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Pharminform AD Replekpharm AD

Project No.: CLOP-REPL-0807 – clopidogrel

CLINICAL STUDY REPORT

A Mono-Center, Open, Two-Sequence Randomized, Two-Way Cross-Over Study of The Bioequivalence of Clopidogrel as a Film-Coated Tablet from One Test Formulation (Klopidogrel 2 x 75mg, film-coated tablets manufactured by Replekpharm AD) and One Reference Formulation (Plavix 2 x 75 mg film-coated tablets manufactured by Sanofi-Aventis), Each Given as a Single Oral Dose to 24 Healthy Subjects in the Fasting State.

Test Drug: Klopidogrel (REPLEKPHARM AD)

Reference Drug: Plavix (SANOFI-AVENTIS.)

Sponsor: Replekpharm AD

CRO: Pharminform AD

Identification code: CLOP-REPL-0807 Phase of study: Bioequivalence

EudraCT-No.: 2007- 005133-11

Study Initiation Date: September 2007

Study Completion Date: March 2008

Principal Investigator: Acad. Prof. PhD. MD. Victor Voicu

Sponsor’s Project Manager: Emilija Spaseska Aleksovska

CRO Authorized Official: Dr. Zlatka Etropolska

This study has been performed in compliance with Good Clinical Practice (GCP), including the archiving of the essential documents.

Date of the report: March 2008

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SYNOPSIS

Name of Sponsor/ Company

REPLEKPHARM AD

Name of CRO

PHARMINFORM AD

Individual Study Table

Referring to Part IV of the Dossier

Name of Finished Product

KLOPIDOGREL

Volume

Name of Active Ingredient

clopidogrel

Page

Title of Study:

A Mono-Center, Open, Two-Sequence Randomized, Two-Way Cross-Over Study of The

Bioequivalence of Clopidogrel as a Film-Coated Tablet from One Test Formulation

(Klopidogrel 2 x 75mg, film-coated tablets manufactured by Replekpharm AD) and One

Reference Formulation (Plavix 2 x 75 mg film-coated tablets manufactured by Sanofi-

Aventis), Each Given as a Single Oral Dose to 24 Healthy Subjects in the Fasting State

Principle Investigator

Acad. Prof. PhD. MD. Victor Voicu

Study centre

National Institute for Aeronautical and Space Medicine within Central Clinical Emergency

Military Hospital

Bucharest, Romania

Publication (Reference) None Studied period (years)

(date of first enrolment) 18.04.2008 (date of last completed) 18.05.2008

Phase of development

Bioequivalence

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Objectives Primary: To compare bioavailability of clopidogrel in healthy volunteers after administration of a single dose of 2 x 75mg, film-coated tablets manufactured by Replekpharm AD as Test formulation and Plavix 2 x 75 mg film-coated tablets manufactured by Sanofi-Aventis as Reference formulation and to assess their bioequivalence.

Clopidogrel itself is inactive. In-vivo, it is being metabolized rapidly. It is metabolized by CYP monoxygeanse (CYP 3A4 and 2B5) to active thiol metabolite (minor). Its major metabolite (85%) is inactive carboxylic acid. The plasma levels of Clopidogrel are difficult to determine as the plasma levels fall immediately (in 2 h!!) due to its major metabolite, inactive carboxylic acid. The levels of active metabolite are also difficult to measure (practically impossible) because it is tightly bound to platelets P2Y12 ADP receptor. The carboxylic acid metabolite is generally used for pharmacokinetic study to determine the exposure. In our study, the carboxylic acid metabolite (inactive) (CCA) was used to determine the exposure to copridogrel. This is due to the fact that the concentration of metabolite will reflect the exposure of parent drug.

Secondary: To monitor possible occurrence of adverse events or adverse drug reactions. Methodology After determining the content of CCA in plasma, based on data for plasma concentration/time, several objective pharmacokinetic parameters will be determined and namely: Primary: Cmax, AUC0-48, AUC0-∞; Secondary: Tmax; Additional: T½, Kel, CL, MRT.

HPLC method for clopidogrel plasma determination was used with the following characteristics:

Lower limit of quantification: 0.2 µg/ml

Lower limit of determination: 0.1 µg/ml

Linearity and determination range: 0.2 – 10 µg/ml

Intra-day precision (relative error): 0.30 – 11.67%

Inter-day precision (relative error): 2.23 – 4.93%

Intra-day accuracy (RSD): 0.06 – 8.64%

Number of volunteers (planned and analysed) 24 Inclusion criteria

• Healthy volunteers, both gender, 18-55 years old

• negative history of past and present diseases;

• normal body temperature, respiratory rate, pulse, arterial blood pressure in supine and

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standing positions, standard 12-lead ECG test;

• normal blood test (hemoglobin, hematocrit, leukocyte count, ESR, blood sugar, electrolytes, creatinin, total bilirubin, ASAT, ALAT);

• normal urine tests (рН, specific gravity, albumin, sugar, bilirubin, sediment).

• negative combined drugs test, test for HCV, HBSAG and HIV. Duration of treatment NA Test product, dose and mode of administration, batch number

KLOPIDOGREL film-coated tablets 75 mg, manufactured by REPLEKPHARM AD

Batch No. 6676;

Shelf-life: 2 (two) years

Analytical certificate No. 6-3156/24.08.2007 Reference therapy, dose and mode of administration, batch number

PLAVIX film-coated tablets 75 mg, manufactured by SANOFI-AVENTIS

Batch No.: 1444;

Shelf-life: 3 years

Batch certificate No. 1444

Analytical certificate No. 6-3157/24.08.2007

Criteria for evaluation

Efficacy Parameter: with without log-transformation

AUC: 0.80 – 1.25 0.8 – 1.20

Cmax: 0.80 – 1.25 0.8 – 1.20

Safety Physical status; appearance of adverse reactions; changes in clinic-laboratory parameters.

Statistical methods

The correspondent 90% confidence intervals for AUC(0-∞), AUC(0-t) and Cmax of the tested preparation as a ratio to the correspondent values of the referent preparation using parametric and nonparametric methods without or with log-transformation of data were calculated. The differences in Tmax of Test and Reference preparations were analyzed by means of a non-parametric analysis of variance at a 90% confidence interval.

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SUMMARY – CONCLUSIONS

Efficacy results

The results from the analysis of the primary and secondary pharmacokinetic parameters are presented on Table S1.

Table S1. Primary and secondary pharmacokinetic parameters (mean values ± SD)

Parameter

AUC(0-∞)

(h.µg/ml)

AUC(0-t)

(h.µg/ml)

Cmax

(µg/ml)

Tmax

(h)

Test preparation

12,05 ÷ 3,839 11,347 ÷ 3,546 4,01 ÷ 1,044 1,18 ÷ 0.53

Reference

preparation 11,74 ÷ 3,921 11,08 ÷ 3,60 4,45 ÷ 1,012 0,81 ÷ 0.22

The mean values of plasma CCAL concentrations versus time for the Test and Reference preparations are shown on Figure S1.

Figure S1. Test and reference plasma CCA concentrations/time curves (mean values)

The results from the bioequivalence assessment between the Test and Reference preparations are presented on Table S2.

0 10 20 30 400

1

2

3

4

5

test

reference

Mean

time (h)

µg/m

l

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LIST OF ABBREVIATIONS

AE: adverse event

ALA alanine amino transferase

ANOVA analysis of variance for nontransformed data

ASAT aspartate amino transferase

AUC(0-t) area under the curve of plasma concentrations from 0 to the last point measured

AUC(total) area under the curve of plasma concentrations extrapolated to infinity

BE bioequivalence

BES Bioequivalance Study

C.I. confidence interval

Clapp total clearance

CCA Clopidogrel carboxylic acid

Cmax Maximum Plasma Concentration

Conc Concentration

CRA. Clinical Research Associate

CRF Case Report Form

CRO Contract Research Organization

CTP Clinical Trial Protocol

C.V. coefficient of variation

DRC Dairy Record Card

GMP Good Manufacturing Practice

GCP Good Clinical Practice

GLP Good Laboratory Practice

h hour

HPLC High Performance Liquid Chromatography

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IS Internal standard

Kel constant of elimination

kg kilogram

LOD Limit of Detection

LOQ Limit of Quantification

Max Maximum

mg milligram

µg microgram

Min Minimum

min minute

ml miililiter

MLIC(1983) Metropolitan Life Insurance Company (1983)

MRT mean residence time

N Number of samples

n number of observations

NS No Sample received for assay

QC Quality Control

RSD relative standard deviation

SD standard deviation

S.E.M. standard error of the mean

St Calibration Standard

t1/2 elimination half-life

Tmax time to reach the maximum plasma concentration

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1 ETHICS

1.1 Independent Ethics Committee (IEC) and Regulatory

The clinical study was initiated after obtaining of the positive statement issued by the National

Ethics Committee for Clinical Trials with Medicinal Products at the Academy of Science in

Medicine of Romania under the Chair of the President Prof. Dr. Sava Dumitrescu. The Ethics

Committee had been given a full set of the trial documentation. The approval was documented

with a Protocol No. 5848/14.02.2008 issued by the Ethics Committee.

Following this procedure an official permission for conducting the trial has been obtained, after

the whole set of required documentation has been submitted to the respective authority – The

National Medicine Agency at the Ministry of Public Health of Romania. The approval of the trial

was in full compliance with current Romanian regulations. The letter of approval was signed by

the President Farm.Pr. Magdalena Badulescu and issued with No.4115/15.04.2008 in favor of

S.C. Biopharmacy and Pharmacol Res SA in Bucharest.

1.2 Ethical Conduct of the Study

The study was conducted in accordance with the “Declaration of Helsinki” (Helsinki, Finland,

June 1964) and the Supplements at Tokyo (Japan, October 1975), Venice (Italy, October 1983),

Hong Kong (September 1989) and Summerset West (Republic of South Africa, October 1996)

and Edinburgh (Scotland, October 2000).

1.3 Patient Information and Consent

Prior to entering the study, at the beginning of the Screening visit and before starting the

screening procedure, each volunteer was given an Informed Consent Form (ICF) and time has

been provided for the volunteer to study the form thoroughly and ask as many questions as might

arise. Each volunteer had the chance to get acquainted in details with the objective, study methods

used, obligations and rights of parties in the course of the study, possible risks and inconveniences

induced by the study and expected results. Each volunteer was given sufficient time to consider

his/her participation in the study, as well as to receive any additional information. Following this

step each volunteer would sign the Informed Consent Form in the presence of the Principal

Investigator or his/her duly authorized colleague.

The Informed Consent Form was signed by both the volunteer and the investigator/representative.

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The Informed Consent Form is duly kept as part of the Clinical Site File under the responsibility

of the Principal Investigator.

A copy of the IC (both in English and Romanian) is provided in Appendix 12.1.3.

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2 INVESTIGATORS AND STUDY ADMINISTRATIVE STRUCTURE

The study was conducted at a centre, situated on the territory of National Institute for

Aeronautical and Space Medicine “Gral.Dr.Victor Anastasiu” with Central Clinical Emergency

Military Hospital “Carol Davila”, with the study team been managed by Acad. Prof. PhD. MD.

Victor Voiku in his capacity of Principal Investigator. The coordinating Study Investigator was

Prof. PhD Constantin Mircioiu. The study team included 6 nurses and 2 lab technicians.

The results of the study were subject to discussion by the Scientific Committee of the Sponsor,

which has made an initial evaluation of the trial results, based on the information provided.

The study has been monitored by Dr. Boyan Doganov from Mediclintrial Support Ltd and Mihai

Manolache from MaxiMax International, Inc. in their capacity of independent monitors.

The safety data was based on the evaluation of the indicators as specified in clinical trial protocol,

while these results have been obtained by the lab physicians and technicians at the Clinical

Laboratory of the National Institute for aerospace medicine, under the management of the Head of

the Laboratory Dr. Simona Berbecar.

The specific evaluation was based on the blood test results obtained at the Biopharmacy &

Pharmacology Research S.A. lab (Bucharest, Romania), under the management of Dr. Stanislav

Yanev, PhD, Department of Drug Toxicology, Bulgarian Academy of Science.

The Sponsor has authorized Pharminform AD, a Contract Research Organization (CRO), to act as

its representative for this trial and manage the trial in compliance with the local and international

regulations and the clinical trial protocol. The CRO has subcontracted for the practical execution

of the trial S.C. & Pharmacology Research S.A. lab, a company in Bucharest, Romania.

A list of all investigators and other individuals who have in any way materially affected the

conduct of the study is provided in Appendix 12.1.4.

The signatures of the Blood sample analyst/biostatistician, Sponsor’s responsible Officer and

CRO’s responsible officers are provided in Appendix 12.1.5.

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3. INTRODUCTION

3.1 Description and Mechanism of Action:

Clopidogrel is an inhibitor of platelet aggregation. A variety of drugs that inhibit platelet function

have been shown to decrease morbid events in people with established cardiovascular

atherosclerotic disease as evidenced by stroke or transient ischemic attacks, myocardial infarction,

unstable angina or the need for vascular bypass or angioplasty. This indicates that platelets

participate in the initiation and/or evolution of these events and that inhibiting them can reduce

the event rate.

Clopidogrel selectively inhibits the binding of adenosine diphosphate (ADP) to its platelet

receptor and the subsequent ADP-mediated activation of the glycoprotein GPIIb/IIIa complex,

thereby inhibiting platelet aggregation. Biotransformation of clopidogrel is necessary to produce

inhibition of platelet aggregation, but an active metabolite responsible for the activity of the drug

has not been isolated. Clopidogrel also inhibits platelet aggregation induced by agonists other

than ADP by blocking the amplification of platelet activation by released ADP. Clopidogrel does

not inhibit phosphodiesterase activity.

Clopidogrel acts by irreversibly modifying the platelet ADP receptor. Consequently, platelets

exposed to clopidogrel are affected for the remainder of their lifespan.

3.2 Pharmacokinetics

Clopidogrel selectively inhibits the binding of adenosine diphosphate (ADP) to its platelet

receptor and the subsequent ADP-mediated activation of the glycoprotein GPIIb/IIIa complex,

thereby inhibiting platelet aggregation. Biotransformation of clopidogrel is necessary to produce

inhibition of platelet aggregation, but an active metabolite responsible for the activity of the drug

has not been isolated. Clopidogrel also inhibits platelet aggregation induced by agonists other

than ADP by blocking the amplification of platelet activation by released ADP. Clopidogrel does

not inhibit phosphodiesterase activity.

Clopidogrel acts by irreversibly modifying the platelet ADP receptor. Consequently, platelets

exposed to clopidogrel are affected for the remainder of their lifespan.

Dose dependent inhibition of platelet aggregation can be seen 2 hours after single oral doses of

Clopidogrel. Repeated doses of 75 mg Clopidogrel per day inhibit ADP-induced platelet

aggregation on the first day, and inhibition reaches steady state between Day 3 and Day 7. At

steady state, the average inhibition level observed with a dose of 75 mg Clopidogrel per day was

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between 40% and 60%. Platelet aggregation and bleeding time gradually return to baseline values

after treatment is discontinued, generally in about 5 days.

After repeated 75-mg oral doses of clopidogrel (base), plasma concentrations of the parent

compound, which has no platelet inhibiting effect, are very low and are generally below the

quantification limit (0.00025 mg/L) beyond 2 hours after dosing. Clopidogrel is extensively

metabolized by the liver. The main circulating metabolite is the carboxylic acid derivative, and it

too has no effect on platelet aggregation. It represents about 85% of the circulating drug-related

compounds in plasma.

Following an oral dose of 14C-labeled clopidogrel in humans, approximately 50% was excreted

in the urine and approximately 46% in the feces in the 5 days after dosing. The elimination half-

life of the main circulating metabolite was 8 hours after single and repeated administration.

Covalent binding to platelets accounted for 2% of radiolabel with a half-life of 11 days.

The administration of Clopidogrel bisulfate with meals did not significantly modify the

bioavailability of clopidogrel as assessed by the pharmacokinetics of the main circulating

metabolite.

Absorption and Distribution: Clopidogrel is rapidly absorbed after oral administration of repeated

doses of 75 mg clopidogrel (base), with peak plasma levels (≡ 3 mg/L) of the main circulating

metabolite occurring approximately 1 hour after dosing. The pharmacokinetics of the main

circulating metabolite are linear (plasma concentrations increased in proportion to dose) in the

dose range of 50 to 150 mg of clopidogrel. Absorption is at least 50% based on urinary excretion

of clopidogrel-related metabolites.

Clopidogrel and the main circulating metabolite bind reversibly in vitro to human plasma proteins

(98% and 94%, respectively). The binding is nonsaturable in vitro up to a concentration of 100

ug/mL.

Metabolism and Elimination: In vitro and in vivo, clopidogrel undergoes rapid hydrolysis into its

carboxylic acid derivative. In plasma and urine, the glucuronide of the carboxylic acid derivative

is also observed.

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4 STUDY OBJECTIVES

The primary objective of the study was to compare bioavailability of the Test and Reference

products and thereby to determine their equivalence.

The secondary objective of the study was to assess the safety of both Test and Reference products

when applied in a single dose of 150 mg (2 tablets x 75 mg).

The purpose of the study, however, was to receive a solid evidence that the Test product can be

introduced into the market place as therapeutically equivalent to the Reference product, based on

the fact that it is i) compared with a specified, approved Reference product; ii) the time-dependent

drug concentrations in blood from the Reference product without any doubt lead to therapeutic

effects; iii) Test and Reference products are both chemically and pharmaceutically equivalent,

with same rate and extent of absorption (i.e. they are bioequivalent); and iv) bioequivalent

products are by inference considered therapeutically equivalent. (Thiessen, JJ).

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5 INVESTIGATIONAL PLAN

5.1 Overall Study Design and Plan: Description (see Graph 10.3.1 in Section 10.3)

The study was a single-center, open, single-dose, randomized, balanced, two-way crossover study

in 24 healthy volunteers with a wash-out period of one week between the study periods.

The Clinical Trial Protocol (CTP), dated 10.09.2007 is provided as Appendix 12.1.1 to this report.

Samples of the Case Report Form (CRF) for the study and the Daily Record Card (DRC) are

provided in Appendix 12.1.2. No other sources of information for this section of the report have

been used.

The volunteers have been randomly divided in 2 groups and treated with Klopidogrel and Plavix

in a two-way, cross-over manner. The dose used was 150 mg of clopidogrel each, which means in

practice 2 film-coated tablets, of any of the products under trial.

The dose has been applied in compliance with the procedures of the protocol, at about 07:30-

08:30 in the morning of day 1 and day not less than 8 for Phase I and Phase II, respectively. The

dose was applied after 10 hours of starving and 1 hour without drinking water, while at the

moment of application a volunteer would take the tablets orally with a glass of water – app. 240

ml, without the tablet being chewed or crashed in any other way. Following the application a

physician would check the mouth cavity for any evidence of the tablet not being swallowed or

chewed, or crashed in any other way.

In total 30 volunteers have been screened for the trial, 24 of them included in the trial phase.

There was no blinding, as the study is open.

No placebo but only active treatments have been applied following a cross-over trial

configuration.

The treatment to each group has been assigned following a randomization scheme, part of the

CTP.

There were 2 study periods, each consisting of 2 days and with a wash-out period of 7 days

between study periods. The study visits were preceded by a Screening visit, 2 days before entry

into study period. There was a Follow-up visit 6 days following the II study period. The

randomization would occur at the Screening visit and was done on a next-to-come basis. A flow

chart of the study including timing is provided in Appendix 12.1.1. The study timing was in

compliance with the protocol and no major deviations have been registered.

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The data processing at the clinic level has been a subject to monitoring by an independent

monitor, a considerable part of the monitoring process being the monitoring of safety data. No

special steering or evaluation committees have been in place, except for a specialized expert body

of the Sponsor.

No interim analysis has been planned and therefore implemented.

5.2 Discussion of Study Design, Including the Choice of Control Groups

As a BES with a typical for this type of trials design, the study does not include a control group,

nor placebo treatment. The volunteers are divided in two groups, equal number of volunteers in

each group, and are treated actively during Phase I of the trial with the Test or Reference drug, in

a random manner (randomization scheme provided in Appendix 12.1.7.).

The groups change their treatment in Phase II in a cross-over manner and thus get the opposite

drug compared to Phase I.

With each subject receiving the same dosages and being its own witness, the number of 24

volunteers is sufficient to evaluate and compare each study medication from a pharmacokinetic

and statistical point of view. The experimental design is suitable for bioequivalence studies.

Men and women volunteers are included in this trial in compliance with the recommendations of

EMEA in CPMP/EWP/QWP/1401/98 item 3.2.1

The number of volunteers is determined based on reference data, where the calculated, following

a log transformation, mean square error (MSE) with ANOVA analysis, has a CV value of 13.3.

This leads to confidence that a number of not less than 18 volunteers is big enough so that with

80% probability it will be possible to determine a 20% difference between the mean values for

AUC of the Test and Reference product with a level of significance α = 0.05.

The number and time-scheme of points for blood collection to determine the bioavailability of

both drugs are based on literature data and the pharmacokinetic profile of CCA. The resulting

AUC curves can be thus well compared and the existence of equivalence can be easily and

reliably assessed.

The wash-out period is selected also in compliance with the pharmacokinetic profile of CCA. It is

long enough so that the blood is cleared from any traces of the active ingredient followed.

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5.3 Selection of Study Population

Volunteers consisting of university students and members of the community at large were used in

the study. The volunteers were recruited from the database of the National Institute for Aeronautic

and Space Medicine with acceptable clinical trial volunteers. The medical check–up was

performed at the National Institute for Aeronautic and Space Medicine. Twenty-four healthy

volunteers, aged 21 - 34, with ideal body weight ±10%, non-smokers as per trial criteria, were

included into study. The volunteers' health condition was established on the base of medical

history, physical examination, biochemical, hematological and serological tests.

Prior to entering the study, the volunteers were informed about the products to be administered

and the possible risk for their health. All of them signed the Informed Consent Form provided

(Appendix 12.1.3).

5.3.1 Inclusion Criteria

A volunteer is eligible for inclusion in the study if he/she:

• has signed of his/her own free will the Informed Consent Form;

• is a man or a women, Caucasian;

• is 18 to 45 years old;

• is with body weight within the normal range for his/her height;

• is a non-smoker or smoker but smoking not more than 5 cigarettes/day;

• is willing to allow all data concerning him/her to be checked by monitor or representative

of the Sponsor;

• is physically and mentally healthy as per medical and standard lab checks.

5.3.2 Exclusion Criteria

A volunteer is not eligible for inclusion in the study and has to be excluded from participation in

case he/she meets any of the following criteria:

• has not signed the Informed Consent Form;

• history, physical examination and the planned lab tests have shown clinically significant

deviations from normal physical status (conditions);

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• results from blood and urine tests, which are outside the reference values, are clinically

significant in view of the Principal Investigator or co-investigators;

• ECG with clinically significant deviations;

• acute infection, suffered within two weeks before the first trial drug application;

• volunteer takes and does not agree to stop the application of other medicinal products,

including antacids and analgesics (like aspirin and paracetamol) from within two weeks

before the first trial drug application till the end of the follow-up examination;

• volunteer is on a special diet (i.e. volunteer is a vegetarian) or has lost more than 5 kg for

the last month as a result of loosing-weight diet;

• volunteer consumes regularly drinks with metilxantines (coffee, tea or coca-cola more

than 0.5 l/day altogether);

• volunteer consumes more than 20 units alcohol per week (one alcohol unit is equal to 0.5 l

beer, 0,2 l wine, 20 g alcohol);

• volunteer does not agree not to consume products from grapefruit within the period of 7

days before the first trial drug application till the end of the follow-up examination;

• volunteer does not agree to stop eating and drinking foods and beverages with

methylxantines like caffeine (coffee, tea, coca-cola, chocolate, etc.) and fruit juices for the

period between 48 hours before the first trial drug application and the last time point of

blood sample collection;

• volunteer has a history of prior:

o hypersensitivity to clopidogel or similar medicinal products;

o hypersensitivity to the excipients of the Test and Reference product;

o hypersensitivity to multiple medicinal products;

o allergic diseases, acute hay fever, asthma;

o drugs and alcohol abuse;

o epilepsy and other seizures;

o mental diseases, i.e. latent or manifested depression,

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o schizophrenia, neurosis;

o respiratory diseases;

o GI tract surgery (excluding appendectomy);

o renal diseases;

o coagulation disorders or acute anemia;

o glucose-6-phosphate dehydrogenises insufficiency;

o chronic treatment or pathology.

• volunteer has metabolic diseases;

• volunteer has history of metabolic disorders related to medicinal products;

• volunteer has blood pressure in recumbent position over 145/90 mm Hg and below 90/50

mm Hg, heart rate under 50 beats/min and over 90 beats/min;

• volunteer suffers diseases that can disturb the course of the clinical trial;

• volunteer has suffered a serious disease within the period of three months before the

screening visit;

• volunteer suffers a disease of the GI tract;

• volunteer has abused with drugs;

• positive tests for AIDS or hepatitis;

• volunteer has donated blood in the period within three months before the first application

of the tested drugs;

• volunteer has participated in a clinical trial with medicinal products during the last three

months before the first application of the tested drugs;

• volunteer cannot be contacted in case of emergency;

• volunteer plans a hospitalization within three months following the first application of the

tested drugs;

• volunteer is an active sportsman and/or intends to take part in sport activities in the course

of the clinical trial;

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• volunteer works during night time;

• volunteer drives vehicles for transportation of people or operates machines with potential;

risk within the period between the first application of the tested drugs and the follow-up

examination.

5.3.3 Removal of Patients from Therapy or Assessment

Participation in this clinical study is planned to be discontinued for any of the following reasons:

• volunteer not willing to obey the requirements of the study protocol;

• volunteer is not available for taking blood samples (more than 2 drop outs);

• in the opinion of the Principal Investigator there is a serious adverse effect as a result of

the study drug;

• for an acute illness during the study, needing medications.

The investigators are supposed to remove a volunteer from the study if it is proven that he/she

does not follow pre-study directions regarding alcohol and drug use, fasting, etc., or in case a

volunteer is not cooperative during the study. Details of reasons for removal of volunteers are to

be recorded and reported to the Sponsor. Every effort is to be made to obtain a complete follow-

up for any withdrawn subject. The withdrawn subject's plasma level data are to be provided in the

final report if the subject has been removed from the study for a drug-related event.

No replacements are planned for this trial. Only completed subjects have been included in the

final statistical analysis. Detailed reasons of dropping-out are recorded in the CRF.

5.4 Treatments

5.4.1 Treatments Administered

Two medicinal products have been applied in this trial in a cross-over manner.

5.4.1.1 Test product

KLOPIDOGREL 75 mg film-coated tablet

Manufacturing Authorization Holder:

REPLEKPHARM AD – Skopje, Republic of Makedonia

5.4.1.2 Reference product

PLAVIX 75 mg film-coated tablet

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Marketing Authorization Holder:

Sanofi Pharma, France

5.4.1.3 Dosage

150 mg, single dose of each product, in each phase of the trial, in the morning (about 7.30-8.00)

of Study Day 1 and not less than 8.

5.4.1.4 Route of administration:

Oral, with one glass (240 ml) mineral water.

All study products were provided and their quality assured by the Sponsor.

5.4.2 Identity of Investigational Products

5.4.2.1 Test product

Klopidogrel film-coated tabl. 75 mg, manufactured by REPLEKPHARM AD Batch No.: 6676

The product was provided by the manufacturer, accompanied by an Analytical certificate

№ 6-3156/6676/24.08.2007.

5.4.2.2 Reference product

Plavix film-coated tabl. 75 mg, manufactured by Sanofi Pharma, France.

Batch No: 1444. The product was provided by the Sponsor, accompanied by an Analytical

certificate № 6-3157/6676/24.08.2007.

5.4.3.3 Identity of investigational products

In compliance with the current GMP and bioequivalence guidelines it is possible to retrace the

composition and the pharmaceutical quality as well as to insure a good traceability of the products

administered in this study.

The used batch numbers of the investigational products are 6676 for the Test product and 1444 for

the Reference product.

The analytical reports for both products are provided in Appendix 12.1.6.

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The packaging bears the following information for the Test and the Reference product,

respectively:

5.4.3 Method of Assigning Patients to Treatment Groups

Allocation of volunteers (assignment to Group I or Group II) was performed after verification by

the investigator of all selection criteria, practically at the beginning of Phase I visit, during

admission in trial center. The randomization numbers were assigned on a first-to-come basis for

all volunteers assessed as eligible to enter the trial.

Treatments were packed in compliance with the randomization list drawn up for the clinical trial

center – National Institute for Aeronautic and Space Medicine. A detailed description of the

randomization method, including its execution, is given in Appendix 12.1.7. A table exhibiting the

randomization codes, patient identifier, and treatment assigned is also presented in this Appendix.

5.4.4 Selection of Doses in the Study

The chosen doses of clopidogrel were single oral administration of a 150 mg dose at Day 1 of

each trial phase (Study Day 1 and not less than 8).

Sanofi-Aventis

Volunteer No.:……….. Study period: ………….. PVAVIX, film-coated tablets

1 package contains 2 tablets for oral application 1 tablet contains 75 mg clopidogrel Dose: Single dose (2 tablets 75 mg each) in the morning Take the tablets with 240 ml water Store at room temperature! Batch №: 1444 Shelf life: 3 years Replekpharm AD ONLY FOR CLINICAL TRIAL CLOP_REPL_0807

Replekpharm AD

Volunteer No.:……….. Study period: ………….. KLOPIDOGREL, film-coated tablets

1 package contains 2 tablets for oral application 1 tablet contains 75 mg clopidogrel Dose: Single dose (2 tablets 75 mg each) in the morning Take the tablets with 240 ml water Store at room temperature! Batch №: 6676 Shelf life:2 years. Replekpharm AD ONLY FOR CLINICAL TRIAL CLOP_REPL_0807

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This dose is the lowest available single dose for the product and also the lowest advised dose for

the treatment of the indicated conditions.

5.4.5 Selection and Timing of Dose for Each Patient

The products were administered under fasting conditions. The dose was applied in the morning,

after at least 10 hours of fasting and 1 hour not drinking water or other beverages. The dose was

applied in the presence of the investigator or co-investigator between 7:30 - 8:30 AM. Drugs were

administered per-os in the form of tablets with 240 ml of water at ambient temperature. The doses

were administered as follows:

Test product: Klopidogrel 2 tablets of 75 mg: The volunteer put the tablets in the mouth

taking care not to chew it. Then the subject swallowed it while drinking

240 ml of water.

Reference product: Plavix 2 tablets of 75 mg: The volunteer put the tablets in the mouth taking

care not to chew it. Then the subject swallowed it while drinking 240 ml of

water.

The volunteers received the tested formulations according the randomization scheme. After

washout period of 7 days the products were applied in a cross-over manner, in compliance with

the randomization scheme.

In the days when the drugs were administered the volunteers received a standard lunch 6.5 hours

after dosing and standard dinner 12,5 hours after dosing. Drinking of water was allowed after 2

hours of drug administration. In the course of the study the volunteers were not allowed to

consume cola drinks, tee, coffee or cocoa.

5.4.6 Blinding

As this is an open study blinding has not been applied.

5.4.7 Prior and Concomitant Therapy

Volunteers were informed that they should have not taken any medicinal products for at least 30

days prior to study and are not supposed to do that during the entire period of the study. They

were specifically reminded that this includes products to treat colds, and also vitamins and antacid

preparations. No concomitant medication was allowed during the study. Each subject was

specifically asked on this issue prior to study drug administration. If a volunteer would apply any

medication during the trial (mainly between Phase I and Phase II, during the wash-out period), the

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investigator would decided whether the subject was permitted to remain in the study depending

on the product used. The name of the product, the daily dose, the route, the dates of

administration and the indication were noted and reported by the investigator in the CRF.

5.4.8 Treatment Compliance

Compliance was evaluated by: (i) direct administration of the compound by the investigator or co-

investigator (following the administration of the study drug, the mouth was checked in order to

confirm the consumption of medication); (ii) by blood sampling for determination the quantity

(concentration) of the active ingredient at different time-points.

5.5 Efficacy and Safety Variables

5.5.1 Efficacy and Safety Measurements Assessed and Flow Chart

5.5.1.1. Efficacy Measurements

The efficacy variables assessed include several pharmacokinetic parameters, namely Cmax, Tmax,

AUC0-48, AUC0-∞ plus some other parameters like T½, Kel, CL, MRT. For this reason blood

samples are tested, collected at specific time-points.

During the study periods, 16 blood samples were drawn at the following time-points for each

phase:

At Day 1 and Day 7: 0(before dosing), 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 24 and 48 h

(Total of blood samples for both phases per subject = 32)

Blood samples were taken from a forearm vein via either a venocath or by direct venepuncture

(only for the sample taken at 24 and 48 hours time-point).

The whole blood samples (5 ml) were centrifuged for 10 min in order to separate plasma; plasma

samples are stored on the temperature of -30o 13, until the HPLC analyzing. The samples were

coded at the analytical laboratory with randomly selected numbers, and the numeration key was

decoded after determine plasma concentrations.

The experimental time was calculated according to the clopidogrel administration schedule. The

clock times of all blood draws were recorded and reported for each subject. Any deviation from

the sampling schedule was recorded in the subject's sampling time sheet. Sampling time sheets for

all subjects were included in the CRF. Considering that pre and post-study blood tests required 20

ml each, the total volume of blood withdrawn over the duration of the study of about 3 weeks did

not exceeded 200 mL.

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The method for measuring the efficacy variables is described in Section 5.5.2. “Appropriateness

of Measurements”. The persons responsible for blood collection are from the clinical team under

the control of the Principal investigator Accad. Dr. Voicu. Blood analysis was done at

Biopharmacy&Pharmacology Research S.A. lab (Bucharest, Romania) by the methodological and

technical assistance of Department for Drug Toxicology, Institute of Neurobiology, Bulgarian

Academy of Science by Analyst Bozhidara Pandova under the supervision and management of

Dr. Stanislav Yanev, Head of Department.

5.5.1.2. Safety Measurements

To monitor safety any adverse events have been actively looked for and reported – both clinical

and laboratory. The monitoring of adverse events was done in compliance with the procedures

and definitions described below.

Definition of an adverse event

Clinical adverse events or serious adverse events are illness, subjective or objective signs or

symptoms that have appeared during the course of a study independently of a causal relationship

to study medication. This includes all events both expected (known pharmacological response)

and unexpected or unwanted occurring during the course of the study. Moreover all events that

occur in relation to a clinical study after the last clopidogrel administration had to be estimated as

an Adverse Event or Serious Adverse Event.

Adverse Events:

• Event related or non-related to study medications,

• Intercurrent illnesses,

• Important abnormal laboratory values, as well as significant shifts form baseline within

the range of normal, which the Clinical Investigator considers to be clinically important.

Serious Adverse Events:

• overdose,

• results in in-patient hospitalization or prolonged hospitalization,

• life threatening,

• temporarily or permanently disabling,

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• fatal outcome.

Classification of Adverse Events

All adverse events were recorded on an adverse event information sheet in the CRF of the

volunteers and graded as mild, moderate, or severe according to the following definitions.

Mild: Causing no limitation of usual activities; the subject may experience slight

discomfort.

Moderate: Causing some limitation of usual activities; the subject may experience annoying

discomfort.

Severe: Causing inability to carry out usual activities; the subject may experience

intolerable discomfort or pain.

Causality/Drug-related Assessment

The Clinical Investigator determined the relationship of any adverse event to study medication

according to the following criteria:

UNLIKELY: An AE is placed in this category when it meets the following criteria:

• It may readily have been produced by other factors than the study medication (e.g. the

subject's clinical state, environmental factors etc.)

• It does not show a known pattern of response to the study medication and/or it does not

follow a time course that can reasonably be associated with the time of administration of

the study medication.

POSSIBLE: An AE is placed in this category when it meets the following criteria:

• It follows a time course that can reasonably be associated with the time of administration

of the study medication.

• It may have been produced by other factors than the study medication.

• It shows a known pattern of response to the study medication.

PROBABLE: This category applies to an AE when it meets three of the following criteria:

• It follows a time course that can reasonably be associated with the time of administration

of the study medication.

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• It is not likely to be produced by other factors than the study medication.

• It disappears or decreases on discontinuation/dose reduction of the study medication.

• It shows a known pattern of response to the study medication.

NOT ASSESSABLE: This category applies to an AE whom relationship to the study medication

can not be assessed due to conflicting data or poor documentation.

Adverse Events Documentation

The recording of every single Adverse Event and/or Serious Adverse Event had to meet special

requirements:

• detailed subject data,

• exact documentation of the event,

• exact description of temporal sequence following drug administration,

• documentation of duration and severity,

• documentation of the results of diagnostic and therapeutic measurements,

• results of a repeated exposure (re-challenge) if possible,

• details to the development and outcome including medical judgment,

• as much data as possible have to be obtained which are important for judgment concerning

the relationship of the adverse event to study drug,

• critical examination of the relationship to study drug.

All adverse events followed this scheme when spontaneously reported by the subject, observed by

the Clinical Investigator or elicited by general questioning.

Registration Procedures of Adverse Events

It was the Clinical Investigator's responsibility to record and report all adverse events which

occurred during the study (including all deviations of laboratory values from normal ranges),

regardless of their relationship to the study medication.

Information about serious adverse event was recorded on the specific sheet of the VRF and was

reported to the Sponsor within one working day. This report contained a detailed description of

the observed symptoms and the contra-active therapy.

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The Clinical Investigator judged the possible causal relationship between the event and the study

drug.

The Clinical Investigator arranged additional examinations at his own discretion to clarify if the

event was connected with the study medication and consulted a specialist if necessary. All adverse

events, serious or not, were followed up and reported regularly to the Sponsor until an outcome

was known.

The Sponsor or its representative was responsible for notification to regulatory agencies.

Several cardiovascular indicators have been monitored as a part of the safety measurements

procedures.

Blood pressure (BP) and heart rate (HR)

Haemodynamic safety was evaluated by measurement of systolic and diastolic (SBP and DBF)

blood pressures and heart rate (HR) using an oscillometric method (Dinamap®) after 5 minutes

supine and then 2 minutes standing. They were measured at:

• the Screening Visit

• the Final visit.

BP was expressed in mmHg and heart rate in strokes/min.

Moreover vital signs were monitored if judged necessary by the physician in charge. If any

abnormalities occurred, measurements continued until the values had returned to within ± 10 % of

baseline value.

Electrocardiogram (ECG)

Cardiovascular safety was also evaluated by means of a standard 12-lead ECG after 5 minutes

rest. The ECGs were recorded at 25 mm/s at:

• the Screening visit

• the Final visit

All ECGs were duly analyzed by a physician. The following parameters were automatically

measured:

• heart rate (bpm)

• PR interval (msec)

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• QRS interval (msec)

• QT and QTc (automated from electrocardiograph) (msec).

In case significant deviations from normal were diagnosed, this has been noted in the CRFs.

A set of laboratory assessments were done, in compliance with the protocol and with the objective

to follow basic body system functions, both at the beginning and at the end of the trial.

Laboratory safety was evaluated by tests performed in fasting condition. The following laboratory

parameters were determined during the initial visit - 2 days before Day 1 of Study phase I.:

Haematology : Haemoglobin, haematocrit, leukocytes, erythrocytes sedimentation rate (ESR),

platelets;

Biochemical: Glucose, creatinine, sodium, potassium, total proteins, albumin, total bilirubin,

ASAT (SGOT), ALAT (SGPT), urea

Urinalysis: Sediment, pH, proteins, glucose, ketone bodies, and blood.

The physician in charge assessed each abnormal value (not within 10% of normal laboratory

values) to determine if it was clinically significant and treatment related. Laboratory values within

the 10% extended normal values were assessed as per they were assumed to be clinically normal.

Treatment related abnormal and clinically significant laboratory values were reported as an

adverse event.

Laboratory safety during the study

The above hematology, blood chemistry and urine screening were performed in fasting condition

once again at the end of the study visit, 8 days after final administration.

The hematology, biochemical and urinalysis have been done at the Clinical Laboratory of the

National Institute for Aeronautic and Space Medicine, as well as the serological tests.

All female volunteers have been tested for pregnancy at the Screening visit in compliance with

the requirements of the CTP.

The reference laboratory ranges are given in Appendix 12.1.8. “Audit certificates”.

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5.5.2 Appropriateness of Measurements

For the assessment of efficacy variables a method was used based on an analyte CLOPIDOGREL

CARBOXYLIC ACID HCl (SynFyne Res., Canada) and an internal standard Ticlopidine

(Sigma).

Analyte: Internal standard:

Clopidogrel carboxylic acid HCl Ticlopidine HCl

(SynFine Research, Inc.) (Sigma)

MF = C15 H15 Cl2 N O2 S MF = C14 H15 Cl2 N S

MW = 343.796 MW = 299.786

5.5.2.1. Summary of method

The method used for Clopidogrel carboxylic acid (CCA) plasma determination was developed

and validated in Department of Drug Toxicology, Inst. Neurobiology, BAS, Sofia based on

modified procedure described by Souri E. et al (Biomed. Chromatography, 20, 12, 1309-1314,

2006).

0.5 ml of plasma was mixed after thawing with 0.5 ml 0.2N HCl and 50 µl of internal standard.

Samples were mixed for 10 sec by means of a vortex mixer and then were applied on

preconditioned SPE columns Strata-X-C 30mg/1mL. The active substances were eluted with 1mL

5% ammonia in 1:1 methanol/acetonitrile to a clean glass tubes and evaporated to dryness under a

stream of nitrogen. The residues was reconstituted in 200 µl freshly prepared mobile phase and

then, a 50 µl aliquot was injected onto the HPLC.

N

OH O

ClS

HCl

N

S

Cl

HCl

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The plasma content of CCA in control and unknown samples is determinate using internal

standard method of calibration curves of chromatographic peaks area ratios of known amounts of

CCA to IS.

5.5.2.2. Quality Assurance

The study was conducted according to the principles of good laboratory practice. In-house

standard operating procedures were applied during all phases of the study. Grades and sequence

numbers of all chemicals used were recorded and equipment used was checked and calibrated

before use. All calculations and data transcriptions were checked by the analyst who performed

the original calculation or transcription. In addition the internal auditor checked results on a daily

basis. Final data was approved by the laboratory director before release. All raw data, validation

data, summaries and reports are stored in the archives of S.C. & Pharmacology Research S.A.,

Bucharest.

All results are presented graphically in Appendix 12.1.10. Documentation of inter-laboratory

standardization methods and quality assurance procedures.

5.5.2.3. Equipment and materials

a. Chemicals

Reagent Grade Supplier Batch. No.

Acetonitril HPLC, spectroscopy LEDA LD0020

Methanol HPLC grade LEDA LD0150

85% o-phosphoric acid Pure for analysis Merck 904

Potassium dihydrogenphosphate Pure for analysis Merck A549471

Tetrahydrofuran HPLC Labscan C2520

Hydrochloric acid Suprapur Merck 100318

Ammonium hydroxide Extrapur Merck 105426

Water was prepared in-house from Millipore water purifier.

b. Reference compounds

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Name Supplier Lot/Batch No.

Clopidogrel carboxylic acid HCl SynFine Research, Inc. S-1229-182A3

Ticlopidine HCl Sigma T6654

Certificate of Analysis are kept on files.

c. Equipment

Glassware Description Supplier

Volumetric flasks

Graduated cylinders

Tubes (2.0 ml)

Grade A

Grade A

Plastic

Alkem

Alkem

Valerus

Chromatographic devices

Description Supplier Lot number

Solid phase extraction columns

Strata-X-C 33 µm Strata S229-42

Chromatographic column

Gemini-C18, 150x4,6 mm, 5µm

Phenomenex Inc …………………

Chromatographic guard columns

Gemini-C18, 4.0x3.0 mm

Phenomenex Inc ……………….

Dispensers Make Supplier

Variable volume

Pipetteman 1000

Pipetteman 200

Multipette

Eppendorff

Eppendorff

Eppendorff

Lab. Equipment Type Manufacturer

Analytical balance KERN 770 Mettler

Top loading balance KERNGJ Mettler

Vortex mixer Heidolph Reax Heidolph

pH meter WTW Beckman

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Centrifuge MPW - 310 Poland

Shaker Thyys10 Germany

SPE positive pressure processor device

Varian CEDEX USA

SPE columns Strata-X-C 30mg/1mL Phenomenex

Magnetic Stirrer MR 3001 K Thermolyne Corporation

5.5.2.4. Plasma treatment

To 0.5 ml of plasma was added 0.5 ml 0.2N HCL and 50 µl of internal standard. Samples

were mixed for 10 sec by means of a vortex mixer and then were applied on preconditioned SPE

columns Strata-X-C 30mg/1mL.

SPE extraction procedure (as proposed by Strata Sample Preparation Method

Development Software version 1.0, 2005, Phenomenex Inc.):

SPE columns: Strata-X-C 30mg/1mL (polymeric SCX/RP sorbent, here used in

SCX mode.

Sample matrix: plasma

Conditioning: 1mL methanol

Equilibration: 1mL 0.1N HCl

Sample load: 0.5 plasma diluted with 0.5 ml 0.2N HCl and 50 µl IS in water.

Wash: 1mL 0.1N HCl

Wash: 1mL methanol

Elute: 1mL 5% ammonia in 1:1 methanol/acetonitrile

The eluent was collected to a clean glass tubes and evaporated to dryness under a stream

of nitrogen. The residues was reconstituted in 200 µl freshly prepared mobile phase and then, a 50

µl aliquot was injected onto the HPLC.

5.5.2.5. Chromatographic conditions

The analytical method is developed on Waters reverse phase liquid chromatography system

equipped with:

• Quaternary pump 600E

• PDA 996 set at 201 nm.

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The following chromatographic conditions apply:

Pre-column:

Gemini-C18, 4.0x3.0 mm, Phenomenex

Analytical column

Gemini-C18, 150x4,6 mm, 5µm, Phenomenex

Working temperature of the analytical column: 25oC

Mobile phase:

• A: Buffer (0.025 M potassium dihydrogenphosphate adjust to pH 3.2 with 85% orto-phosphoric acid; B: acetonitril; C: tetrahydrofuran; A/B/C (80/18/2); final pH=3.5

Flow rate: 1.0 ml/min

Detection: UV=202 nm

Injection volume: 50 µl

Instrument control and integration is performed on Pentium 566 computer by Waters Empower

software and the chromatograms are stored and reproduced by the same system.

5.5.2.6. Calculations

During the validation procedures all data was subjected to regression analysis using different

regression equations. The equation that gave the best results, based on accuracy for the entire

validation range was selected for the calculation of concentrations of CCA in unknown samples.

For this study a linear regression equation without weighting was found to be the most suitable to

cover the dynamic range.

5.5.2.7. Analytical method validation

Methods employed by Dept. Drug Toxicology undergo a three-phase validation prior to use. A

pre-study validation is performed during the developmental stages of a new method. This is

followed, if necessary, by a confirmatory revalidation performed immediately prior to

commencement of trial sample analysis. During the actual analysis of the study samples the

within-study validation is performed on a daily basis to monitor actual performance of the method

over the analytical period of the particular study. Methods used for these validations are based on

FDA Guidance for the Industry, Bioanalytical method validation (May 2001) and the SOP of

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Biopharmacy&Pharmacology Research S.A. lab and Dept. Drug Toxicology (GLP certificate

attached).

a. Validation Parameters

Definitions and Acceptance Criteria:

Specificity and selectivity: Selectivity of the analytical method will be determinate by the

comparing the chromatograms of diluents, CCA standards, blank plasma and spiked plasma

samples. In chromatogram of diluents and blank plasma sample there should not be any

interfering peak at retention times of CCA and IS, while retention times of CCA standards should

correspond to retention times of the same component in spiked plasma samples.

The validation of specificity and selectivity of analytical method is done by System suitability

test. Waters Empower software provides these data automatically under the criteria of European

Pharmacopoeia:

• Precision of the detector response: RSD of the detector response for 6 injections of

standard solution is <1%.

• Resolution factor (Rs) and Capacity Factor (k’) between peaks of CCA and an unknown

peak is >2.

• Tailing (symmetry) factor for CCA is <2.0.

• The number of plate count (N) as measure for column efficiency is for CCA >2000.

Linearity and range of determination: Linearity will be performed at 10 concentration points

(excluding blank values) in the concentration range from 0.2 to 10 mcg/ml for CCA, in 3 different

days. Spiked plasma samples will be prepared and analysed as described in analytical procedure.

Linear regression equation, slope and intercept, correlation coefficient and coefficient of

determination will be calculated. The results will be also present graphically.

Criterion: R2 > 0.999

Sensitivity: two parameters define the sensitivity of the method, the Limit of Quantification

(LOQ) and Limit of Detection (LOD) The LOQ is that concentration of CCA which can be

quantitatively determined with accuracy and precision better than RSD < 20%, and a signal-to-

noise ratio better than 10:1. The LOQ will be determined using 6 replicate determinations. The

LOD is the concentration of analyte that can be reliably differentiated from background levels but

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not quantitated with sufficient accuracy or precision. A signal-to-noise ratio of between two and

three is generally acceptable.

Recovery: indicates losses incurred during sample processing. Recoveries of more than 80% are

normally required however reproducible recoveries lower than this is acceptable.

Criterion: minimum 80%

Accuracy:

Intraday accuracy

Intraday accuracy will be determinate by 5 times replicate analysis of plasma samples at 3

different concentration levels of CCA, first nearby LOQ. The concentrations will be: 0.4,

1 and 8 mcg/ml. Spiked plasma samples will be analysed as described in analytical

procedure. From 5 determinations RSD for CCA will be calculated.

Criterion: RSD (N=5) < 20% at low concentration level (LOQ)

RSD (N=5) < 15% at middle and high concentration level

Accuracy of injection

Accuracy of injection will be determined by 6 consecutive injections of standard solution

of CCA. The content of CCA in mcg/ml plasma will be calculated for each sample against

the standard curve. From 6 determinations RSD for retention time and area for CCA will

be calculated.

Criterion: RSD < 1.0%.

Precision:

Repeatability

Repeatability (intra - day precision) will be determinate by measuring individually prepared 5

spiked plasma samples at 3 different concentration levels of CCA: low (0.4 mcg/ml), middle (1

mcg/ml) and high (8 mcg/ml). Spiked plasma samples will be analysed as described in analytical

procedure. The content of CCA in mcg/ml plasma will be calculated for each sample against the

standard curve. From 5 determinations the average value and relative error will be calculated.



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Intermediate precision (formally known as ruggedness)

Intermediate precision will be determinate by measuring 5 individually prepared spiked plasma

samples at 3 different concentration levels of CCA: low (0.4 mcg/ml), middle (1 mcg/ml) and

high (8 mcg/ml), in 3 different days. Spiked plasma samples will be analysed as described in

analytical procedure. The content of CCA in mcg/ml plasma will be calculated for each sample

against the standard curve. From 15 determinations, the average value and relative error will be

calculated.



Stability testing will be performed using 3 spiked plasma samples at two different concentrations:

low (0.4 mcg/ml) and high (8 mcg/ml) for CCA. Spiked plasma samples will be analysed after

following storage conditions:

• immediately (reference samples)

• after tree freeze/thaw cycles

• long term stability (standard samples stored at –28oC were tested after 1 month)

• Autosampler stability test (after 24h)

Criterion: relative error +/- 20% at low concentration level (LOQ)

relative error +/- 15% at high concentration level

Robustness (the capacity of the method to remain unaffected by small deliberate variations in

method parameters) of the method will be demonstrated by means of statistical experimental

design (DoE) varying the following factors:

• mobile phase pH, column temperature, buffer molarities, organic phase volume, volume of

mobile phase modifier, flow rate.

Criterion: Resolution factor (Rs) between peaks of CCA and IS is >2.

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5.5.3 Primary Efficacy Variables

According to the obtained plasma concentrations/time data of CCA the following

pharmacokinetic parameters were calculates using software KINETICA™ 4.2 (Innaphase

corporation, USA) (see Section 5.7.1.3.).

Primary parameters: AUC(0-t) and AUC(0-∝) (area under the curve of the plasma concentrations

until the last sampling time and infinity), Cmax (maximum plasma concentration).

Secondary variable: Tmax (time of reaching the maximum plasma concentrations).

Additional variables: Kel (elimination rate constant), MRT (mean residence time), Clapp

(apparent clearance) and t½ (elimination half-life).

5.5.4 Drug Concentration Measurements

The CCA concentrations in plasma were determined with High Performance Liquid

Chromatography, using PDA detector according to the method of Souri E. et al (2006).

All used chemicals for quantification of CCA in plasma, were purchased by Sigma. The standard

CCA HCl was obtained by SynFine Res., Canada

The chromatograph system and PDA-spectrometric data were controlled by a special software

program Waters Empower.

The method was specific for CCA since no interfering peaks are appearing at the chromatogram.

The method had linear response for the concentration levels from 0.2 to 10 µg/ml CCA. The

lower limit of quantification was taken to be 0.2 µg/ml with C.V. = 4.9% for N=5.

The sample collection times and periods in relation to the timing of drug administration is

described in Section 5.5.1.1. “Efficacy measurements”. The details concerning relation of drug

administration and sampling to ingestion of food, posture and the possible effects of concomitant

medication/alcohol/ caffeine/nicotine is addressed in Section 5.4. “Treatments”. The biological

sample measured, the handling of samples and the method of measurement used is described in

Section 5.5.1.1. “Efficacy measurements”, while the internal assay validation documentation is

presented in Section 5.5.2. “Appropriateness of measurements” and 5.6. “Data Quality

Assurance”. The respective graphs in support of the validation are presented in Appendix 12.1.10

“Documentation on inter-laboratory standardization methods and quality assurance procedures”.

No other factors are believed important in assessing pharmacokinetics.

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5.6 Data Quality Assurance

The trial was implemented according to the European directives about the Good Clinical Practice

in Europe (“Note for guidance on the investigation of bioavailability and bioequivalence",

London 2001, CPMP) and ICH E3 guideline.

The procedures described in the protocol are subject to Standard Operating Procedures as

described in the Standard Operating Procedure manual of Pharminform AD.

Designated personnel from the Sponsor was responsible for maintaining quality control and

quality assurance systems with the written SOP's to ensure that the trial was conducted and data

generated, documented and reported in compliance with the protocol, GCP and the applicable

regulatory requirements.

The quality assurance of the results and based on them the data used in the study is done through

the implementation of pre-study and within-study validation techniques and measurements,

meetings of representatives of the Sponsor and authorized CRO with study team to ensure

compliance with the requirements of the protocol and monitoring of the center and documentation

by an independent monitor.

5.6.1 Study validation techniques and measurements

5.6.1.1 Pre-study and within-study validation

a. Pres-study validation procedures

For the pre-study validation, calibration standards and quality control samples covering the

validation range, which exceeds the expected dynamic range for the actual study samples, were

prepared. These were then processed in sequences, according to the actual analytical method, over

a period of several days. The following table summarizes the samples analyzed to obtain the data

required. The results of the pre-study validation are presented on the following pages.

Table 5.6.1.1.1. Summary of the validation procedures:

SAMPLES PROCESSED DATA REQUIRED 1 Blank plasma extracts Specificity 2 Samples for calibration curves Regression equations

3 Samples for Quality Controls

Intra-day accuracy and precision Inter-day precision Linearity Signal to-noise ratios (LOQ and LOD)

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4 Pure solutions in mobile phase and blank plasma

Freeze-thaw stability over three cycles Recovery Robustness

Preparation of calibration standards and quality controls

Dynamic Range: After a single oral dose of 150 mg of CLOPIDOGREL, maximum

plasma levels for CCA in some volunteers of approximately 7-9 mcg/ml are to be

expected. A validation range approximately from 0.2 to 10 mcg/ml was therefore chosen

to allow determination of CCA plasma levels in the largest possible interval.

Procedure: Stock solutions of CCA (in methanol) and ticlopidine (in methanol) were

prepared in concentration of 1.0 mg/ml. The IS (ticlopidine) working solution was with

concentration 100µg/ml in water.

For method validation 10 calibration standards and 3 quality control samples were prepared.

They were prepared by dissolving CCA stock solution in water and then in drug-free human

plasma to produce the initial working calibration standards and quality controls. Thus prepared

samples were store in small volume (0.5 ml) in a freezer set at -28oC. External calibration

working standards were diluted with mobile phase.

Table 5.6.1.1.2. Preparation of Calibration Standards samples

Standard number

CCA concentrations

in plasma (mcg/ml)

CCA concentrations in water (mcg/ml)

(working solution)

Volume taken from the working solutions

of CCA (ml)

Diluted with plasma until (ml)

S10 0.2 2 1.0 10 S9 0.4 4 1.0 10 S8 0.6 6 1.0 10 S7 0.8 8 1.0 10 S6 1 10 1.0 10 S5 2 20 1.0 10 S4 4 40 1.0 10 S3 6 60 1.0 10 S2 8 80 1.0 10 S1 10 100 1.0 10

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Preparation of the samples for Quality Control (QC) The samples for Quality controls

were prepared by everyday dilution of the corresponding CCA standard with blank plasma to

reach final concentration of 0.4, 1 and 8 mcg/ml. Two injections were done for one run from

every concentration.

Full documentation of inter-laboratory standardization methods and quality assurance procedures,

plus results is provided under Appendix 12.1.10 “Documentation on inter-laboratory

standardization methods and quality assurance procedures”.

b. pre-study validation results

Specificity and selectivity

Selectivity was performed by recording the chromatograms of diluents, standards of CCA, blank

plasma and spiked plasma samples. In the chromatogram of diluents and blank plasma samples

there were no peaks matching that of CCA and IS (Rt between 5.8 - 6.1 min and 7.20 – 7.66 min

correspondingly).

The method selectivity is demonstrated with several representative chromatograms, where in

Figure 12.1.10.1.1 are demonstrated overlay chromatograms from blank drug free plasma sample

and blank plasma sample spiked with 0.2 mcg/ml CCA. Figure 12.1.10.1.2 represents

chromatogram from volunteer plasma sample at 1.0h after the oral intake of 150 mg

CLOPIDOGREL. It can be seen that CCA peak is well separated from the probable unknown

peaks.

The parameters of System suitability (Fig. 12.1.10.1.3) for CCA showed the following results:

Precision of the detector response for CCA: 0.3%

Resolution factor (Rs) for ticlopidine: 4.637 (SD 0.108)

Capacity factor (k’): 2.404 (SD 0.003)

Symmetry factor (tailing): 1.315 (SD 0.022)

The number of theoretical plates (N) for CCA is > 7022.

The number of theoretical plates (N) for ticlopidine > 6670.

Linearity and range of determination

Linearity was performed at 10 concentration points for CCA (excluding blank values) in

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concentration range from 0.2 to 10 mcg/ml, run in 3 different days. Spiked plasma samples were

prepared and analysed as described in analytical procedure. Linear regression equation,

coefficient of regression, slope of regression line and intercept were calculated. The results for

CCA are presented in Table 5.6.1.1.3. The graphical presentation of linearity is shown in the Fig.

12.1.10.1.1.

Table 5.6.1.1.3. Intercept, slope and r2 in 3 different days (CCA)

day intercept slope r2 1 0.004688 0.9968 0.9994

2 -0.004688 1.003 0.9997

3 -0.004477 1.002 0.99988

mean 0,004617 1,0006 0,99966

SD 0,000121 0,00332

C.V. (%) 2,63 0,332

In the concentration range of 0.2 to 10 mcg/ml the dependency of peak area ratios of CCA/IS to

concentration of CCA is linear with regression line presented as y = bx + a where y is the average

peak area ratios while x represents concentration of CCA, b is the slope, a the intercept and r2 is

the coefficient of determination.

Accuracy

• Accuracy (intraday)

Intraday accuracy was determined by 5 times replicate analysis of plasma samples at 3 different

concentration levels of CCA: 0.4, 1 and 8 mcg/ml.. Spiked plasma samples were analysed as

described in analytical procedure. From 5 determinations average value and RSD for CCA were

calculated. The repeatability results are presented in Table 5.6.1.1.4.

Table 5.6.1.1.4. Intraday accuracy

sample 0.4 mcg/ml

1 mcg/ml

8 mcg/ml

CCA CCA CCA 1 0.39 1.089 8.198

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2 0.411 1.09 8.113

3. 0.429 1.053 8.113

4 0.404 1.091 8.191

5 0.417 1.107 8.148

mean 0.4102 1.086 8.1526

SD 0.0145 0.0198 0.0409 RSD (%) 8.6470 1.6851 0.0615

Both criteria: RSD (N=5) < 20% at low concentration level (LOQ) and RSD (N=5) <15% at

middle and high concentration levels were fulfilled.

Accuracy of injection

Accuracy of injection was performed by 6 consecutive injections of standard solution of CCA (6

mcg/ml) (Table 5.6.1.1.5). The calculated RSD for Rt and Area were 0.0% and 0.3%

correspondingly (Fig. 12.1.10.1.8).

Table 5.6.1.1.5. Accuracy of detector response

Sample CCA (6 mcg/ml)

Retention time (min)

Area (AU)

1 2 3 4 5 6

6.232 6.231 6.235 6.234 6.233 6.233

4237035 4243341 4256334 4244262 4270331 4264965

Mean SD %RSD

6.233 0.001 0.0

4252711.334 13256.427

0.3

Criterion RSD to be < 1.0% is fulfilled.

Precision

Intraday precision (Repeatability)

Intraday precision was determined by measuring individually prepared 5 spiked plasma samples

at 3 different concentration levels of CCA: low (0.4 mcg/ml), middle (1 mcg/ml) and high (8

mcg/ml). Spiked plasma samples were analysed as described in analytical procedure. The content

of CCA in mcg/ml plasma was calculated for each sample against the standard curve. From 5

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determinations the average value and relative error were calculated. The results are presented in

Table 5.6.1.1.6.

Table 5.6.1.1.6. Intraday precision

added (mcg/ml) added (mcg/ml) added (mcg/ml) 0.4 1 8

sample found (mcg/ml)

relative error (%)

found (mcg/ml)

Relative error (%)

found (mcg/ml)

Relative error (%)

CCA CCA CCA CCA CCA CCA 1 0.392 -2.00 1.149 14.90 8.613 7.66 2 0.443 10.75 1.122 12.20 8.469 5.86 3 0.443 10.75 1.151 15.10 8.583 7.29 4 0.438 9.50 1.151 15.10 8.289 3.61 5 0.434 8.50 1.164 16.40 8.11 1.37

mean 0.43 7.50 1.15 14.74 8.41 5.16

SD 0.02 0.02 0.21 RSD (%) 11.67 1.17 0.30

Both criteria: RSD (N=5) < 20% at low concentration level (LOQ) and

RSD (N=5) <15% at middle and high concentration levels were fulfilled.

Intermediate precision (formally known as ruggedness)

Interday precision were determined by measuring 5 individually prepared spiked plasma samples

as in 4.1. at 3 different concentration levels of CCA: low (0.4 mcg/ml), middle (1 mcg/ml) and

high (8 mcg/ml), in 3 different days. Spiked plasma samples were analysed as described in

analytical procedure. The content of CCA in mcg/ml plasma was calculated for each sample

against the standard curve. From 15 determinations the average values and relative error were

calculated. The results are presented in Table 5.6.1.1.7.

Table 5.6.1.1.7. Inter day precision

added (mcg/ml) added (mcg/ml) added (mcg/ml) 0.4 1 8


relative error (%)

found (mcg/ml)

Relative error (%)

found (mcg/ml)

Relative error (%)

CCA CCA CCA CCA CCA CCA 1/1 0.392 -2.00 1.149 14.90 8.613 7.66 1/2 0.443 10.75 1.122 12.20 8.469 5.86

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1/3 0.443 10.75 1.151 15.10 8.583 7.29 1/4 0.438 9.50 1.151 15.10 8.289 3.61 1/5 0.434 8.50 1.164 16.40 8.11 1.37 2/1 0.404 1.00 1.093 9.30 8.058 0.72 2/2 0.403 0.75 1.095 9.50 8.163 2.04 2/3 0.417 4.25 1.146 14.60 8.082 1.03 2/4 0.415 3.75 1.154 15.40 8.274 3.42 2/5 0.437 9.25 1.163 16.30 8.282 3.53 3/1 0.395 -1.25 1.143 14.30 8.253 3.16 3/2 0.391 -2.25 1.130 13.00 8.248 3.10 3/3 0.402 0.50 1.170 17.00 8.207 2.59 3/4 0.398 -0.50 1.146 14.60 8.369 4.61 3/5 0.389 -2.75 1.166 16.60 8.607 7.59

mean 0.413 3.35 1.143 14.29 8.307 3.84

SD 0.020 0.024 0.185 RSD (%) 4.928 2.067 2.230

Both criteria: RSD (N=15) < 20% at low concentration level (LOQ) and

RSD (N=15) <15% at middle and high concentration levels were fulfilled.

Sensitivity:

LOQ (limit of quantification): The lowest concentrations of CCA which corresponded of

criteria of precision, accuracy and signal-to-noise ratio 10:1 (RSD of the lowest

concentration of CCA from the calibration curve to be < 20%; see Fig 12.1.10.1.3) were

found out to be 0.2 mcg/ml (Fig. 12.1.10.1.1).

LOD (limit of detection): Upon the accepted criterion (signal-to-noise ratio 3:1) the LOD

for CCA was determined to be around 0.1 mcg/ml.

Recovery

The recovery of the analyte from plasma was quantified at tree different concentrations over the

calibration range used (0.6, 2 and 10 mcg/ml). This was performed using six individually spiked

plasma samples at each concentration assayed, and comparing CCA peak so obtained with those

of aqueous solutions of similar concentrations. The results are presented in Table 5.6.1.1.8. and

5.6.1.1.9.

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Table 5.6.1.1.8. Recovery of CCA spiked to plasma samples

Concentration (mcg/ml)

Area for InSt (n=6)

Area for ExSt (n=6)

Analytical yield(%)

10 8076333 8210775 98,36

2 1480479 1580454 93,67

0.6 520108 544127 95,59 Mean yield 95,87

Table 5.6.1.1.9. Recovery of IS spiked to plasma samples

Concentration of CCA(mcg/ml)

Area for InSt (IS)(n=6)

Area for ExSt (IS)(n=6)

Analytical yield(%)

10 3886791 3647823 106,55 2 3552749 3679404 96,56

0.6 3689170 3948551 93,43 Mean yield 98,85

The mean analytical yield of CCA and IS spiked to plasma samples as compared with CCA and

IS injected directly to HPLC system is around 95,87% and 98,85% correspondingly.

Stability:

The samples from pre-study method validation were stored in dark at -28oC.

Stability testing were performed using 3 spiked plasma samples at two different concentrations of

CCA: low (0.4 mcg/ml) and high (8 mcg/ml). Spiked plasma samples were analysed after

following storage conditions:

immediately (reference samples)

after tree freeze/thaw cycles

long term stability (standard samples stored at –28oC were tested after 1 month)

Autosampler stability test (after 24h)

The results of stability are presented in Tables 5.6.1.1.10, 5.6.1.1.11 and 5.6.1.1.12.

Table 5.6.1.1.10. Stability during freeze/thaw cycles

added (mcg/ml) added (mcg/ml) 0.4 8

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sample found

(mcg/ml) relative

error (%) found

(mcg/ml) Relative error (%)

CCA CCA CCA CCA 1 0.398 -0.50 8.178 2.23 2 0.423 5.75 8.266 3.33

Imm

3 0.428 7.00 8.039 0.49 mean 0.416 4.08 8.161 2.01

1 0.414 3.50 8.149 1.86

2 0.39 -2.50 8.18 2.25

Cycle 3

3 0.374 -6.50 8.327 4.09 mean 0.393 -1.83 8.219 2.73

Table 5.6.1.1.11. Long term stability (standard samples were frozen on February 1st and tested

after 1 month on February 28th)

added (mcg/ml) added (mcg/ml) 0.4 8


relative error (%)

found (mcg/ml)

Relative error (%)

CCA CCA CCA CCA 1 0.398 - 8.178 - 2 0.423 - 8.266 -

Imm

3 0.428 - 8.039 - mean 0.416 - 8.161 -

1 0.434 8.50 8.299 3.74

2 0.398 -0.50 8.107 1.34

After 3

months 3 0.414 3.50 8.219 2.74

mean 0.415 3.83 8.208 2.6

Table 5.6.1.1.12. Autosampler stability (after 24h)

Standard First injection Found (mcg/ ml)

Second injection (after 24 Found (mcg/ ml)

0.404 0.443 0.403 0.443

0.4 mcg/ml

0.415 0.438 Mean 0.407 0.441 SD 0.007 0.003

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C.V. (%) 1.635 0.654

8.253 8.583 8.248 8.289

8 mcg/ml

8.369 8.11 Mean 8.290 8.327 SD 0.068 0.239

C.V. (%) 0.826 2.868

According to the results presented in Tables 5.6.1.1.10, 5.6.1.1.11 and 5.6.1.1.12, CCA added to

plasma samples are stable after tree freeze/thaw cycles. Long-term stability of spiked plasma

samples (after 1 month stored at –28oC) is satisfactory. CCA concentrations did not change

significantly in samples left for 24h in the autosampler.

Robustness:

The factors that could have a potential influence on chromatographic separation were examined as

follows:

Table 5.6.1.1.13. HPLC factors and their levels with regards to the robustness test

Code Factor Unit Limits Level (-1) Level(+) Nominal A pH value - ± 0.2 units 3.3 3.7 3.5 B Temperature oC ± 12% rel. 22 28 25 C Buffer molarities mM ± 8% rel. 23 27 25 D Organic phase % ± 10% rel. 16.2 19.8 18 E Modifier (THF) % ± 10% rel. 1.8 2.2 2.0 F Flow ml/min ± 10% rel. 0.9 1.1 1.0 G Dummy - ± 1 -1 +1 0

For the determination of the influence of these six factors, a Plackett-Burman test plan with 8

experiments was chosen using Design Expert 7.1software.

Table 5.6.1.1.14. Plackett-Burman test plan for n= 8

Exp. Factor A B C D E F G 1 + + + - - + - 2 - + + + + - - 3 - - + + - + + 4 + - - + + + + 5 - + - - + + + 6 + - + - + - - 7 + + - + - - - 8 - - - - - - +

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The experiments were carried out in a randomized order (as proposed by the software) and the

target values (Rs) were determined by Waters Empower software (Table 5.6.1.1.15).

Table 5.6.1.1.15. Determined target values “resolution Rs”

Experiment Response (resolution Rs) 1 7.144 2 2.697 3 2.382 4 5.786 5 2.216 6 6.507 7 6.808 8 2.198

Analysis of variance and effects were calculated for the analysis of the experimental design

(Tables 9e) along with a graphical analysis of the results with standardized Pareto diagrams.

Table 5.6.1.1.16. Calculated effects for each individual factor and their 95% confidential interval

Factor Resolution Rs Effect P-value A: pH 2.507 0.0933 B: Temperature -1.94 0.1576 C: Buffer -2.255 0.1033 D: Organic phase 1.149 0.1188 E: Modifier -2.421 0.0945 F: Flow 1.87 0.1355

It is evident that the method used is with significant robustness to small deviation in

chromatographic conditions.

Conclusion:

The HPLC analytical method for CCA determination in spiked human plasma showed parameters

of precision, accuracy, linearity, limits of quantification and detection and specificity in the limits

of validation criteria. Additionally the stability of the analyte was evaluated. The results of this

validation show that CCA can be analyzed in human plasma in the concentration range 0.2 to 10

mcg/ml according to the method described with sufficient reliability for pharmacokinetic studies.

c. within-study validation procedures

A total of 768 frozen plasma samples were received by Biopharmacy&Pharmacology Research

S.A. lab (Bucharest, Romania) on between the 5th and 18 of May 2008 for the CLOPIDOGREL

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study (Study No.: CLOP_REPL_0807). The samples were analyzed here with the

methodological and technical supervision of specialists from Dept.Drug Toxicology,

Inst.Neurobiology, BAS, Sofia, Bulgaria. The samples were stored in a freezer set at -20°C and

were analyzed in sequencees by the method described in validation report between the 6th and

18th of May 2008. Each sequences consisted of seven calibration standards and three triple

quality control samples, together with study samples from both phases of two volunteers.

Preparation of Calibration Standards and Quality Control Samples: Calibration

standards and quality control samples were prepared as described previously (see table 1

of Pre-study Validation Report). For the analysis of the actual trial samples, the calibrators

and quality controls used are listed on Table 5.6.1.1.17.

Table 5.6.1.1.17. Calibration Standards and Quality Controls used

Standard No. Concentrations of CCA (mcg/ml)

Quality control (No.) Concentrations of CCA (mcg/ml) (ng/ml)

S1 10 QC3 0.4

S2 6 QC2 1

S3 4 QC1 8

S4 2

S5 0.8

S6 0.6

S7 0.2

Sequential coding

Standards/Controls identification

In the analytical laboratory each Standard/Control Sample received a simplified name. The correspondence of the names with the tube labels is given below:

Standard Samples

Sx_mno

where:

- x represents the number of the standard (from 1 to 7)

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- mno represents the number of the sequence (from 001 to 012)

Control Samples

QCuPv_abc

where:

- u represents the number of the control (from 1 to 3)

- v represents the number of the sequence (from 1 to 12)

- abc represents the control’s set number within the sequence (from 001 to 003)

Sample code description

The real plasma samples, arrived from the clinical center were labeled with:

- name of the drug;

- code of the study;

- phase of the study;

- volunteer number;

- sample number (from 01 to 16, corresponding to the sampling order).

The analyses have been performed in parallel on the samples of the two phases

following, in ascending order, the progressive subject numbering and the sample

numbering within the same sample set.

The analyst was not informed about the type of administered treatment in the phases of

the study.

In the analytical laboratory each sample received a simplified name. The

correspondence of the names with the tube labels is given below:

CLRxymno

where:

- xy represents the number of the volunteer (from 01 to 24)

- mno represents the number of the sample (from 001 to 032)

Samples codes description

Sample code Phase I (collection)

Phase II (collection)

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CLRxy001 1 -

CLRxy002 - 1

CLRxy003 2 - CLRxy004 - 2 CLRxy005 3 - CLRxy006 - 3 CLRxy007 4 - CLRxy008 - 4 CLRxy009 5 - CLRxy010 - 5 CLRxy011 6 - CLRxy012 - 6 CLRxy013 7 - CLRxy014 - 7 CLRxy015 8 - CLRxy016 - 8 CLRxy017 9 - CLRxy018 - 9 CLRxy019 10 - CLRxy020 - 10 CLRxy021 11 - CLRxy022 - 11 CLRxy023 12 - CLRxy024 - 12 CLRxy025 13 - CLRxy026 - 13 CLRxy027 14 - CLRxy028 - 14 CLRxy029 15 - CLRxy030 - 15 CLRxy031 16 - CLRxy032 - 16

Linearity: Based on the results of the pre-study validation a linear regression equation

was used throughout. The reproducibility of the coefficient of determination (r2)

throughout the study, as shown in table 1, is indicative of the method's stability (Table

5.6.1.1.18.).

Table 5.6.1.1.18. Regression Equations

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Sequence Date Volunteer No. Linear Regression Equations

Coefficients of Determination

(r2)

1 08.05.2008 1, 5 Y = 0,1930X + 0,00258 0,998949

2 09.05.2008 6, 7 Y = 0,2136X + 0,001817 0,999569

3 10.05.2008 8, 9 Y = 0,2059X + 0,00899 0,999417

4 10.05.2008 11, 12 Y = 0,2171X + 0,002693 0,999066

5 11.05.2008 13, 14 Y = 0,2163X + 0,001197 0,999811

6 12.05.2008 16, 17 Y = 0,2189X + 0,000898 0,999898

7 12.05.2008 18, 19 Y = 0,2404X + 0,002799 0,999184

8 13.05.2008 21, 23 Y = 0,2543X - 0,004157 0,998388

9 14.05.2008 24, 2 Y = 0,2008X + 0,001544 0,999647

10 15.05.2008 3, 4 Y = 0,2081X + 0,001426 0,999714

11 15.05.2008 10, 20 Y = 0,2254X + 0,001784 0,999629

12 16.05.2008 15, 22 Y = 0,2277X + 0,0009 0,999906

Mean Y = 0.2189X + 0.002565 0,999432

Calibration Curve Data: The results of the back-calculated calibration standards have

shown that the mean precision and accuracy had to be acceptable across the whole

calibration range and all prescribed acceptance criteria are met. For a sequence to be

acceptable, at least five of the seven calibration standards must be within ±15% or ±20%

(<3xLOQ), Sensitivity was also maintained throughout the study, which can be seen from

the precision and accuracy of standards with low concentrations (Table 5.6.1.1.19.).

Table 5.6.1.1.19. Calibration curves data

Sequence S1 (10

mcg/ml)

S2 (6.0

mcg/ml)

S3 (4.0

mcg/ml)

S4 (2.0

mcg/ml)

S5 (0.8

mcg/ml)

S6 (0.6

mcg/ml)

S7 (0.2

mcg/ml) 1 10,15 5,83 3,87 2,03 0,79 0,59 0,20 2 10,01 5,90 4,13 1,96 0,84 0,64 0,19 3 9,89 6,12 4,12 1,95 0,83 0,57 0,21

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4 9,93 6,20 3,83 2,07 0,81 0,63 0,20 5 9,99 6,00 3,97 2,11 0,80 0,57 0,20 6 9,96 6,05 4,02 2,05 0,79 0,57 0,18 7 9,99 6,16 3,81 1,94 0,83 0,61 0,19 8 10,20 5,74 3,86 2,02 0,83 0,63 0,22 9 9,95 6,00 4,12 2,00 0,82 0,63 0,19 10 9,92 6,10 4,07 2,01 0,76 0,56 0,21 11 9,96 6,14 3,91 1,98 0,82 0,58 0,22 12 10,03 5,99 3,93 2,02 0,83 0,57 0,22 N 12 12 12 12 12 12 12

Mean 10,00 6,02 3,97 2,01 0,81 0,60 0,20

SD 0,093 0,139 0,120 0,051 0,023 0,030 0,014

C.V.,% 0,93 2,31 3,02 2,54 2,80 5,04 6,76

Quality Control Data

Three quality control samples were assayed in triplicate with each sequence (tested at the

beginning, between and the end of each volunteer samples). All acceptance criteria for precision

and accuracy were within the prescribed limits indicating that the method functioned acceptably

throughout the study period. For a sequence to be acceptable, six of the nine controls, including at

least one at each concentration, must be within the acceptance range.

Sequence QC1 (8.0 mcg/ml)

QC2 (1.0 mcg/ml)

QC3 (0.4 mcg/ml)

1 8,24 8,22 8,04

1,038 1,014 1,023

0,397 0,404 0,400

2 7,91 8,01 8,11

1,074 1,069 1,128

0,390 0,391 0,385

3 7,98 8,11 8,12

1,079 1,128 0,968

0,392 0,388 0,385

4 8,11 8,12 8,23

0,970 0,957 1,003

0,351 0,303 0,395

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5 8,05 8,02 8,07

1,093 1,058 1,110

0,405 0,390 0,425

6 7,94 8,06 8,04

1,077 1,088 1,115

0,394 0,406 0,412

7 7,99 8,01 8,00

1,078 1,066 1,030

0,423 0,403 0,430

8 8,12 8,42 8,32

1,102 1,113 1,185

0,402 0,405 0,409

9 8,10 8,09 8,09

1,184 1,164 1,139

0,406 0,401 0,397

10 8,19 8,16 8,17

1,042 1,500 1,003

0,410 0,416 0,418

11 8,14 8,11 8,04

1,072 1,077 1,007

0,402 0,424 0,423

12 7,93 7,90 8,14

0,954 1,048 1,217

0,393 0,428 0,402

N 36 36 36 Mean 8,09 1,08 0,400

SD 0,11 0,10 0,023 C.V.,% 1,35 8,89 5,649 Spiked

concentration 8 mcg/ml 1.0 mcg/ml 0.4 mcg/ml

Accuracy bias (%) 1.125 8,0 0.0

The data presented here indicate that the method used for the determination of CLOPIDOGREL

CARBOXYLIC ACID in plasma samples generated during the clinical phase of the study was

suitably validated prior to analysis and remained consistent throughout the period of analysis. The

analyte was also stable in plasma under the conditions and term of storage. The results are

therefore suitable for pharmacokinetic analysis.

5.6.1.2. Meetings with Investigators

The CRO has organized 1 meeting with the Investigators’ team before the study. At the meeting

the team was introduced to the medicinal products subject of testing in this study. Another

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meeting has been organized by the CRO, where in the form of a training session, to ensure the use

of standard terminology and the collection of accurate, consistent, complete, and reliable data.

The team has been given all study documentation and received detailed explanations on issues

from the CTP, CRF, IB. It has been announced that a central laboratory for all paraclinical tests

will be used and the procedures of blood sampling and processing at the center have been

discussed in details..

5.6.1.3. Monitoring

The CRO has contracted an independent monitors from Mediclintrial Support Ltd and MaxiMax

International Co. The company has good experience in monitoring. The monitoring has been

effected by Mihai Manolache and he has provided 3 monitoring reports for his visits before,

during and at the end of the study. The monitoring protocols are provided in Appendix 12.1.8.

5.7 Statistical Methods Planned in the Protocol and Determination of Sample Size

5.7.1 Statistical and Analytical Plans

5.7.1.1. Initial description /Population description

Demographic characteristics (age, weight, height) were tabulated by subject and summarized by

descriptive statistics (mean, standard deviation, standard error of the mean, coefficient of

variation, ranges, n).

Haemodynamic parameters (blood pressures and heart rates supine and standing), heart rate,

laboratory parameters (haemotology, biochemistry, enzymology) recorded on screening, were

tabulated by subject and summarized by descriptive statistics (mean, standard deviation, standard

error of the mean, coefficient of variation, ranges, n).

Conformity with inclusion and non-inclusion criteria (in particular ECG recordings,

cardiovascular parameters, clinical examination, urinary parameters) was checked and the results

were reported on individual tables.

Medical history and concomitant treatment, if any, were tabulated by subject.

Duration of each period of treatment, wash-out, screening, and end of study visit duration were

listed by subject. Number of drug units prescribed and given or not was listed by subject.

5.7.1.2. Analysis of pharmacokinetic data

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For all the pharmacokinetic parameters, descriptive statistical data were calculated: mean value,

median value, minimum, maximum and standard deviation. The ratio between the calculated

medians with the corresponding 25-75% distribution levels for every pharmacokinetic parameters

of the test and reference drug was represented by Box-Whisker plots.

Statistical comparisons in pharmacokinetic variables for AUC and Cmax were done by analysis of

variance (ANOVA) using the model for a two-period, two-treatment crossover design as proposed

by Grizzle (1965) by a computer program EquivTest 2.0 (Statistical Solutions). This model takes

into account sources of variation due to subjects, treatments, sequence and periods.

The information obtained from ANOVA served to calculate the correspondent 90% confidence

intervals and 90% Westlake symmetrical intervals for AUC(0-∝), AUC(0-t) and Cmax of the tested

preparation as a ratio to the correspondent values of the referent preparation using parametric and

nonparametric methods with log-transformation of data. The same confidence intervals were

calculated also by the method of Hauschke and Steinijans (1992) (a distribution independent

approach) as well as by the Schuirmann’s parametric test and the nonparametric tests of Hodges-

Lehmann and Wilcoxon-Mann-Whitney.

As a discrete variable Tmax was analyzed by means of a non-parametric analysis of variance

(Wilcoxon rank sum test) at a 90% confidence interval.

Using the multiplicative model, the acceptance ranges for bioequivalence after log-transformation

of the data, should be: for AUC-ratio and for Cmax-ratio the 90% confidence interval should lie

within a range of 0.80 - 1.25. Using the additive model, the acceptance ranges for bioequivalence

without log-transformation of the data for Tmax difference should lay between ± 2 hours.

5.7.1.3. Pharmacokinetic variables calculations and principle equations

Based on the obtained plasma concentrations/time data of CCA the following

pharmacokinetic parameters were calculates using software KINETICA™ 4.2 (Innaphase

corporation, USA) in noncompartmental mode.

Primary parameters: AUC(0-t) and AUC(0-∞) (area under the curve of the plasma

concentrations until the last sampling time and infinity), Cmax (maximum plasma concentration).

Secondary variable: Tmax (time of reaching the maximum plasma concentrations).

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Additional variables: Kel (elimination rate constant), MRT (mean residence time), Clapp

(total clearance) and t1/2 (elimination half-life).

The following principle equations have been used:

The individual as well as the mean plasma concentrations with the standard deviations are

reported at each time point for all preparations administered.

AUC(0-∞) has been calculated from measured data points by means of linear trapezoidal

rule up to last sampling point Clast and extrapolated to infinity by adding Clast/β, β denoting

the last-squares estimate at the terminal elimination constant and Clast denoting the

concentration value at tlast estimated by terminal logarithmic linear regression.

The peak plasma concentration (Cmax) and the time to the peak plasma concentration

(Tmax) were taken directly from the measured data.

The elimination half-life (t1/2) was calculated as t1/2=ln(2)/(-b) where b was obtained as the

slope of the linear regression of the in-transformed plasma concentrations versus time in

the terminal phase of the plasma curve.

The mean residence time (MRT) was also calculated derived from AUC(0-∞) and the

elimination constant.

The corresponding elimination rate constant (Kel) and plasma clearance (Clapp) were also

calculated.

5.7.1.4. Audits and Inspections

According to national regulations, the Health Authorities could made at any moment an

inspection in order to verify the application of regular requirements and the respect of the Good

Clinical Practice.

By signing the protocol, the investigator has authorized an access to all files of the study to audit

team of the Clinical Quality Assurance Department of Sopharma AD and to inspectors of Health

Authorities.

In case of an inspection by the Health Authorities, the following points might be controlled:

the overall study organization;

the qualifications of the staff responsible for conducting the study;

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the quality of the equipment;

the informed consent forms;

the acquisition of the Ethics Committee's opinion;

the method for dispensing and storing drug supplies;

the conduct of the study (data collected);

the archiving of the study documents.

Pharminform AD have to be notified on future inspection by the authorities. Pharminform AD

should inform Sopharma AD and authorize Sopharma AD to participate at this inspection.

Pharminform AD and its team will be available during the auditors visit, giving them access to the

technical site and the study material kept at the CRO.

The audit team can than evaluate the accuracy and the appropriateness of data and insure that

clinical study would be carried out accordingly to the Good Clinical Practice and to regulations in

force.

The subject's anonymity should be safeguarded and data checked during the audits should remain

confidential. The auditors, linked by the professional secret, could not divulge any personal

information.

In the course of these audits or inspections, data appearing in CRFs should be compared to the

original files. The totality of the documentation to be archived by the investigator as well as the

accounting of study products should be equally verified.

By the time this report has been prepared, no audits or inspections have been initiated neither by

regulatory authority, nor by any other inspecting or auditing institution locally or world-wide.

5.7.2 Determination of Sample Size

The number of volunteers needed for the study was calculated taking into account published data

for clopidogrel carboxylic acid bioavailabilty for AUC(0-24) (Souri E. at al., Biomed.Chromatogr.,

2006, 20, 1309-1344). After log-transformation of the data, the MSE from ANOVA analysis

showed value for CV about 13.3%. This value showed that a number of at least 18 volunteers is

needed to be fulfill the 20% difference criteria between the mean values for AUC of test and

reference preparation with power α = 0.05.

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The calculation was done using software Statmate 2.0 (Graphpad) by equation:

N ≥ 2δ2/∆2 (t(1-α)/2 + t(1-β))2

where t = t value ; δ = MSE ; 1-β = 0.80; α = 0.05 and ∆ = 0.2 (20%)

5.7.3. Statistical Analysis Plan

The relative bioavailability of the test condition with respect to the reference condition has been

determined from the individual ratios of the independent parameters Cmax, Tmax, AUC(0-t) and

AUC(0-∞) after the corresponding administrations.

For all the pharmacokinetic parameters, descriptive statistical data were calculated: mean value,

median value, minimum, maximum and standard deviation. The ratio between the calculated

medians with the corresponding 25-75% distribution levels for every pharmacokinetic parameters

of the test and reference drug was represented by Box-Whisker plots.

Statistical comparisons in pharmacokinetic variables for AUC and Cmax were done by analysis of

variance (ANOVA) using the model for a two-period, two-treatment crossover design as proposed

by Grizzle (1965) by a computer program EquivTest 2.0 (Statistical Solutions). This model takes

into account sources of variation due to subjects, treatments, sequence and periods.

The information obtained from ANOVA served to calculate the correspondent 90% confidence

intervals for AUC(0-∞), AUC(0-t) and Cmax of the tested preparation as a ratio to the correspondent

values of the referent preparation using parametric and nonparametric methods with log-

transformation of data. The same confidence intervals were calculated also by the method of

Hauschke and Steinijans (1992) (a distribution independent approach) as well as by the

Schuirmann’s parametric test and the nonparametric tests of Hodges-Lehmann and Wilcoxon-

Mann-Whitney.

As a discrete variable Tmax was analyzed by means of a non-parametric analysis of variance

(Wilcoxon rank sum test) at a 90% confidence interval.

Using the multiplicative model, the acceptance ranges for bioequivalence after log-transformation

of the data, should be: for AUC-ratio and Cmax-ratio: the 90% confidence interval should lie

within a range of 0.80 - 1.25. Using the additive model, the acceptance ranges for bioequivalence

without log-transformation of the data for Tmax difference should lay between ± 2 hours.

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5.8 Changes in the Conduct of the Study or Planned Analyses

There have been no changes in the conduct of the study or planned analyses (e.g., dropping a

treatment group, changing the entry criteria or drug dosages, adjusting the sample size) instituted

after the start of the study should be described.

6. STUDY PATIENTS

6.1 Disposition of Volunteers

Twenty eight (28) male and women Caucasian volunteers have been recruited for participation in

the trial. Among the 28 volunteers recruited for the trial, twenty four (24) were included in the

study.

Below is a diagram representing the disposition of volunteers.

Out of 24 eligible for enrollment volunteers 12 have been randomized for Test product (green

color) and 12 for Reference product (blue color) during the First Study period. All patients

completed First Study period and entered Wash-out period. Following this period both groups

have changed treatment in a cross-over fashion at the entry of Second Study Period. All patients

completed this period as well. None have been withdrawn. Following 6 days after the Second

Study Period all volunteers appeared for their Follow-up (Final visit). There have been no adverse

events registered at this visit and thus all 24 patients can be assessed as eligible for their data to be

used for evaluation based these criteria.

In Appendix 12.2.1 a listing of all volunteers discontinued from the study but before enrollments

are presented with the specific reason for discontinuation noted.

6.2 Protocol Deviations

A protocol violation was defined as any infringement of the protocol selection criteria.

A protocol deviation was defined as any departure from the protocol design or procedures after

the subject had entered the trial.

The study was carried out in compliance with the protocol without any protocol deviation which

might have likely affect the pharmacokinetic of the treatments.

Thus no deviations related to study inclusion or exclusion criteria, conduct of the trial, patient

managements or patient assessment has been neither described, nor documented.

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Pharminform AD Replekpharm AD Project No.: CLOP-REPL-0807 – clopidogrel

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7. EFFICACY EVALUATION

7.1 Data Sets Analyzed

As the number of subjects who were randomized and entered is the same with the number of

subjects who completed each phase of the study, the 24 subjects were assayed and included in the

pharmacokinetic evaluation.

No patients have been excluded neither from the study, nor from the efficacy analysis.

7.2 Demographic and Other Baseline Characteristics

The anthropometric data of the 24 volunteers are given on Table 10.1.1. in Section 10.1.

Demographic data. The values are presented on Table 7.2.1 below:

Table 7.2.1. Summary of anthropometric data

age (years)

height (cm)

weight (kg)

average 24,7083 175,125 67,4167 median 23 178 66,5

SD 3,49508 9,5294167 9,63651 minimum 21 158 55 maximum 34 190 83

No relevant concomitant illness necessitating concomitant medication was noted at inclusion, as

presented on Table 10.1.2. in Section 10.1. Demographic data.

7.3 Measurements of treatment compliance

All the subjects received the single oral administration at dose 2x75 mg at Day 1 of each Study

Phase under the supervision of the investigator or the co-investigator. The treatment compliance

was perfect, because the administration of the compound was ensured by the pharmacist and the

drug quantization in blood samples was detected.

No deviation between real time and theoretical time of drug administration did occur during the

study.

The protocol provided for at the most 4 days between the Screening visit and the first Study

Phase, then at least 7 days between each phase. These theoretical periods were respected.

No concomitant treatment was given during the study.

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7.4 Efficacy results and tabulations of individual patient data

7.4.1 Analysis of efficacy

Figures 7.4.1.a and 7.4.1.b illustrate the mean plasma concentration time-course of CCA obtained

after the administration of 150 mg CLOPIDOGREL as KLOPIDOGREL® formulation (treatment

T = test) and as the reference formulation PLAVIX® (treatment R = reference) in the twenty four

healthy young male volunteers, in linear (a) and semi-logarithmic (b) scale.

Figure 7.4.1.a

Figure 7.4.1.b

0 10 20 30 400

1

2

3

4

5

test

reference

Mean

time (h)

µg/m

l

0 5 10 15 20 25 30 35 40 45-3

-2

-1

0

1Mean (log)

Test

Reference

time (h)

log

( µg/

ml)

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Mean pharmacokinetic parameters of CCA are presented on Tables 7.4.1.1. below:

Table 7.4.1.1.a

Preparation KLOPIDOGREL (TEST)

Parameter n Mean Median Min Max SD AUC(0-∞) 24 12,05 10,88 7,38 23,22 3,839 AUC(0-t) 24 11,347 10,025 7,18 20,78 3,546 Cmax (µg/ml) 24 4,01 4,2 2,01 6,03 1,044 tmax (h) 24 1,187 1 0,5 0,5379 0,5379 Kel (1/h) 24 0,182 0,145 0,048 0,402 0,1099 t½ (h) 24 5,66 4,78 1,73 14,58 3,766 Clapp (mg/(µg/ml.h)) 24 13,5 13,78 6,46 20,32 3,672 MRT (h) 24 5,809 4,825 2,97 15,5 2,8867

Table 7.4.1.1.b

Preparation PLAVIX (REFERENCE)

Parameter n Mean Median Min Max SD AUC(0-∞) 24 11,74 11,305 6,67 24,91 3,921 AUC(0-t) 24 11,08 10,665 6,58 23,45 3,6 Cmax (µg/ml) 24 4,45 4,285 2,61 6,34 1,012 tmax (h) 24 0,812 0,75 0,5 0,2242 0,2242 Kel (1/h) 24 0,2128 0,171 0,052 0,568 0,1317 t½ (h) 24 4,81 4,075 1,22 13,4 3,272 Clapp (mg/(µg/ml.h)) 24 13,97 13,275 6,02 22,48 4,1142 MRT (h) 24 5,036 4,355 2,3 9,45 2,1746

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7.4.2 Statistical/analytical issues

7.4.2.1 Statistical analysis and bioequivalence trial of tested treatment versus treatment of reference for CLOPIDOGREL.

The results of the statistical analysis of the pharmacokinetic parameters of CCA between the

KLOPIDOGREL formulation and the reference formulation PLAVIX are resumed on Table

7.4.2.1. below:

Table 7.4.2.1.

Used test for the statistical comparison Tmax Cmax AUC(0-t) AUC(0-∞)

Friedman test (χ2) N.S. - - - ANOVA

Treatment - N.S. N.S. N.S. Subject S,(p<0.001) S. (p<0.001) S. (p<0.001) Period N.S. N.S. N.S.

Power of the study 0.998 >0.999 >0.999 Bioequivalence test -

90% standard confidence interval

0.834 ÷ 0.966 0.948 ÷ 1.099 0.942 ÷ 1.111

Two one-sided T-tests (Schuirmann)

can conclude equivalence



Geometric mean ratio T/R

0.900 1.023 1.027

The results showed bioequivalence for the Tmax, Cmax, AUC(0-t) and AUC(0-∞) parameters.

7.4.2.2. Concentration/time data

Individual plasma CCA concentrations are given in Tables 12.2.5.1 and 12.2.5.2 in Appendix

12.2.5. For each subject the plasma concentration/time curves representing the test and reference

formulations tested are plotted. The linear and logarithmic graphs of the mean-value curves of

both formulations over the entire period of measurement are also given in Appendix 12.2.5.

The results of the pharmacokinetic parameters obtained after application of two drugs containing

CCA in a single dose of 150 mg (2 tablets KLOPIDOGREL® of 75 mg and 2 tablets PLAVIX® of

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75 mg), correspond to literature pharmacokinetic data obtained after single oral dose

administration of Clopidogrel - 150 mg.

7.4.2.3. Pharmacokinetic parameters and Bioavailability

AUC(0-∞) and AUC(0-t)

Tables 10.2.1 and 10.2.2 in Section 10.2. “Efficacy data” reflect the results of the calculation of

the area under the plasma concentration-time curve. Since the calculated portion of the

extrapolated value for AUC between the last sampling point (48th hour) and infinity is 5.76% and

5.17% for the test and reference preparations respectively, it may be concluded that the last

sampling point has been chosen correctly. The statistical analysis shows that, with respect to

AUC(0-∞) and AUC(0-t), there are significant inter-individual differences among the individual

trial subjects (p≤0.001) and no effect of the period of intake of the test or reference preparation

(Appendix 12.1.9.).

Since the value for the point estimator for AUC(0-∞) and AUC(0-t) in all four tests used,

was close to 1.0, and the 90% confidence intervals lie within the range of 80-125%, it may be

asserted that the two preparations are bioequivalent with respect to these parameters.

Table 7.4.2.3.1. 90% Confidence Intervals for AUC(0-∞) of CCA

Test Point estimator 90% Confidence interval

ANOVA 1.027 0.942 ÷ 1.111

ANOVA-log 1.031 0.951 ÷ 1.118 Schuirmann’s permutation test 1.0308 0.9506 ÷ 1.1176

Hauschke 1.019 0.949 ÷ 1.091

Table 7.4.2.3.2. 90% Confidence Intervals for AUC(0-t) of CCA

Test Point estimator 90% Confidence interval

ANOVA 1.023 0.948 ÷ 1.099

ANOVA-log 1.024 0.956 ÷ 1.097 Schuirmann’s permutation test 1.0242 0.9563 ÷ 1.0969

Hauschke 1.017 0.957 ÷ 1.064

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Cmax

The results of the calculated maximal plasma concentration of CCA are shown in Table 10.2.3. in

Section 10.2. “Efficacy data”. They show that there are significant inter-individual differences

with respect to this parameter (Appendix 12.1.9.).

The test preparation reaches almost the same mean maximal plasma concentrations as those of the

reference preparation: 4.01 and 4.45 µg/ml respectively. By virtue of this, the values of the 90%

confidence interval in all four tests used were within the range of the criterion for bioequivalence

(between 80 and 125%).

Table 7.4.2.3.3. 90% Confidence Intervals for Cmax of CCA

ANOVA 0.900 0.834 ÷ 0.966 ANOVA-log 1.123 1.047 ÷ 1.204 Schuirmann’s permutation test 0.8902 0.8302 ÷ 0.9544

Hauschke 0.901 0.835 ÷ 0.966 ANOVA 0.900 0.834 ÷ 0.966

Tmax

The preparations are bioequivalent with respect to the time needed to reach the maximal plasma

concentration – Tmax: 1,187h and 0,812 h for the test and reference preparations,

correspondingly (Table 10.2.4. in Section 10.2. “Efficacy data”, Appendix 12.1.9). Test

preparation shows differences in the upper time boundaries because of the significantly delayed

absorption of the drug by volunteer number four in the second period. This delay of absorption is

due not to the drug formulation but possibly to physiological reasons of this particular volunteer

in the second period of the study. The delay of absorption do not reflect in smaller extend of

absorption (no changes in the AUC and Cmax values).

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Table 7.4.2.3.4. 90% Confidence Intervals for Tmax of CCA

Test Point estimator 90% Confidence interval Wilcoxon 0.250 0.125 ÷ 0.500

Kel, t1/2, MRT, Clapp

The data of the calculations for the other pharmacokinetic parameters are reflected on

Tables 10.2.5. through 10.2.8. They show that there is no significant difference neither in the total

extent of the biological action (MRT) or in the rate of elimination of CCA from the two

preparations.

7.4.3 Tabulation of individual response data

Individual response data is presented in Appendix 12.2.6.

7.4.4 Efficacy conclusions

The comparative statistical analysis of the pharmacokinetic parameters reflecting the extent and

the continuance of the biological action of tested preparations (AUC and MRT) obtained in a

cross-over design trial in healthy volunteers from the plasma concentration/time curves showed

that they fulfill the statistical criteria for bioequivalence.

The test for treatment x period interaction was also not statistically significant at the 10% level,

which enabled treatment comparisons to be made.

Comparing the rate of absorption of both preparations, the time to reach the maximal plasma

concentration as well as the peak plasma concentrations itself, no differences could be expected in

the rate and the time of the onset of their biological effects.

From the results of the ratio analysis for AUC, Cmax and Tmax obtained under defined

conditions equivalence of the KLOPIDOGREL 75 mg (REPLEKPHARM AD) - Test and

PLAVIX® 75 mg (SANOFI-AVENTIS) - Reference formulations can be concluded with

respect to the rate and extent of absorption. Thus, in view of the clinical use, both

formulations are exchangeable without restrictions.

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8. SAFETY EVALUATION

The medication was well tolerated in all cases without any side effects found in volunteers,

registered as a result of volunteers’ complains or found with the support of objective

measurements.

Vital signs showed no marked changes throughout the study. No serious adverse events occurred.

Clinical laboratory and hematology parameters checked at the beginning and the end of the study

were in the normal range of values, while deviations from normal were assessed as insignificant

from both clinical and study perspective.

8.1 Extent of exposure

In summary the extent of exposure to test and reference products is the following:

Volunteer Duration of exposure Treatment

Each volunteer as per randomization scheme

one single administration KLOPIDOGREL film-coated tablets 2 x 75 mg, manufactured by REPLEKPHARM AD Batch No. 6676;

Each volunteer as per randomization scheme

one single administration PLAVIX film-coated tablets 2 x 75 mg, manufactured by SANOFI-AVENTIS Batch No.: 1444

The extent of exposure to Test and Reference products can be characterised in details as follows.

● Duration: All volunteers have received a single dose from both Test and Reference

products at the beginning of each study period in compliance with the randomization

scheme applied. (Appendix 12.1.7. Randomization scheme). The dose was applied in

the morning under fasting conditions. Test and Reference products have been applied to

all 24 volunteers, in a cross over manner as per the randomization scheme..

● Dose: A single dose of 150 mg of both products has been applied.

● Drug concentration: Drug concentration data is presented in Appendix 12.2.5.

8.2 Adverse events

8.2.1 Brief summary of adverse events

No adverse event experience can be described for this study.

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8.2.2 Display of adverse events

N/A

8.2.3 Analysis of adverse events

N/A

8.2.4 Listing of adverse events by volunteer

N/A

8.3 Deaths, other serious adverse events, and other significant adverse events

N/A

8.3.1 Listing of deaths, other serious adverse events and other significant adverse events

N/A

8.3.2 Narratives of deaths, other serious adverse events and certain other significant adverse

events

N/A

8.3.3 Analysis and discussion of deaths, other serious adverse events and other significant

adverse events

N/A

8.4 Clinical laboratory evaluation

Tables 10.3.2. (a through d) in Section 10.3. Safety data, present the paraclinical (laboratory)

parameters at the Screening visit. Of the initial biological abnormalities detected at screening,

none was considered as clinically significant by the Principal investigator.

The urinalyses were normal in all volunteers. The urinalysis results (detection of glucose, ketone,

proteins, haemoglobin, and leukocytes in urine) at the inclusion were negative for all volunteers

and are presented also on Tables 10.3.2. (a through d) in Section 10.3. Safety data.

The laboratory results from the Final visit are presented on Tables 10.3.3. (a through d) in Section

10.3. Safety data.

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8.4.1 Listing of individual laboratory measurements by volunteer

All individual laboratory data is presented in Appendices 12.3.1. and 12.3.2, page 7 and 27 of

each CRF and also on Tables 10.3.1. and 10.3.2. (a through d for both tables) in Section 10.3.

8.4.2 Еvaluation of each laboratory parameter

Summary evaluation of each laboratory parameter followed is presented for both Screening and

Final visit on Table 10.3.4. (a and b). Deviations of parameters by patient and visit are presented

in Tables 8.4.2.1 and 8.4.2.2. below.

Table 8.4.2.1 Deviation of laboratory parameters at the Screening visit

Biochemical parameters

Normal range M/F

Volunteer number

Out of range Values

None Hematological Parameters ESR 1-10 mm 2 22 mm ESR 1-10 mm 5 18 mm ESR 3-15 mm 14 30 mm ESR 3-15 mm 16 38 mm

Table 8.4.2.2 Deviation of laboratory parameters at the Final visit


Normal range Volunteer number

Out of range values

ALAT 17 – 79 UI/L 2 16 UI/L ASAT 14-36 UI/L 3 70 UI/L Creatinine 0.7-1.2 mg/dL 3 0.6 mg/dL Albumin 3.5-5 g/dL 9 5.2 g/dL Hematological Parameters Hemoglobin 12.0-16.0 g/dl 10 11.8 g/dl Erythrocytes 4.0-5.4 x 106/µl 10 3.91 x106/µl Leucocytes 4-11x103/µl 11 11.7 x103/µl ESR 1-10 mm 2 21 mm ESR 1-10 mm 5 19 mm ESR 3-15 mm 8 36 mm ESR 3-15 mm 14 42 mm ESR 3-15 mm 16 28 mm

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8.4.2.1 Laboratory Values Over Time

The summary changes in clinical laboratory parameters are analyzed on Table 10.3.5.

8.4.2.2 Individual Volunteer Changes

Individual volunteer changes in laboratory parameters are presented on Table 10.3.7 in Section

10.3. “Safety data”. All changes have been assessed as insignificant from both clinical and study

perspective, but for volunteers’ safety and well-being it has been decided that in 2 cases a

notification will be made to the general practitioner of the volunteer, namely for volunteers 4 and

24.

8.4.2.3 Individual Clinically Significant Abnormalities

No biological abnormality clinically significant occurred after the studied drugs administrations.

Some biological abnormalities observed at the inclusion as well as at the end of study assessments

are not clinically significant.

8.5 Vital signs, physical findings and other observations related to safety

The results from the follow up of cardiovascular system parameters and the clinical assessment

are presented on Tables 10.3.1. (a and b) and 10.3.6. respectively. The second table presents a

comparison for clinical assessment between Final and Screening visit. Such comparison for

cardiovascular parameters is presented on Table 10.3.1.c.

8.6 Safety conclusions

The overall safety evaluation of the Test product can be described as excellent based on the results

of this study. There have been no AE registered and the laboratory measurements deviations were

insignificant both at Screening and Final visit.

9. DISCUSSION AND OVERALL CONCLUSIONS

9.1. Discussion of the methodology

The objective was to evaluate and compare the relative bioavailability, and therefore the

bioequivalence of REPLEKPHARM AD- KLOPIDOGREL 2x75 mg formulation versus a

reference formulation PLAVIX, following 2x75 mg administration under fasting conditions. The

study was carried out according to the protocol. All the pharmacokinetic and safety assessments

were performed as planned in the protocol.

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9.2. Discussion of pharmacokinetics

From the 24 subjects included in this study, 24 were analyzed and included in the

pharmacokinetic and statistical analysis for the clopidogrel.

After the administration of 2x75 mg clopidogrel as KLOPIDOGREL formulation and as the

reference formulation PLAVIX, the mean plasma concentration time-courses of CCA present the

same pharmacokinetic profiles with minor differences between the two formulations.

The peak plasma concentration is bioequivalent between the KLOPIDOGREL formulation and

the reference formulation (respectively 4,01 ÷ 1,044 µg/ml and 4,45 ÷ 1,012 µg/ml). The peak

plasma concentration of CCA is attained at about 0.8 – 1.1 hours for the both formulations. The

AUC parameters showed that the AUC(0-∞) of CCA (12,05 ÷ 3,839 h.µg/ml and 11,74 ÷ 3,921

h.µg/ml for the test and the reference formulation, respectively) are bioequivalent after the

administration of the KLOPIDOGREL formulation and after the administration of the PLAVIX

formulation

The statistical analysis of the half life of elimination, clearance, rate of elimination and mean

residence time showed no significant difference between the values of CCA after the

administration of the KLOPIDOGREL formulation and after the administration of the reference

formulation PLAVIX.

As conclusion, the REPLEKPHARM AD- KLOPIDOGREL formulation dosed at 2x75 mg is

bioequivalent for clopidogrel (Tmax, Cmax, AUC(0-t) and AUC(0-∞) parameters) to the reference

formulation PLAVIX after a single oral administration of 2x75 mg clopidogrel. The peak plasma

concentration of CCA is identical between the two formulations (Tmax and Cmax) and the AUC

parameters are bioequivalent to the reference formulation.

9.3. Discussion of safety:

All the subjects included in this study (24 subjects) were considered for the safety analysis.

No death, no serious adverse event or adverse event did occur during the study.

The safety analysis shows that the treatments were well tolerated.

From cardiovascular safety point of view, the cardiovascular data, blood pressures, heart rates and

electrocardiogram parameters, didn't show clinically significant changes for all the subjects.

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From the laboratory safety, the biological assessments showed no clinically significant

abnormalities at the end of study.

9.4. General Conclusion:

The objective of the present study was to evaluate and compare the relative bioavailability, and

therefore the bioequivalence of REPLEKPHARM AD- KLOPIDOGREL 2x75 mg formulation

versus a reference formulation PLAVIX, following 2x75 mg administration under fasting

conditions. The results presented herein showed that the criteria used to estimate the

bioavailability and the bioequivalence of the REPLEKPHARM AD formulation and the reference

formulation were fulfilled. In fact, the 90% confidence interval of the relative mean Cmax and

AUC parameters as well as the ratio of the geometric means were strictly within the acceptance

range of 80 - 125% for CCA.

Therefore, it can be concluded that the REPLEKPHARM AD- KLOPIDOGREL 2x75 mg

formulation is bioequivalent for the drug clopidogrel to the reference PVAVIX following

2x75 mg administration under fasting conditions.

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10. TABLES, FIGURES AND GRAPHS REFERRED TO BUT NOT INCLUDED IN

THE TEXT

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10.1 Demographic data

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10.2 Efficacy data

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10.3 Safety data

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10.3.1 Displays of adverse events

Except for some deviation in the laboratory parameters, which were considered unsignificant and

not documented as adverse events, no other adverse events have been neither found nor

documented for the volunteers enrolled in this study.

10.3.2 Llistings of deaths, other serious and significant adverse events

There has been no cases of deaths, serious adverse events or significant adverse events.

10.3.3 Narratives of deaths, other serious and certain other significant adverse events

N/A

10.3.4 Abnormal laboratory value listing (each patient)

The abnormal laboratory values by volunteer are presented on Tables 10.3.4.1 and 10.3.4.2.

below, both at Screening and Final visits respectively:

Table 10.3.4.1 Abnormal laboratory values by volunteer at Screening visit.

Volunteer number

Biochemical parameters Normal range M/F Out of range Values

2 ESR 1-10 mm 22 mm 5 ESR 1-10 mm 18 mm 14 ESR 3-15 mm 30 mm 16 ESR 3-15 mm 38 mm

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Table 10.3.4.2 Abnormal laboratory values by volunteer at Final visit.

Volunteer number


Normal range Out of range values

2 ALAT 17 – 79 UI/L 16 UI/L 3 ASAT 14-36 UI/L 70 UI/L 3 Creatinine 0.7-1.2 mg/dL 0.6 mg/dL 9 Albumin 3.5-5 g/dL 5.2 g/dL 10 Hemoglobin 12.0-16.0 g/dl 11.8 g/dl 10 Erythrocytes 4.0-5.4 x 106/µl 3.91 x106/µl 11 Leucocytes 4-11x103/µl 11.7 x103/µl 2 ESR 1-10 mm 21 mm 5 ESR 1-10 mm 19 mm 8 ESR 3-15 mm 36 mm 14 ESR 3-15 mm 42 mm16 ESR 3-15 mm 28 mm

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11. REFERENCE LIST

● Chow, S-C., Liu, J-P. (1992): Design and Analysis of Bioavailability and Bioequivalence Studies. Published by

Marcel Dekker Inc., New York.

● Declaration of Helsinki, The Word Medical Association, Inc. October 1996

● Guidelines for Good Clinical Practice CPMP/ICH/135/95, 17 January1997 (ICH-GCP) First amendment September

1997

● Jeoung M.K., K.S.Kim, C.S.Kim, N.H. Kim, Y-B. Chung, J.T. Hong and D.-C. Moon, J.Liquid

Chromatography&Related Technologies, 28, 1299-1309, 2005.

● Medicinal products and pharmacies in Human medicine act (with amendments State Gazette, issue 30/2.4.1998 and

issue date 4.2.2000) (Section IV).

● J.Macek, P.Ptacek, J.Klıma, J. Chromatography B, 736, 231-235, 1999.

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