/2 , and Kel was reported for cases that do not exhibit a linear
relationship in the terminal elimination phase in the log plasma concentration versus
time profile.
4.8.2 Statistical Analysis
Statistical analyses were performed on Metformin using the WinNonlin Version 5.2.
4.8.2.1 Summary Statistics
Arithmetic means, standard deviations and coefficients of variation was calculated for
the parameters listed in section 4.28.1.2. Additionally, geometric means were
calculated for AUC0- and Cmax.
4.8.2.2 Analysis of Variance (ANOVA)
The log-transformed pharmacokinetic parameters (Cmax, and AUC0-) was analyzed
using a GLM ANOVA model with the main effects of treatment.
The main effects was tested at the 0.05 level of significance against the residual error
(mean square error/MSE) from the ANOVA as the error term.
Each analysis of variance will include calculation of least-square means, the difference
between the adjusted formulation means and the standard error associated with the

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difference. The above analysis was done using the WinNonlin Version 5.2 version.

4.8.2.3 Ratio Analysis and 90% Confidence Intervals

Differences of LSMs were calculated for log-transformed, AUC0- and Cmax. The
differences were of the form: - Test-Reference. Ratios of means were expressed in
percentage by taking the anti-log value of difference of LSM. The geometric mean
values was reported.
Consistent with the two one-sided tests for bioequivalence, 90% confidence intervals
for the difference between drug formulation least-square means (LSM) were calculated
for the parameters AUC0- and Cmax using log-transformed data. The confidence
intervals was expressed as a percentage relative to the LSM of the reference
formulation
4.8.2.4 Acceptance Criteria for Bioequivalence
The geometric least square mean ratios and the 90% CI of Cmax, AUC0-t and AUC0- for
the test product and the reference standard of the study drug metformin were
considered, for evaluating bioequivalence. To be considered bioequivalent, ratios and
confidence interval should lie within the following acceptance range.

Table 19: Acceptance Criteria for Bioequivalence

Acceptance Range of ln-transformed data

Sr. No. Primary Parameters

For Ratios (%) For 90% CI (%)

1. Cmax 80.00-125.00 80.00-125.00

2. AUC0-t 80.00-125.00 80.00-125.00

3. AUC0- 80.00-125.00 80.00-125.00

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4.9 Study Documentation

4.9.1 Report Format
The final report was written according to the ICH E3 Guideline (Structure and Content
of Clinical Study Reports).
4.9.2 Supplementary Documentation
The following documents were maintained at the CRO site:
a) Raw Data File
List of Study personnel responsibility
Master List of Subjects
Subject Admission Details
Record of Informed consent obtainment and volunteer queries
Record of Subject Compensation.
Protocol Training record
Record of Sample Separation And Storage
Sample transfer record.
Raw Data forms accountability list
b) Case Report Form (CRF)
Zero Hour Blood Sampling Forms
Record of Drug Administration
All Blood Sampling Forms
Record of Vitals
Record of Medical Examination
Record of Subject well being
Adverse Event Forms
Meal consumption Record
Subject Discharge
Subject compliance report
Record of Reason for Subject Withdrawal/dropout
c) Trial Master File
Clinical Summary Report
Quality Assurance Statement
IEC Approval Letter
IEC Approved Protocol with Appendices
Documentation of Informed Consent Procedure
Informed Consent Document (ICD)
Randomization Schedule
Investigational Drug Product Receipt
Certificate of Analysis
Investigational Drug Product Acknowledgment
Investigational Drug Product Identification and Dispensing Details

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Adverse Events Reporting Forms

SOP / Protocol Deviations Details (If any)
d) VSR (Volunteer Screening Record) File
History and General Physical and Systemic Examination Forms
ECG
Chest X ray Reports
Laboratory Test Reports
Inclusion and Exclusion Criteria check
e) Chromatograms of the last 20% of the total number of subjects analyzed were
submitted with the final report.

4.10 Archives
All raw data generated in connection with this study, together with the original copy of
the final report, was retained in the archival area of Auriga Research Limited for at
least 2 yrs after the expiration date of the batch as per CDSCO.

4.11 Publication Policy

The publication policy will be at the discretion of the sponsor. If published the
subjects identity will not be revealed.

4.12 Quality assurance audits

In process and Periodic review were carried out by the QA department to confirm that
deviations if any, from approved protocol or SOPs are adequately documented. In the
present study no deviation was reported.

4.13 Study monitoring / Audit from sponsor

A monitor or auditor appointed by the sponsor may meet the Investigators and visit the
study facilities at any time in order to maintain current knowledge of the study through
review of the records, comparison with source documents, observation and discussion
of the conduct and the progress of the study. Prior to the start of the study, the Principal
Investigator was contacted and informed of any impending visits and the frequency of
such visits. At each visit the Investigator had assisted the study monitor in terms of
reviewing and verifying those records associated with the study.
4.14 Deviations
In the present study no deviation was reported.

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4.15 Figure 6: Flowchart of Bioequivalence Study

Flowchart of Bioequivalence Study

1) 1) Preparation of Protocol & Appendices

Check Approval Status in FDA & DCGI

Label of Drug Monograph from FDA site
Calculate blood sampling time points according to half life
Calculation of volunteer compensation
Protocol Preparation
Review of Draft Protocol by Study Investigators
Completing signature page

2) IEC for Review and Approval

Inform IEC about Initiation of Clinical Study

Take the approval of Study Protocol

3) Screening

Screening of Volunteers (Lab and Medical Examination)

within 21 days prior to study
Healthy volunteers enrolled.

4) Study Procedures

Period I

Admission Day (Day 1)

ICD Presentation
Provide ample time to volunteers to understand the ICD
Obtain the consent of volunteers on ICF.
Check in of the volunteers
Vital measurements & Medical examination
Dinner as per study meal menu

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Dosing Day (Day 2)

Cannulation
Predose blood sample
Dosing of the study drug
Collection of blood samples as per the phlebotomy schedules
Vital measurements & Medical examination (During Study)
Reporting of any AE/ADR (if any)
Discharge Day (Day 3)
Medical examination (Before Discharge)
Discharge with 30% compensation
Check out of the volunteers
Washout period
Washout period of 07 days

Period II

Same as Period I
Discharge with remaining 70% compensation

5) Post Study Procedures

Plasma separation
Analysis of drug level in plasma samples
Statistical & PK Analysis
Preparation of Study Reports
Submit the Study Reports to Regulatory body, Sponsor and IEC
Archive the study documents

5. Results and Discussion

5.1 Demographic and Other Baseline Characteristics

Hippocrates Independent Ethics Committee (HIEC), New Delhi has given the approval
to conduct this study and allowed to enroll twelve subjects for this particular study

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based on ethical reason. All the enrolled twelve subjects were of age between 18 and
45 years, weight within 15% of the ideal Height-Weight chart of Life Insurance
Corporation (LIC) of India (see Annexure V). Table 19 &20 describes the demographic
characters like gender, age, height, weight and diet of the enrolled volunteers.

Table 20: Subjects Demographic Profile

Subject Age Height Weight BMI Smoking Date of

Sex Diet Screening
No. (yrs) (cm) (kg) (kg/m2)

Non- Non- 26-10-09

1 Male 22 165 62 22.77
Veg Smoker

Non- Non- 26-10-09

2 Male 27 171 70 23.93
Veg Smoker

Non- Non- 26-10-09

3 Male 23 170 64 22.14
Veg Smoker

Non- 24-10-09
4 Male 36 169 64 22.40 Veg
Smoker

Non- Non- 26-10-09

5 Male 30 160 60 23.43
Veg Smoker

Non- Non- 24-10-09

6 Male 20 163 54 20.32
Veg Smoker

Non- Non- 24-10-09

7 Male 20 163 50 18.81
Veg Smoker

Non- Non- 24-10-09

8 Male 20 153 50 21.35
Veg Smoker

Non- Non- 24-10-09

9 Male 19 161 53 20.44
Veg Smoker

Non- Non- 24-10-09

10 Male 35 153 56 23.92
Veg Smoker

Non- Non- 24-10-09

11 Male 31 166 64 23.22
Veg Smoker

12 Male 37 166 69 25.03 Non- Non- 26-10-09

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Veg Smoker

Table 21: Summary of Demographics of the study volunteers

Age (yr) Height Weight

(cm) (kg)

N 12 12 12

Mean 26.66 163.33 59.66

SD 6.879 5.898 6.958

CV (%) 25.79 3.61 11.66

Range 19-37 153-171 50-70

5.2 Clinical Laboratory Evaluation

Screening examination was conducted within 21 days prior to study. During screening,
medical history of each volunteer was taken. Laboratory tests (including Hematology,
Biochemistry Serology and Urine examination) were also performed for each
volunteer. After passing the screening examination, healthy volunteers were enrolled in
the study.

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Table 22: Clinical Laboratory Evaluation

Normal Range Subject Numbers

Test Name

1 2 3 4 5 6 7 8 9 10 11 12

HAEMOGLOBIN (Hb) 13 - 17 13.0 13.7 13.3 13 13.5 13.3 13.1 13 15.7 14.9 13.6 13.3

TLC 4 - 10 5.3 6.1 8.8 4.5 8.8 7.1 9.5 9.4 8.6 8.6 6.1 9.1

NEUTROPHIL 40 - 80 65 57 70 40 70 66 50 62 57 62 72 61

LYMPHOCYTE 20 - 40 28 35 26 40 24 28 39 33 38 30 22 30

EOSINOPHIL 01 - 06 4 5 1 3 2 1 3 1 2 3 2 6

MONOCYTE 02 - 10 3 3 3 2 4 5 3 4 3 5 4 3

BASOPHIL <01 02 0 0 0 0 0 0 0 0 0 0 0 0

PLATELET COUNT 150 - 410 193 380 202 273 226 156 210 157 219 156 165 178

GLUCOSE (FASTING) 74 - 100 84 81 83 90 80 100 79 79 93 90 88 93

BLOOD UREA NITROGEN 07 - 18 11 7 9 7 12 13 7 7 10 7 8 11

CREATININE 0.9 - 1.3 1.0 1.1 0.9 0.9 0.9 1 0.9 1.3 1.1 1 1.1 1.3

TOTAL BILIRUBIN 0.0 1.0 0.8 0.3 0.4 0.5 0.6 0.4 0.7 0.3 0.4 0.5 0.3 0.3

S.G.O.T(AST) 15 - 37 30 29 26 26 37 28 18 35 23 20 23 30

S G.P.T(ALT) 30 - 63 59 43 47 39 56 58 35 52 40 42 63 41

ALKALINE PHOSPHATASE 50 - 136 97 98 132 64 131 93 121 111 127 87 97 125

TOTAL PROTEIN 6.4 - 8.2 7.6 7.9 8 7.5 7.9 7.3 6.4 8.2 8.1 6.7 8 8.1

TOTAL CHOLESTEROL <200 103 182 151 159 195 122 92 161 150 175 115 148

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SEROLOGY

V.D.R.L NR NR NR NR NR NR NR NR NR NR NR NR NR

HIV (AIDS) antibody !&II NR NR NR NR NR NR NR NR NR NR NR NR NR

HBsAg (Qualitative) NR NR NR NR NR NR NR NR NR NR NR NR NR

Anti Hepatitis C Virus (Qual) NR NR NR NR NR NR NR NR NR NR NR NR NR

Out of range parameters (Highlighted) considered as Insignificant or Clinically Insignificant), NR: Non Reactive

Table 23: Urine Analysis

Subject Numbers

Test Name

1 2 3 4 5 6 7 8 9 10 11 12

Physical
Examination

Quantity (ml) 25 25 25 25 25 25 25 25 25 25 25 25 25

Colour N/AP PY PY PY PY PY PY PY PY PY PY PY PY

Appearance N/AP CLEAR CLEAR CLEAR CLEAR CLEAR CLEAR CLEAR CLEAR CLEAR CLEAR CLEAR CLEAR

Chemical
Examination

Protein Not detected ND ND ND ND ND ND ND ND ND ND ND ND

Glucose Not detected ND ND ND ND ND ND ND ND ND ND ND ND

pH 6 6 6.5 6 6 6 7 7 5.5 6 5.5 5.5

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Microscopic
Examination

WBC(Pus Cells)
/HPF 0-5 0-1 0-1 0-1 0-1 0-1 0-1 0-1 0-1 0-1 0-1 0-1 0-1

RBC's Not detected ND ND ND ND ND ND ND ND ND ND ND ND

Casts N/AP ND ND ND ND ND ND ND ND ND ND ND ND

Crystals N/AP ND ND ND ND ND ND ND ND ND ND ND ND

Epithelial Cells 0-5 0-1 0-1 0-1 0-1 0-1 0-1 0-1 0-1 0-1 0-1 0-1 0-1

Bacteria Not detected ND ND ND ND ND ND ND ND ND ND ND ND

Bile salt Not detected ND ND ND ND ND ND ND ND ND ND ND ND

Bile pigment Not detected ND ND ND ND ND ND ND ND ND ND ND ND

Others Specify ND ND ND ND ND ND ND ND ND ND ND ND

SP.Gravity 1.003 - 1.035 1.015 <=1.005 <=1.005 <=1.005 <=1.005 1.010 <=1.005 <=1.005 1.010 <=1.005 <=1.005 <=1.005

PY: Pale Yellow, DY: Deep Yellow, ND: Not detected

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5.3 Dropout and Withdrawn Subjects

All the enrolled subjects successfully completed this study, and no

dropout/withdrawal was reported.

5.4 Protocol Deviation

5.4.1 Blood Sample Collection

Majority of the post-dose samples were collected within 2 minutes from the
scheduled sampling time in all the periods of the study as per SOP. None of the
deviations were observed in blood sample collection.

Table 24: Blood Sample Collection

S. No. Subject No Period No Time Point Early/ Delay * Reason

(Hr) /missing
samples

None

*Reason

A. Difficulty with veins

B. Late due to preceding draw
C. Late due to medical event
D. Subject arrived late
E. Subject did not report for ambulatory sample

5.4.2 Deviation In plasma Samples Storage Condition

The plasma samples were stored at -20C or colder till the completion of the time
period and then transferred to the bioanalytical facility as per the SOP and were stored
at (-8010)C or colder until analysis. None of the deviations were observed in
plasma storage condition.

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5.5 Graphical Representation

Figure 7: Mean plasma concentration vs. time curve of Test and Reference Metformin Hydrochloride (SR) 500mg tablet formulations

600

500

400

300
R
T

200

100

0
0 5 10 15 20 25
Time (hr)

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Figure 8: Mean of Log plasma concentration of Test and Reference Metformin Hydrochloride (SR) 500mg tablet formulations

6.5

6.0

5.5

R
T
5.0

4.5

4.0
0 2 4 6 8 10 12 14 16 18
Time (hr)

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5.6 Estimation of Pharmacokinetic Parameters

The rate of absorption was determined by Cmax and tmax. The adequacy of the
sampling time was determined by the ratio (AUC0-t / AUC0-) %. The half life of
elimination t1/2 and the rate of elimination Kel were used to further characterize the
pharmacokinetics outcome of this study. The extent of absorption was determined by
using AUC0-t and AUC0- of Metformin hydrochloride SR 500 mg.

AUC0-t, AUC0- and Cmax values of Metformin hydrochloride SR 500 mg were used
for bioequivalence estimations.

Pharmacokinetic parameters like Cmax, tmax, t , AUC0-t, AUC0- and Kel were
calculated using plasma concentration vs. time profile of both investigational products
using WinNonlin Version 5.2. These data were statistically analyzed and the
bioequivalence of test and reference formulations were proved.

Administration of the reference preparation, tablet Dibeta SR as a single dose in the

fasting state produced the maximum plasma concentration of 621.10 126.65ng/ml
(Cmax) at the time 4.83 1.46hour (tmax) whereas the test preparation of tablet
Metformin hydrochloride SR as a single dose in the fasting stage produced the
maximum plasma concentration of 621.18 112.09ng/ml (Cmax) at the time 5.25
1.65hour (tmax).

Administration of the reference preparation, tablet Dibeta SR produced the plasma

elimination half life t1/2 3.82 0.44hr. whereas the test preparation of tablet Metformin
hydrochloride SR produced plasma elimination half life t1/2 3.78 0.66hr.

Administration of the reference preparation, tablet Dibeta SR produced the area under
plasma concentration time curve (AUC0-t) 5768.22 1425.27ng hr/ml, whereas the
test preparation of tablet Metformin hydrochloride SR produced the area under
plasma concentration time curve (AUC0-t) 5924.45 1290.10ng hr/ml.

When administered as a single dose in the fasting state, the reference preparation,
tablet Dibeta SR, produced the area under plasma concentration time curve up to
infinity (AUC0-) 5960.311439.23ng hr/ml, whereas the test preparation of tablet
Metformin hydrochloride SR produced the area under plasma concentration time
curve (AUC0-) 6159.181354.45ng hr/ml.

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When administered as a single dose in the fasting state, the reference preparation,
tablet Dibeta SR, produced AUC0-t /AUC0- (%) of 96.71 2.00%, whereas the test
preparation of tablet Metformin hydrochloride SR produced AUC0-t /AUC0- (%) of
96.23 1.20

Administration of the reference preparation, tablet Dibeta SR produced the plasma

elimination Constant (Kel) 0.18 0.021/hr, whereas the test preparation of tablet
Metformin hydrochloride SR produced plasma elimination Constant (Kel) 0.19 0.03
1/hr.

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Table 25: Pharmacokinetic Parameters of Test Product and Reference Standard of the Study Drug
AUC0-t/AUC0-
AUC0-t (ng hr/ml) AUC0- (ng hr/ml) Cmax (ng/ml) tmax (hr) Kel (1/hr) T1/2 (hr) (%)

Subject
No. Test Reference Test Reference Test Reference Test Reference Test Reference Test Reference Test Reference

1 8037.08 6606.88 8498.24 6711.47 827.81 531.61 6.00 4.00 0.22 0.19 3.20 3.65 94.57 98.44

2 4680.02 4654.37 4841.34 4931.23 551.22 540.63 5.00 6.00 0.24 0.18 2.90 3.82 96.67 94.39

3 8128.28 6468.62 8407.66 6561.09 709.38 811.57 4.00 4.00 0.13 0.20 5.20 3.46 96.68 98.59

4 6153.68 5714.70 6375.59 5852.69 619.93 568.32 4.00 5.00 0.17 0.18 4.18 3.80 96.52 97.64

5 6995.02 4111.06 7163.32 4211.99 665.41 533.14 5.00 4.00 0.20 0.21 3.42 3.25 97.65 97.60

6 5464.39 5983.10 5705.61 6509.81 697.61 636.21 7.00 4.00 0.24 0.16 2.85 4.21 95.77 91.91

7 5595.07 6487.58 5926.66 6746.39 552.37 520.93 6.00 8.00 0.20 0.17 3.54 4.16 94.40 96.16

8 5479.86 2699.60 5641.54 2798.27 521.69 503.15 4.00 3.00 0.17 0.18 4.02 3.89 97.13 96.47

9 4315.02 5383.26 4418.55 5495.35 401.81 587.10 3.00 6.00 0.18 0.18 3.94 3.80 97.66 97.96

10 4887.00 8212.56 5179.73 8302.02 653.97 915.92 6.00 5.00 0.19 0.23 3.72 3.06 94.35 98.92

11 4695.21 6385.60 4831.00 6613.97 551.74 674.38 4.00 3.00 0.17 0.17 4.07 4.13 97.19 96.55

12 6662.79 6511.29 6920.94 6789.42 701.22 630.25 9.00 6.00 0.16 0.15 4.39 4.66 96.27 95.90

Mean 5924.45 5768.22 6159.18 5960.31 621.18 621.10 5.25 4.83 0.19 0.18 3.79 3.82 96.24 96.71

SD 1290.10 1421.27 1354.45 1439.23 112.10 126.66 1.66 1.47 0.03 0.02 0.67 0.44 1.21 2.00

CV(%) 21.78 24.64 21.99 24.15 18.05 20.39 31.59 30.35 17.43 11.86 17.60 11.55 1.26 2.07

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The various mean pharmacokinetic parameters estimated for both the Reference and
Test Product for Metformin Hydrochloride SR 500 mg are described in tables given
below:

Table 26: Primary Pharmacokinetic Parameters of Test and Reference product

of the Study Drug
Mean SD

Parameters Test Reference

AUC0-t (ng
5924.45 1290.10 5768.22 1425.27
hr/ml)

AUC0-(ng
6159.181354.45 5960.311439.23
hr/ml)

Cmax (ng/ml) 621.18 112.09 621.10 126.65

Table 27: Secondary Pharmacokinetic Parameters of Test and Reference

product of the Study Drug
Mean SD

Parameters Test Reference

tmax (hrs) 5.25 1.65 4.83 1.46

AUC0-t/AUC0-
96.23 1.20 96.71 2.00
(%)

t1/2 (hr) 3.78 0.66 3.82 0.44

Kel (1/hr) 0.19 0.03 0.18 0.02

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5.7 Confidence Intervals

As shown in the Table given below, the 90% confidence intervals were constructed
for the ratios of the geometric least square mean of ln-transformed pharmacokinetic
parameters Cmax, AUC0-t and AUC0- for test and reference formulations.
Bioequivalence was concluded, as the 90% confidence intervals for Cmax, AUC0-t and
AUC0- fall within the acceptable range of 80125%.

The 90% confidence interval for ln-transformed Cmax was 89.13%-112.44%. The
90% confidence interval for ln-transformed AUC0-t was 87.460%-123.850%.The
90% confidence interval for ln-transformed AUC0- was 88.290%-123.870%.

The Mean T/R ratio (%) for ln-transformed Cmax, AUC0-t and AUC0- were
100.110%, 104.080% and 104.580% respectively. Table given below describes the
Geometric mean value; 90% confidence Interval and T/R ratio (%) of the ln-
transformed parameters.

Table 28: Pharmacokinetic Bioequivalence Parameters of Test and Reference

product of the Study Drug
Pharmacokinetic Geometric Mean Point 90 % Confidence
Parameter Estimator (%) Interval
Reference Test Test/Reference Lower Upper
AUC0-t (ng hr/ml) 5574.334 5801.763 104.080 87.460 123.850
AUC0-(ng hr/ml) 5765.003 6028.953 104.580 88.290 123.870
Cmax (ng/ml) 610.745 611.410 100.110 89.130 112.440

5.8 Analysis of Variance

Analysis of variance (ANOVA) was performed (=0.05) on the untransformed and

ln-transformed pharmacokinetic parameters Cmax, AUC0-t and AUC0-.
Factors included for Analysis of variance (ANOVA) model were randomization
sequence, subjects included in that particular sequence, period and treatment effect.
These factors for both untransformed and ln-transformed pharmacokinetic
parameters were found to be statistically insignificant, as shown in the Table 29
below.

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Table 29: Summary of ANOVA Results obtained from Test and Reference
product of the Study Drug
Log Transformed Cmax Analysis-ANOVA
Source DF S.S M.S.S F stat P value
Sequence 1 0.008 0.008 0.110 0.753
Subject (Seq) 9 0.645 0.072 4.070 0.024
Treatment 1 0.007 0.007 0.410 0.537
Period 1 0.062 0.062 3.500 0.094
Error 9 0.159 0.018 -- --
Total 21 0.880 0.166 8.090 1.408
Log Transformed AUC0-t Analysis-ANOVA
Source DF S.S M.S.S F stat P value
Sequence 1 0.002 0.002 0.040 0.855
Subject (Seq) 9 0.418 0.046 3.040 0.057
Treatment 1 0.005 0.005 0.300 0.598
Period 1 0.002 0.002 0.110 0.751
Error 9 0.138 0.015 -- --
Total 21 0.563 0.070 3.490 2.262
Log Transformed AUC0- Analysis-ANOVA
Source DF S.S M.S.S F stat P value
Sequence 1 0.001 0.001 0.030 0.867
Subject (Seq) 9 0.412 0.046 3.110 0.053
Treatment 1 0.005 0.005 0.310 0.590
Period 1 0.001 0.001 0.050 0.822
Error 9 0.133 0.015 -- --
Total 21.00 0.552 0.067 3.500 2.332
DF = Degree of freedom: S.S = Sum of Squares: M.S.S = Mean Sum of square,
F = M.S.S/Error M.S.S, P = Probability

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5.9 Safety Assessment

5.9.1 Vital Signs Measurements

Sitting blood pressure, radial pulse rate, oral temperature and respiration rate was measured after
admission, prior to dosing and at 2,6,12, 18 and 24 hours post-dosing in each period. Vital signs
were measured with the subject in sitting posture and within 45 minutes of the scheduled time as
per the SOP. No major fluctuation(s) in sitting blood pressure, pulse rate and body temperature
was observed in any subjects in both the periods of the study.

Table 30: Deviations in Vital Signs Measurements

S.No. Subject Period No Time Point 2, 6, Early/ Delay * Reason

No 12, 24, 48 hrs /missing
(1& 2)

BP, Pulse rate, Temp and No abnormalities

Respiration rate

Reason *

A: Subject did not come for ambulatory visit

B. subject arrived late

C. Subject withdrawn

D subject dropped out

5.9.2 Medical Examination

Whole body medical examinations of the subjects were conducted by a medically qualified
person on duty after subject admission, prior to dosing of study drug, once in every 12 hours post
dose in in-house part of the study and at discharge. No major complaints were observed in any
subjects in both the periods of the study.

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5.9.3 Assessment of Well Being

Subjects well being were assessed at admission, prior to dosing and once in every 4 hrs post dose
(except at 16 and 20 hrs), at discharge in each period and at each ambulatory visit. No major
complaints were observed in any subjects in both the periods of the study.

Blood glucose level was monitored at the interval of 1 hrs after drug administration till 4 hrs of
drug administrations.

5.9.4 Evaluation of Blood Glucose

Blood glucose was monitored by finger prick method at the interval of 1 hr after drug
administration till 4 hrs. post dose.
Table 31: Blood glucose monitoring Period I
Blood Glucose Concentration (mg/dl) Post Drug Administration in
Hour

Subject No. 1 2 3 4

1 166 186 112 105

2 142 121 110 108

3 125 121 115 105

4 127 123 120 110

5 186 154 120 118

6 119 129 110 105

7 162 146 153 142

8 108 127 152 122

9 105 140 105 110

10 141 157 116 110

11 166 157 130 120

12 188 190 140 120

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Table 32: Blood glucose monitoring Period II

Blood Glucose Concentration (mg/dl) Post Drug Administration in
Hour

Subject No. 1 2 3 4

1 141 130 110 110

2 120 110 108 112

3 137 120 116 110

4 130 120 110 110

5 170 126 104 100

6 137 129 116 110

7 116 112 108 104

8 170 140 120 110

9 182 160 140 110

10 187 160 140 110

11 119 110 108 100

12 122 118 110 104

5.9.5 Adverse Events (AE)

Adverse Event is defined as any untoward medical occurrence in a patient or clinical

investigation subject administered a pharmaceutical product and which does not necessarily have
a causal relationship with this treatment. An adverse event (AE) can therefore be any
unfavourable and unintended sign (including an abnormal laboratory finding), symptom, or
disease temporally associated with the use of a medicinal (investigational) product, whether or
not related to the medicinal (investigational) product

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The study subjects were asked to inform the clinical staff of occurrence of any AEs immediately
once experienced. Furthermore, the clinical staff was instructed to check on the subjects for the
occurrence of any AE at specified time intervals (before dosing and 4.00, 8.00, 12.00 and 24.00
hrs from study drugs administration) and to notify immediately to the Clinical Investigator.
Table below describes the incidence of adverse events observed from the time of admission of
drug till the time of discharge. No Adverse Event was reported in both the Periods of the Study.

Table 33: Incidence of Adverse Events

Body System / Adverse Event Period 1 Period 2

Lactic Acidosis - -

Abdominal discomfort - -

Diarrhea - -

Gas - -

Headache, - -

Indigestion - -

Nausea - -

Vomiting - -

Weakness - -

Others - -

5.9.6 Safety Results

Both the formulations (Test and Reference) were well tolerated. No AE/SAE/Death was reported
during the conduct of study.

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5.10 Discussion

Bioequivalence study is usually carried out, by comparing the in vivo rate and extent of drug
absorption, of test and reference formulation, in healthy subjects.

The present study was an open label, balanced, randomized, two treatments, two periods, two
sequence, single dose, cross over bioequivalence study under fasting condition.

The following steps were incorporated into the study to minimize the bias:

Volunteer admission was dependent on satisfactory fulfillment of the inclusion and

exclusion criteria as mentioned in the study protocol approved by the ethics committee.
Subjects were randomly assigned to the two possible sequences (test-reference or
reference- test) for the administration of test and reference formulation.
Bioanalytical Investigators were kept blinded to the randomization schedule throughout
the study duration.

In this particular study, study participants received test and reference products on separate
occasions, as a single dose, with random assignment to the two possible sequences of product
administration. Samples of plasma were analyzed for drug concentrations, and pharmacokinetic
parameters were obtained from the resulting concentrationtime curves. These pharmacokinetic
parameters were then analyzed statistically to determine whether the test and reference products
yielded comparable values.

The statistical methodology was based on the calculation of a 90% confidence interval for the
ratio (or difference) between the test and reference product pharmacokinetic variable averages.
The limits of the observed confidence intervals were within a predetermined range for the ratio
(or difference) of the product averages.

An Analysis of variance (ANOVA) test was performed (=0.05) on the untransformed and ln-
transformed pharmacokinetic parameters Cmax, AUC0-t and AUC0-. Factors included for
Analysis of variance (ANOVA) model were randomization sequence, subjects included in that

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particular sequence, period and treatment effect. These factors for both untransformed and ln-
transformed pharmacokinetic parameters were found to be statistically insignificant.

Since, this was a DCGI submission study, so all the target parameters were chosen in accordance
with Guidelines for Bioavailability and Bioequivalence Studies issued by Central Drugs
Standard Control Organisation (CDSCO), March 2005.

The study was conducted as per the IEC approved study protocol, CDSCO BA/BE Guidelines,
March 2005, ICH-GCP Guidelines, Schedule Y to the Drugs and Cosmetic Rules, Indian GCP
Guidelines issued by CDSCO, GLP, Ethical Guidelines for Biomedical research on human
subjects issued by Indian Council of Medical Research (ICMR Guidelines) and Declaration of
Helsinki.

Hippocrates Independent Ethics Committee (HIEC), New Delhi has given the approval to
conduct this study and allowed to enroll twelve subjects for this particular study. HIEC were
regularly informed regarding the updates of this study.

Food and Drug Administration (FDA) provided the draft guidance on Metformin Hydrochloride
SR 500 mg for conducting the bioequivalence study (FDA Draft Guidance on Metformin, 2008).
It described that bioequivalence study of Metformin should be conducted in fasting condition
and design should be single-dose, two-way, crossover in-vivo in normal healthy males and
females, general population. On the basis of these considerations, a single-dose, two-treatment,
two-period, two- sequence crossover bioequivalence study on healthy human male normal
subjects was adopted.

As a result to its crossover design, no separate control group was necessary. 500 mg Metformin
Hydrochloride (SR) dose was expected to be reasonably well tolerated. It was also expected to
provide adequate plasma concentrations to be measured for assay.

The choice of the blood sampling time schedule was intended to cover the expected time of the
peak plasma concentrations of metformin, which was at around 7(4-8) hour (Drugs @ FDA,
2008). It was also extended for sufficient time to be able to obtain a reliable estimate of the
extent of absorption. The last sample collection was at 24.00 hours post-dose, which was more

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than 3 times the maximum reported half-life of metformin 6.2 hours (Drugs @ FDA, 2008), and
covered at least 80% of the AUC0-.

Pharmacokinetic parameters like Cmax, tmax, t. AUC0-t, AUC0-, & Kel were calculated using
plasma concentration vs. time profile (actual time of sample collection) data of both
investigational products in individual subjects using WinNonlin Version 5.2. These data were
statistically analyzed and described.

Cmax is the measure that represents the major drug concentration observed and is directly
proportionate to the total drug amount absorbed by the body. The Cmax is generally higher when
the extent of drug bioavailability is greater. When comparing drug products, t max can be used as
an approximate indication of drug absorption rate. The value for tmax will become smaller
(indicating less time required to reach peak plasma concentration) as the absorption rate for the
drug becomes more rapid (Shargel & Andrew, 2004).

When pharmacokinetics parameters of the test drug of Sponsor were compared with reference
drug Dibeta SR 500 mg tablet formulation, it was found that Sponsor generic version have
almost same pharmacokinetic parameters.

Administration of the sponsors generic tablet produced the maximum plasma concentration of
621.18 112.09 ng/ml (Cmax) at the time 5.25 1.65 hour (tmax) whereas Dibeta SR tablet
(reference drug) peak concentrations were typically 621.10 126.65 ng/ml at the time 4.83 1.46
hour (tmax) following a single and a repeated 500 mg once daily dose, respectively. In adults,
peak plasma concentrations were achieved 7 (4-8) hour after administration of the oral tablet
(Drugs @ FDA, 2008).

The geometric least square mean ratios and the 90% CI of Cmax, AUC0-t and AUC0- for the test
product and the reference standard of the study drug were considered, for evaluating
bioequivalence. To be considered bioequivalent, ratios and confidence interval should lie within
the acceptance range of 80-125%.
Since, Pharmacokinetic Bioequivalence Parameters (Cmax, AUC0-t, AUC0-) of the Test Product
and the Reference Standard of the Study Drug were within the acceptance range of 80-125%. So
this study proves that, the test product Metformin hydrochloride (SR) 500 mg tablet is
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bioequivalent to the reference standard Dibeta SR tablet (containing Metformin hydrochloride

(SR) 500 mg).
6. Conclusion

The present study was an open label, balanced, randomized, two treatment, two period, two
sequence, single dose, cross over bioequivalence study under fasting condition; conducted at
Auriga Research Ltd., New Delhi. Metformin Hydrochloride (SR) 500 mg tablet was the Test
formulation and Dibeta SR tablet [containing Metformin Hydrochloride (SR) 500 mg] was used
as the Reference standard.

As per CDSCO BA/BE Guidelines, the minimum number of subjects for bioequivalence study
should not be less than sixteen unless justified for ethical reasons. Hippocrates Independent
Ethics Committee (HIEC), New Delhi has given the approval to Auriga Research Ltd. to conduct
this study and allowed to enroll twelve subjects for this particular study based on ethical reason.

The 90% confidence interval for ln-transformed pharmacokinetic parameters (Cmax, AUC0-t and
AUC0-) of the Test and Reference formulation of the Study drug Metformin Hydrochloride (SR)
500 mg were 89.13%-112.44%, 87.46%-123.85%, 88.29%-123.87% respectively. The Mean
Test/Reference ratio (%) for ln-transformed Cmax, AUC0-t and AUC0- were 100.110%, 104.080%
and 104.580% respectively.

Results showed that ln-transformed pharmacokinetic parameters (Cmax, AUC0-t and AUC0-) were
within the acceptable range of 80125%. So, the present study concludes that the Test
formulation Metformin Hydrochloride (SR) 500 mg tablet of Sponsor is bioequivalent to
Reference standard Dibeta SR tablet [containing Metformin Hydrochloride (SR) 500 mg] of
Torrent Pharmaceuticals Ltd., India.

7. References

1) Guidance for Industry: Bioavailability and Bioequivalence Studies for Orally Administered
Drug Products-General Considerations. U.S. Department of Health and Human Services,
Food and Drug Administration (FDA), Centre for Drug Evaluation and

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Research(CDER),March2003. Available at http://www.fda.gov/cder/guidance/index.html.

[Cited on 2009 Sep 6]
2) Spilker B, Spilker. Guide to Clinical Trials, 1st ed. Amazon: Lippincott Williams &
Wilkins; 1991. p. 347
3) Park K. Preventive and Social Medicine. 20th ed. Jabalpur: M/s Banarsidas Bhanot; 2009.
Ch. 6, Epidemiology of Chronic Non-Communicable diseases and Conditions; p. 341-45.
4) Dipiro JT, Talbert RL, Yee GC, Matzke GR. Pharmacotherapy: A Pathophysiologic
Approach, 6th ed. New York: MCGRAW-HILL Medical Publishing Division; 2005.
Ch.72, Diabetes Mellitus; p. 1334.
5) Manual for Good Bioavailability and Bioequivalence Practices, Volume I Module 1:
Clinical Phase. 1st ed. Brazil; 2002. p. 7. Available at http://www.anvisa.gov.br /index.html.
[Cited on 2009 Oct 28]
6) Rang HP, Dale MM, Ritter JM, Flower RJ. Rang and Dales Pharmacology, 6th ed.
Elsevier: Churchill Livingstone; 2007. Ch.56, Drug discovery and development; p.781-84.
7) Brahmankar DM, Jaiswal SB. Biopharmaceutics and Pharmacokinetics A Treatise, 1st ed.
Delhi: Vallabh Prakashan; 1995. Ch. 9 Pharmacokinetics: Basic Considerations; p. 212-15.
8) Manual for Good Bioavailability and Bioequivalence Practices, Volume I Module 3:
Statistical Phase. 1st ed. Brazil; 2002. p. 6-8. Available at http://www.anvisa.gov.br
/index.html. [Cited on 2009 Nov 11]
9) Brahmankar DM, Jaiswal SB. Biopharmaceutics and Pharmacokinetics A Treatise, 1st ed.
Delhi: Vallabh Prakashan; 1995. Ch. 10 Compartment Modeling; p. 233-35.
10) Brahmankar DM, Jaiswal SB. Biopharmaceutics and Pharmacokinetics A Treatise, 1st ed.
Delhi: Vallabh Prakashan; 1995. Ch. 9 Pharmacokinetics: Basic Considerations; p. 218-19.

11) U.S. Department of Health and Human Services Food and Drug Administration Center for
Drug Evaluation and Research (CDER). Guidance for Industry Bioavailability and
Bioequivalence Studies for Orally Administered Drug Products General Considerations.
Available at
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidan
ces/ucm070124.pdf [Cited on 2009 Nov 18]

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12) Manual for Good Bioavailability and Bioequivalence Practices, Volume I Module 3:
Statistical Phase. 1st ed. Brazil; 2002. p. 13-19.
13) BCC Research. World Pharmaceutical Market, Report Code: PHM037A, Published: April
2009.Available at http://www.bccresearch.com/report/PHM037A.html. [Cited on 2010 Apr
3]
14) Central Drugs Standard Control Organization, Directorate General of Health Service,
Ministry of Health & Family Welfare, Government of India. Guideline for Bioavailability
& Bioavailability Studies, March 2005. Available at http://www.cdsco.nic.in/ [cited 2009
Aug 19]
15) Clinical Research Organization-India. Available at http://www.neeman-
medical.com/resources/clinicalresearch/dmc/edc.pdf [Cited 2010 April 19]
16) DiPiro JT, Talbert RL, Yee GC, Matzke GR. Pharmacotherapy: A Pathophysiologic
Approach, 6th ed. New York: MCGRAW-HILL Medical Publishing Division; 2005.
Ch.72, Diabetes Mellitus; p. 1335-36.
17) World Health Organization. Definition, diagnosis and classification of diabetes Mellitus
and its complications-Report of a WHO Conclusion. Geneva: World Health Organization;
1999.
18) DiPiro JT, Talbert RL, Yee GC, Matzke GR. Pharmacotherapy: A Pathophysiologic
Approach, 6th ed. New York: MCGRAW-HILL Medical Publishing Division; 2005.
Ch.72, Diabetes Mellitus; p. 1337-42.
19) Tripathi KD. Essentials of Medical Pharmacology, 6th ed. New Delhi: Jaypee Brothers;
2008. Ch. 19, Insulin, Oral Hypoglycaemic Drugs And Glucagon; p. 266-70.
20) DiPiro JT, Talbert RL, Yee GC, Matzke GR. Pharmacotherapy: A Pathophysiologic
Approach, 6th ed. New York: MCGRAW-HILL Medical Publishing Division; 2005.
Ch.72, Diabetes Mellitus; p. 1343-56.

21) FDA label of reference listed drug of Metformin hydrochloride SR. Available at
http://www.accessdata.fda.gov/scripts/cder/drugsatfda/. [Cited on 2009 Nov 15]
22) Michael RM, Dean TE, Sumit RM, James DL, Bhagra S, Pardeep S J, Mark CP, John J V,

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John RP, Finlay AM. Diabetes care. 2010 Jun; 33(6): 1213-8
23) Donnelly LA, Morris AD, Pearson ER. Diabetes, obesity & metabolism. 2009 Apr; 11(4):
338-42.
24) Shelley RS, Nicholas SB, Justin AK, Edwin ES. The American journal of medicine. 2008
Feb;121(2): 149-157.e2
25) Information available on Toxnet drug information site. Available at
http://www.toxnet.nlm.nih.gov/ [Cited on 2009 Nov 17]

Annexure-I: Ethics Committee Approval Letter

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Annexur-II: Study Design

Period I 0 hr Sampling Hrs

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 12.0 18.0 24.0

Screening
Incl./Excl.
Critaria

2 3 4 5 6 7 8 9 10 11 12 13 14
Day -21 to 0 Predose

Day 0 Blood Samples

Study Administration of Day 1 to 2 Study

Initiation Test/Reference Drug Continued

At least 07 days wash out

Period II same as Period I

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ANNEXURE III: TIMETABLE OF EVENTS

The following is a representative time schedule for one subject assuming that the study
medication was administered at 09:00. There was a gap of 07 days between two
periods. Timings for other subjects were uniformly staggered:

Day Time Approximat Events

Relative to e Time
Dosing
Informed Consent procedure (only in period-I),
- 18.00 to
0 18:00-22:00 Admission of the subjects, Vitals Measurement /
12.00 hr
Assessment of well being / Clinical Examination
0 - 12.00 hr 22:00 Dinner
0 - 10.50 hr 22:30 Bed time
1 -2.5 hr 05:30 Wake up call
Vitals Measurement / Assessment of well being /
1 -1.50 hrs 07:30 Clinical examination / Insertion of Cannula / pre-
dose blood sampling
1 0.00 hr 09:00 Dosing
1 0.25 hr 09:15 Blood sample collection
1 0.50 hr 09:30 Blood sample collection,
1 0.75 hr 09:45 Blood sample collection
1 1.00 hr 10:00 Blood sample collection
1 1.25 hr 10:15 Blood sample collection,
1 1.50 hr 10:30 Blood sample collection
1 2.00 hr 11:00 Blood sample collection, vitals measurement
1 2.50 hr 11:30 Blood sample collection
1 3.00 hr 12:00 Blood sample collection
Blood sample collection, Assessment of well being
1 4.00 hr 13:00
and Lunch
1 6.00 hr 15:00 Blood sample collection, Vitals measurement
1 8.00 hr 17:00 Blood sample collection, Assessment of well being
1 9.00 hr 18:00 Snacks
Blood sample collection, Vitals measurement /
1 12.00 hr 21:00 Assessment of well being / Clinical Examination/
Removal of cannula
1 13.00 hr 22:00 Dinner,
Blood sample collection, Vitals measurement /
2 24.00. hr 09:00
Assessment of well being / Clinical Examination
Ambulatory Samples / Vitals measurement /
3 48.00 hr 09:00 Assessment of well being / Safety sample in Period
II only

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ANNEXURE-IV: SCHEMATIC REPRESENTATION OF COURSE OF

EVENTS
The following time-and-events table illustrates the planned schedule of assessments:

Activity Screening Period 01 Period 02 Post-study

safety
assessment

Review of
X X - -
inclusion/exclusion criteria

Demographic examination X - - -

Clinical examination X X X -

Blood sample for

X - - X
Biochemistry

Blood sample for

X - - X
Haematology

Blood sample for serological

X - - -
examination

Urine analysis X - - -

Electrocardiogram X - - -

Chest X-Rays* X - - -

Concomitant medications X X X -

Adverse Events monitoring - X X X

*Chest X-Ray will be taken for those volunteer whose X-Ray was taken more than 6
months prior to the dosing of Period 01.

**X refers that the corresponding activity has been done during the assessment.

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ANNEXURE-V: HeightWeight Chart for NonMedical Cases for Men and

Women
(Life Insurance Corporation of India)

Weight( Age (Years)

Kg)
18-22 23-27 28-32 33-37 38-42 43-45
Height
(cm) 1
Ideal weight range in Kg
Min Max Min Max 2 M Max Min Max Min Max Min Max
i
n
140-145 41 52 42 53 43 56 43 57 43 58 43 59

146-150 43 54 43 56 44 59 45 60 45 60 45 61

151-155 45 56 44 58 45 62 47 63 47 64 47 64

156-160 48 59 48 61 48 65 50 67 50 67 50 68

161-165 51 62 51 65 51 69 53 70 53 71 53 72

166-170 54 66 54 69 55 73 56 75 56 75 56 76

171-175 58 71 58 73 58 78 60 80 60 80 60 81

176-180 61 75 62 78 62 83 64 85 64 86 64 87

181-185 65 80 65 83 66 88 63 90 68 92 68 93

186-190 69 85 69 89 70 94 72 96 72 97 72 98

2.1 CHART OF OVER & UNDER WEIGHT

Height (cm) 145 150 155 160 165 170 175 180 185

Over Weight (Kg) 60 62.5 66 70 74.5 79 83.5 88.5 94

Under Weight (Kg) 34.5 37 39.5 42 44.5 47.5 50.5 54 57.5

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ANNEXURE-VI: SRL Lab Values

Sr.No. Name of the Tests Normal Range Unit Significant test value Below Range Above Range
1 Haemoglobin (Hb) 13-17 mg/dl 20% 10.4 17
2 Total Leukocytes Count (TLC) 4000-10000 /cumm 20% to 20% 3200 12000
3 Neutruphil 40-80 % 20% to 20% 32 96
4 Lymphocytes 20-40 % 20% to 20% 16 48
5 Eosinophil 1.0-6.0 % 50% 1 9
6 Monocytes 2.0-10.0 % 50% 2 15
7 Basophil 0.0-2.0 % 50% 0 3
8 Platelet Count 150-410 Thousand/cumm 20% 120 410
9 Blood Sugar (F) 47-100 mg/dl N/AP 74 100
10 Total Cholesterol 00.00-200 mg/dl 20% 200 286.8
11 Blood Urea (BUN) 07-18 mg/dl 20% 7 21.6
12 Creatinine 0.9-1.3 mg/dl > Normal Range 0.9 1.3
13 Total Bilirubin 0.0-1.0 mg/dl 20% 0 1.2
14 S.G.O.T. (AST) 15-37 50% 20% 15 55.5
15 S.G.P.T. (ALT) 30-65 50% 20% 30 97.5
16 Alkaline Phosphatase 50-136 50% 20% 50 204
17 Total Protein 6.4-8.2 mg/dl 20% 5.1 8.5
18 V.D.R.L. None Reactive N/AP N/AP None Reactive None Reactive
19 H.I.V. & H.C.V. None Reactive N/AP N/AP None Reactive None Reactive
20 HBsAg None Reactive N/AP N/AP None Reactive None Reactive

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21 Drugs of Abuse (Urine) Negative N/AP N/AP Negative Negative

22 Breath Analysis (Alcohol) Negative N/AP N/AP Negative Negative

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 Abstract

ANNEXURE -VII: INFORMED CONSENT FORM (ENGLISH)

I _________________________________________ hereby willingly agree to participate in the
following clinical research study:

Study Title: An open label, balanced, randomized, two treatment, two period, two sequence,
cross over, bioequivalence study of a single dose of Metformin Hydrochloride (SR) 500 mg
tablet of Sponsor Cipla Ltd and Dibeta SR (containing Metformin Hydrochloride (SR) 500 mg)
tablet of Torrent Pharmaceuticals Ltd, India, in healthy human adult male subjects under fasting
condition.

Please check Box ( / X ) *

1. I confirm that I have read and understood the information provided to me regarding the study [ ]
and have had the opportunity to ask questions.

2. I declare that there has been no compulsion or monetary inducement in my agreeing to be

subject for this project, which I do of my free will. [ ]

3. The general purpose of the experiment has been explained to me and I am convinced that it is [ ]
for the benefit of science and mankind.

4. I am aware of my responsibilities as a clinical trial participant.

[ ]
5. I have also been told about the risks involved.
[ ]
6. I understand that the Sponsor of the clinical trial, other working on the Sponsor's behalf, the
Ethics Committee and the regulatory Authorities will not need my permission to look at my [ ]
health records both in respect of the current study and any further research that may be
conducted in relation to it, even if I withdraw from the trial. I agree to this access however, I
understand that my identity will not be revealed in any information released to the third parties
or published
[ ]
7. I have also been told that I have to undergo following procedures during the experiment

Collection of blood samples

[ ]
Collection of urine samples

Ingestion of Drugs

To remain under observation for prolonged periods

To remain fasting for prolonged periods

[ ]
8.I agree not to restrict the use of any data or results that arise from this study provided such a
use is only for scientific purpose

9. I have informed my family regarding my participation in this trial and I understand that I can [ ]
withdraw from the study at any time. I have been informed that i will be compensated for my
participation

10.I agree to take part in the above study [ ]

 Abstract

* To be filled by Study Volunteer

Any Query __________________________________________________________________

Volunteers Name Volunteers Signature/ Date

Name of the Person Obtaining ICF Signature / Date of the Person obtaining
ICF

Witnesss Name Witnesss Signature/ Date

Clinical Investigators Name Clinical Investigators Signature

with date
 Abstract

ANNEXURE-VIII: Study Meals Menu with Recipe

1. Dinner (Admission Day)

S.No. Food Item Quantity Cal. CHO Prot. Fat

(g) /(ml) /
No. (K Cal) (g) (g) (g)
Kala Chana
1 Curry 125 145 18.1 8.2 4.4
2 Paneer Curry 180 163 5.4 8.7 12
3 Curd 100 60 3 3 4
4 Chapati 4 x 35 273 55.4 9.7 1.4
5 Rice 100 86 19.6 1.7 0.1
6 Aloo Beans 125 132 24.6 1.7 3
7 Kheer 100 169 25.4 5.7 5
Total 1028 151.5 38.7 29.9
Total
Calories 606 154.8 269.1
% of
Calories 58.95 15.05837 26.17704

2. Lunch (Dosing Day)

S.No. Food Item Quantity Cal. CHO Prot. Fat

(g) /(ml) /
No. (K Cal) (g) (g) (g)
1 Chana Masala 125 169 27.6 7.6 3.1
2 Palak Paneer 150 141 8.3 9 8.1
3 Aloo Gobhi 125 108 17.6 3.9 2.3
4 Mix Raita 150 72 5.3 3.2 4
5 Chapati 3X35 205 41.6 7.3 1
6 Rice 150 129 29 2.5 0.1
Vanilla
7 Icecream 1 small cup 145 11.5 2.5 10
8 Banana 110 128 29.9 1.3 0.3
Total 1097 170.8 37.3 28.9
Kcal 683.2 149.2 260.1
% of Total
Kcal 62.3 13.6 23.7
 Abstract

3. Snacks (Dosing Day)

S.No. Food Item Quantity Cal. CHO Prot. Fat

(g) /(ml) /
No. (K Cal) (g) (g) (g)
1 Frooti 200 80 20 0 0
Cheese
2 Toast 95 292 35.5 12 12
Total 372 55.5 12 12.0
Total
Calories 222 48 108
% of
Calories 59.7 12.90 29.03

4. Dinner (Dosing Day)

S.No. Food Item Quantity Cal. CHO Prot. Fat

(g) /(ml) /
No. (K Cal) (g) (g) (g)
1 Dry Urad 125 133 20.5 7.5 2.3
2 Pea Paneer 150 151 7.4 8 10
3 Aloo Methi 125 162 25.8 2.8 5.3
4 Raita (mix.) 140 75 6.3 3.6 4
Vegetable
5 Rice 250 235 48.6 4.1 2.7
6 Chapati 4x 35 273 55.4 9.7 1.4
7 Salad 60 0 0 0 0
Total 1029 164 35.7 25.7
Total
Calories 656 142.8 231.3
% of
Calories 63.8 13.88 22.48
 Abstract

ANNEXURE-IX: Reporting of Adverse Event

Subject No. Study Drug Study No.

Period No. Date of Dosing Time of Dosing

ADVERSE EVENT

Adverse Event Start Date Reporting Date and Time Method of Reporting:
and Time Spontaneous
Date: Date: Observed
Elicited
Time: Time:
Description of Adverse Event Including Medication and Advice

Resolution: Yes No If Yes, Resolution Time & Date:

Completed by: _______________ Date & Time:

___________

This section is to be filled in the last either by Clinical Investigator or Principal Investigator
Please choose and mark one of each below to rate the event
Adverse Event End Date and Time: No. of follow-up sheets used:
Seriousness Frequency Severity Relationship to Action taken Outcome
1 = Serious 1 = isolated 1 = mild Study drug 1 = none 1 = recovered
2 = Non 2 = intermittent 2 = moderate 1 = certain 2 = medication without sequelae
Serious 3 = continuous 3 = severe 2 = probable / therapy 2 = recovered
4 = unknown 4= Life- likely 3 =procedure with Sequelae*
Expectedne threatening 3 = possible 4 = hospitalization 3 = ongoing
ss: 4 = unlikely 5 = other* 4 = death
1= Expected 5 = unclassifiable 5 = other*
2= 6= unclassified
Unexpected

Reviewed by: _______________ Date: ___________

 Abstract

ANNEXURE-X: Reporting of Serious Adverse Event

Subject No. Report Date Phase of study:

Study Drug Study No. Period No.

Report Type: Initial Follow-Up

If Follow-up Date of Previous Report(s):
Investigator Name & Address: Sponsor Name & Address:

A. Subject Information
Enrollment No.: Gender: Male Female Date of Birth:
Age: (completed years) Height: (cm) Weight: (kg)
B. Study Drug (s)
Drug (generic/proprietary Route Duration of Therapy Indication of
name) and Dosage Form / From To Treatment
Dose(e.g. Tab 10 mg OD)

C. Adverse Reaction
Adverse Event Start Date / Time: Adverse Event End Date / Time:
OR Ongoing
Duration of Adverse Event: (days/hours/minutes)
Adverse Event Summary: Diagnosis (if known)

Include clinical history of event, associated signs & symptoms, alternative etiologies being
considered, medical management and relevant past medical history below or attach
additional pages if necessary.

Completed By: _______________ _______________

Reviewed By: ________________ _______________

(Principal Investigator)

Continued.
 Abstract

Serious Adverse Event Reporting Form (Continued)

Subject No. Report Date Phase of study:

Study Drug Study No. Period No.

List relevant abnormal lab results below

Investigations Done to
confirm reaction:

Intensity
Mild Moderate Severe Life- threatening
Please indicate SAE category from the following choices:
Death Persistent or significant disability or incapacity Congenital anomaly or birth defect
life threatening Inpatient hospitalization or prolongation of existing hospitalization
Important medical event (Any event that, based upon appropriate medical judgment, may jeopardize the patient and
may require medical or surgical intervention to prevent one of the outcomes listed above).

Action taken Details of Medication Therapy (If Applicable):

None
Medication therapy
Procedure Hospitalization Other*

Outcome Relationship to Study drug

Recovered without sequelae Recovered with Certain Probable / likely Possible Unlikely
Sequelae* Ongoing Death Other*
Inaccessible / unclassifiable Conditional / unclassified

Comments:

Completed By: _______________

___________

Reviewed By: _______________

___________

(Principal Investigator)
Abstract