DRAFT GUIDELINES FOR BIOAVAILABILITY / BIOEQUIVALENCE STUDIES ON CONVENTIONAL AND  EXTENDED RELEASE DOSAGE FORMS

 

CONTENTS

 

NAMES OF COMMITTEE MEMBERS

INTRODUCTION

DEFINITIONS AND CLASSIFICATIONS

ORGANIZATION PREMISES AND FACILITIES

PROTOCOL AND STUDY DESIGN FOR CONVENTIONAL RELEASE DOSAGE FORMS

PROTOCOL AND STUDY DESIGN FOR EXTENDED RELEASE DOSAGE FORMS

METHODOLOGY FOR CONDUCT OF STUDY

ANALYTICAL METHODOLOGY AND VALIDATION

INVITRO DISSOLUTION

STATISTICAL EVALUATION FOR CONVENTIONAL DOSAGE FORMS

STATISTICAL EVALUATION FOR EXTENDED RELEASE DOSAGE FORMS

WAIVER REQUIREMENTS

DOCUMENTATION

APPENDIX 1:- IN-VITRO IN-VIVO CORRELATION

APPENDIX 2 :- ADVERSE DRUG REACTION

                                                APPENDIX 3 :- GOOD LABORATORY PRACTICE

 


 

1.      INTRODUCTION

 

1.1  Objective and Scope of the Guidelines

 

These guidelines are proposed to be an annexure to Schedule Y of the Drugs & Cosmetics Act, 1940.  It describes in vivo bioavailability / bioequivalence studies and in vitro dissolution testing recommended to applicants intending to submit Abbreviated New Drugs Applications (NDAs) for conventionals and extended release dosage forms administered orally.

 

These guidelines spell out the standards on procedures, norms, design, conduct, interpretation and evaluation of data from these studies.

 

These studies should be conducted for oral dosage forms which are systematically absorbed. For formulations existing in multiple strengths  of a given dosage form, bioequivalence studies may be conducted on the highest strength only, provided the

lower strenghts have excipients identical  to the highest strength and their dissolution profile matches that of the innovator and shows linear dissolution kinetics.

 

In those conditions where a suitable method for determining active drug is not available, it may be possible to obtain an indirect indication of bioavailability and bioequivalence by comparing the pharmacodynamic responses of the formulations.

 

1.2 Justification for an E.R. formulation

 

An ER dosage form could be considered for development if any of the following conditions exist for a drug:

·          The half-life of a drug is short requiring frequent dosing.

·          Maintenance of blood concentration within a narrow therapeutic range is desired.

·          High peak to trough concentration ratio leads to increased dosage requirement, reduced efficacy or increase in the side effects.

·          If the half-life of the drug is >12 hours, then development of an ER formaulation is justified only on the basis of reducing side effects or to minimize peak/trough   variation for drugs with narrow therapeutic index.

 

1.3 Need for Bioequivalence Studies for E.R. products

It was considered that guidelines different from those for conventional release formulation are required for ER dosage forms because of greater chances of intersubject variability and dose dumping. In addition,  there is a possibility of greater accumulation when the drug is given in repeated doses at the recommended dosage intervals.

 

 

 

 

In vivo bioequivalence studies recommended for approval of extended release products are designed to ensure that

 

·          The product meets the extended release label claim made for the E.R. preparation.

·          The  product does not release the active drug substance at a rate and extent leading to dose dumping.

·          There is no significant difference between the performance of the E.R. product and the reference product following single dose, dosing to steady state (depending on the study design) and food  effect.

 

Following is the check list of activities, which should  be evaluated during a bioequivalence study by the auditors.

 

                      Facilities where study is conducted

                      Description of general facilities (floor space, bench space per employee, etc)

                      Comments on general surroundings of the room

                      Can the laboratory and clinical facility appear adequate to support the workload

                      Does the laboratory have standard operating procedures available to all personnel

                      Are copies of standard operating procedures available to all personnel

 

Personnel

                      No and type of employees

                      Qualification, training and experience of staff

                      Assess the ability of the staff to perform the reported tests

 

Volunteers For Study

                      Time of arrival of the subjects at the clinical centre

                      Status of subjects on arrival, predose activities, adherence to acceptance/ exclusion criteria as listed in protocol

                      Time of drug administration

                      Bleeding time

                      Observation of vital signs of the subject during predose and post dose

                      Adverse reactions and monitoring of adverse reactions

                      Deviations from protocol in subject status, time of blood collection, etc.

                      Status of subjects after the completion of the study

                      Post study monitoring of the subjects

 

Sample Handling

                      Samples storage history

                      Is a sample custodian available to receive the samples and maintain a track record for the same

                      Equipment available for storing blood/urine samples

                      Does the storage equipment hold a temperature recording device

                      Is there adequate separation of samples to avoid sample mix-up (mixing samples of one study to another study)

                      For stored samples are the sample labels and identification tags intact.

 

1.4 Labelling requirements

 

         i.            “Extended Release” dosage form

       ii.            Frequency of administration

      iii.            Time of administration and relationship to food where applicable.

 

 

 

 

 

 

2.      DEFINITIONS

 

 

2.1   BIOAVAILABILITY

 

Bioavailability is a measure of the rate and extent of absorption of the active form or forms of a drug from its formulation as reflected by the concentration - time curve of the administered drug in systemic circulation.

 

2.2   BIOEQUIVALENCE

 

Two formulations of a drug are said to be bioequivalent if the rate and extent to which the drug reaches the systemic action after administration of their respective formulations are statistically comparable. In general, two products may be said to be bioequivalent if 90% Westlake’s confidence interval for Cmax, Tmax, mean  AUC(0-t) is within ± 20% of that of the reference product. The margin may be reduced to ± 10% for drugs with a very low therapeutic index, such as antiarrhythmic, antiepileptic and anticoagulant drugs.

 

2.3  PHARMACEUTICAL  EQUIVALENTS

 

"Pharmaceutical equivalents" mean drug products that contain identical amounts of the identical active drug ingredient, i.e., the same salt or ester of the same drug, in identical dosage forms, but do not necessarily contain the same ingredients, and that meet the identical compendial or other applicable standard of identity, strength, quality and purity, including potency and where applicable, content uniformity, disintegration time and/or dissolution rates.

 

2.4   PHARMACEUTICAL ALTERNATIVES

 

Pharmaceutical alternatives mean drug products that contain identical therapeutic moiety, its precursor, but not necessarily in the same amount or dosage form or as the same salt or ester. Each such drug product individually meets either the identical or its own respective compendial or other applicable standard of identity, strength, quality and purity, including potency and where applicable, content uniformity, disintegration times and/or dissolution rates.

 

 

2.5  THERAPEUTIC EQUIVALENTS

 

A medicinal product is therapeutically equivalent with another product if it contains the same active substance or therapeutic moeity and, when administered to the same individual, shows the same efficacy and toxicity as that product, whose efficacy and safety has been established.

 

 

 

2.6  CONVENTIONAL/IMMEDIATE RELEASE DOSAGE FORMS

 

A conventional dosage form is a formulation or a dosage form from which the active drug is released immediately  following administration.

 

2.7  MODIFIED RELEASE DOSAGE FORMS

 

A modified release dosage form is defined as one for which the drug release characteristic of a time course and/or location are chosen to accomplish therapeutic or convenience objectives not offered by conventional dosage form such as solutions, ointments and promptly dissolving forms.

Should the following be defined from USP

Modified release dosage forms may be recognized as :

 

(i)   Extended Release Dosage Forms and

 

(ii)   Delayed Release Dosage Forms

 

 

2.7.1  EXTENDED RELEASE DOSAGE FORMS

 

An extended  release dosage form is defined as one that allows at least a two-fold  reduction in dosing  frequency as compared to that drug presented as a conventional dosage form.

 

The terms controlled release, prolonged action, sustained release and programmed release etc., are used synonynously with extended release.

 

2.7.2  DELAYED RELEASE DOSAGE FORMS

 

A delayed  release dosage form is defined as one that releases a drug (or drugs) at a time other than promptly after administration, e.g., enteric coated products.

 

2.8  Cmax

 

This is the maximum drug concentration achieved in systemic circulation following drug administration.

 

 

2.9  Tmax

 

It is the time required to achieve maximum drug concentration in systemic circulation.

 

 

 

2.10  AREA UNDER THE CURVE (AUC)

 

Area under the curve is the total area under the biological fluid (serum, blood, etc.) concentration-time curve as  determined by the Trapezoidal rule.

 

2.10.1 AUC ( 0- t)

Area under the plasma concentration - time curve to the last quantifiable concentration to be calculated using trapezoidal rule.

 

 

2.10.2 AUC ( 0-t)

Area under the plasma concentration – time curve to the first dosing interval to be calculated under trapezoidal rule.

 
 


This area (AUC( 0-t)) is formed by joining the point on X axis at t to the beginning of the rising arm of the concentration curve at “A”.

 

The calculation of this area (AUC(0-t)) is possible by using trapezoidal rule for the area upto point “B” and then subtracting the area of  the triangle formed by “tAB” .

 

2.11   PHARMACODYNAMIC EVALUATION

 

It is a measurement of effect  on a (patho) physiological process as a function of time, after administration of two different products to serve as a basis for bioequivalence assessment.

 

2.12  ANALYSIS OF VARIANCE (ANOVA)

 

ANOVA is a statistical technique to identify sources of variance and estimate the degree of variability. In most bioavailability studies, there are three readily identified sources of variance namely formulation (Treatment), subject and period; hence it is a 3-way ANOVA.

 

2.13  WESTLAKE’S SYMMETRICAL CONFIDENCE INTERVAL

 

Westlake’s symmetrical confidence interval  is an interval symmetrical about the  hypothesized value, viz., 0 for difference in mean values of pharmacokinetic parameters and 1 for the ratio of mean values of the same pharmacokinetic parameters.

 

2.14  DISSOLUTION

 

Dissolution is defined as the process by which a drug substance from a dosage form dissolves in  a dissolution medium to yield a solution.

 

 

2.15 VALIDATION OF ANALYTICAL METHOD

 

Validation of an analytical method is the process by which it is established, by laboratory studies, that the performance characteristics of the method or process meet the requirements for the intended application.

 

2.16  DRUG DELIVERY SYSTEM

 

Drug delivery system is defined as a therapeutic system which releases drug at pre-determined rate for a fixed time either systematically or to a specified target organ.

 

2.17  ELIMINATION  HALF LIFE

 

The half life  (t½) is the time it takes for the plasma concentration or the amount of drug in the body to be reduced by 50%.

 

2.18      PHARMACOKINETICS

 

Study of the kinetics of absorption, distribution, biotransformation and excretion of drugs.

 

2.19      FIRST ORDER PROCESS

 

Process in which the rate of reaction is proportional to the  amount present is known as first order process.

 

2.20      FIRST ORDER ABSORPTION

 

Whenever the abssorption of a drug from a formulation is  depended on the concentration of the drug at the sight of release, it is called first order absorption (i.e., a fixed % of the available drug is being absorbed per unit time).

 

2.21  FIRST ORDER ELIMINATION

 

Whenever the amount of drug eliminated at any time is dependent upon the concentration of plasma at that time , is known as first order elimination.

 

2.22  LINEAR (FIRST ORDER) PHARMACOKINETICS

 

Whenever the pharmacokinetic parameters of a drug do not change with different doses , it is called first – order or linear pharmacokinetic.(Thus seminogarithmic plot of plasma concentration-time curve is linear).

 

2.23    ZERO ORDER PROCESS

 

Processes in which the rate of reaction is independent of the amount present is known as zero-order process.

 

2.24    ZERO ORDER ABSORPTION

 

Whenever the absorption of a drug from a formulation is independent of the concentration of the drug, it is called zero-order absorption.(i.e. the drug is absorbed from the formulation at a constant rate).

 

2.25    NON – LINEAR (ZERO ORDER) PHARMACOKINETICS)

 

Whenever the pharmacokinetic parameter of  drug change with different doses, it is called zero-order or non-linear pharmcokinetics. (The semilogarithmic plot of plasma concentration-time curve is non-linear).

 

Non-linearity in pharmacokinetics (i.e. changes in such parameters as clearance,volume of distribution and half-life as a function of dose or concentration of drug) usually is due ot saturation of protein binding, hepatic metabolism or active renal transport of the drug.

 

2.26  STEADY STATE

 

An equilibrium state where the rate of the drug input is equal to the rate of elimination during a given dose interval.

 

2.27  t(TAU)

 

The dosing interval (period between 2 successive doses as per recommended regimen).

 

2.28  (Cmin) SS

 

Mean trough level during steady state.

 

2.29  (Cmax)SS

 

Mean peak level during steady state.

 

2.30  (Cavg)

 

Cavg = (AUC)ss/t

 

2.31  AUC (0 –t)

 

Area under curve during first dosing interval in multiple dose study.

 

2.32     AUC 0-t (Single dose)

 

AUC0-t = Area under the plasma concentration - time curve to the last quantifiable concentration to be calculated using trapezoidal rule.

The graphical representation of AUC 0-t is as follows :

t = Last quantifiable point

 

2.33  AUCss

 

Is the area under the curve from t=0 to t=t during a dosing interval at steady state.

 

 

2.34 DEGREE FLUCTUATION (% FLUCTUATION)

 

It is the  ratio of (mean peak levels during steady state - mean trough level during steady state) to mean trough level during steady state, expressed as percentage

 

100  X [ (Cmax)ss - (Cmin)ss / (Cmin)ss ]

 

Though this formula depends on direct observations, it relies on two values, one of thich (Cmax)ss is difficult to estimate accurately unless many blood samples are taken within dosing interval in which case the following formula may be used.

 

 

 

% Fluctuation = [AUC(above Cavg) + AUC (below Cavg)]  X 100

                           ------------------------------------------------

                                                  AUCss

  

 

 

2.35 FLUCTUATION INDEX ( % PEAK-TROUGH FLUCTUATION)% PTF

 

 

% PTF = [ (Cmax) SS - (Cmin)SS ]/ Cavg

                                             Where (Cavg = AUCss/t)

                                                                  Therefore,

               

% PTF =[(Cmax)SS – (Cmin)SS]  x t / AUC ss

 

 

 

 

2.36  BIBLIOGRAPHY:

 

1)      Draft Guidelines for Bioavailability/Bioequivalence studies for conventional products

 

2)  Modified Release - Guidelines of the Expert Advisory Committee of the                

 

     Health Protection Branch of the Department of Health & Welfare, Canada 

 

     John Ruedy.

 

3) The Pharmacological Basis of Therapeutic. Vol - I Eighth edition -

 

    Goodman  & Gilman

 

4) Preclinical Drug Disposition A laboratory handbook-

 

    Francis L.S. Tse; James M. Jaffe.

 

5) Dissolution, Bioavailability & Bioequivalence - Hamed M.Abdou.

 

6) Clinical Pharmacokinetics :  Concepts and Applications – Rowland  & Tozer

 

 

 

 

 

3.      ORGANIZATION PREMISES AND FACILITIES FOR

      BIOAVAILABILITY / BIOEQUIVALENCE CENTER

 

3.1  LEGAL IDENTITY

 

 Legal identity implies that the body, conducting the bioequivalence / bioavailability “study” , or the parent organization to which it belongs, shall be registered with an appropriate statutory body.

 

    3.2  IMPARTIALITY CONFIDENTIALITY INDEPENDENCE AND

      INTEGRITY

 

The center shall:

a.      have managerial staff with the authority and the resources needed to discharge their duties.

b.      have arrangements to ensure that its personal are free from any commercial, financial and other pressures which might adversely affect the quality of their work.

c.       be organized in such a way that confidence in its independence of judgement and integrity is maintained at all times.

d.      have documented policies and procedures, where relevant, to ensure the protection of its clients’ confidential information and proprietary rights.

e.      not engaging any activity that may endanger the trust in its independence of judgement and integrity in relation to its activity.

f.        have documented policies and procedures for the safety of human rights and the use of human subjects in research. Should the ICMR guidelines be referred to?.

g.      have documented policies and procedures for research integrity including procedures dealing with and reporting possible misconduct in research and science.

 

3.3 ORGANISATION AND MANAGEMENT

 

The center shall be organized in such a way that its permanent and temporary staff are able  meet the functional requirements that are stated below.

 

a.      It should have a chief investigator who has overall responsibility of all technical operations.

b.      The ‘center’ should have  identified persons to perform the following functions.

(i)                  Clinical Pharmacological Unit (CPU) management

(ii)                Analytical Laboratory Management

(iii)               Data Handling and Interpretation

(iv)              Documentation and Report Preparation

(v)             Liaison between the center, the client and Drug Controller Office.

 

3.4  DOCUMENTED STANDARD OPERATING PROCEDURES

 

The center shall establish and maintain a quality system appropriate to the type, range and volume of its activities. All operations conducted at the center shall have authorized documented procedures available to the personnel for ready reference. The procedures covered will be as follows :

 

a.      Good clinical and laboratory practices

b.      Maintenance of working standards (pure substances) and documentation.

c.       Procedure for withdrawal, storage and handling of biological sample.

d.      Procedure for maintenance, calibration and validation of instruments.

e.      Safety procedure for

(i)                  Emergency situations

(ii)                Handling of biological fluids

(iii)               Laboratory hazards

f.        Disposal procedures for clinical samples

g.      Documentation of CPU observations, volunteer data and analytical data

h.      Volunteer  screening procedures

i.         Volunteer recycling procedures

j.        Usage of clinical disposable

Are the above SOPs sufficient?

 

3.5 PREMISES AND FACILITIES

 

The center shall have adequate number of personnel having necessary training and experience to carry out the following specialized functions :

 

a.      Supervision

b.      Health care for volunteers

c.       Collection handling and storage of biological samples

d.      Analysis of samples

e.      Laboratory cleaning and maintenance

f.        Administration

g.      Waste management and disposal

h.      Documentation and preparation

i.         Data collection, analysis and interpretation

 

3.6 CLINICAL PHARMACOLOGICAL UNIT

 

It must have additional space and facilities to house at least 12 volunteers as per the general norms of a general hospital.

Additional space and facilities should also be provided for the following :

 

a.      Office and clerical duties

b.      Sample storage

c.       Control sample

d.      Wet chemical laboratory

e.      Instrumental Laboratory

f.        Radio Immuno – Assay room (optional)

g.      Library

h.      Computers (Optional)

i.         Microbiological laboratory (Optional)

j.        Documentation room

 

 

4.      PROTOCOL AND STUDY DESIGN FOR CONVENTIONAL

     DOSAGE FORMS.

 

 

4.1 PERSONS RESPONSIBLE

 

The  study protocol shall contain the following elements :

The names of the principal investigator(s), other persons responsible for conducting the study, and the names of the laboratory and the clinical pharmacology unit where the study will be conducted, should be mentioned. Complete official address of the premises of the laboratory and contact phone numbers of the person in-charge of the

Study, with time for contact (in case of more than one phone numbers), should also   

be mentioned.

 

 The name of the sponsor and / or other person responsible for monitoring the study should be mentioned along with the complete official as well as residential addresses and phone numbers with contact times.

 

4.2 OBJECTIVE OF THE STUDY

 

To compare the bioavailability of one or more pharmaceutically equivalent  formulations and to assess the degree of bioequivalence within and between the formulations been tested.

 

All the subsequent applicants should conduct bioequivalence by comparing the test product with the innovator’s product as another product permitted by the D.C.(I). For Bioequivalence studies, the innovator’s product should be used as a reference product because :

(i)                  its clinical efficacy and safety profile is usually well documented in extensive trials;

(ii)                statistical tests of significance never prove the equivalence of two products, but rather the absence of non-equivalence at an acceptable level of risk (the significance level)

 

4.3 ETHICAL CONSIDERATIONS

Should it be in accordance with GCP guidelines?

All studies shall be conducted in accordance with the Helsinki Declaration (reference)

There should be an ethical committee to approve the study and ensure that the study is carried out as per scientific and ethical norms. The ethical committee should be consist of qualified personnel including at least a clinical pharmacologist, a consultant physician, a social worker and a person with legal background. Sufficient and relevant data on animal studies and human studies should be made available to the ethical committee. If for any reason, the approved protocol need amendments, the amended protocol should be approved by the ethical committee and the Drugs Controller (India). Finally, the amended and approved protocol should be submitted with the report.

If the formulations under study, are likely to adversely affect the health of the volunteers, e.g. anticancer drugs, oral hypoglycemic drugs, etc., healthy volunteers should not be enrolled in the study. Patients  should be enrolled in such cases. An informed and written consent form of the volunteer must be obtained and kept on record. The participating volunteers should be provided with full information about the study in a language understandable to them.

 

The information to the volunteer should include :

a.      Description of the drug

b.      Procedure of the study, i.e. mode of administration, number of blood samples, collection of any biological specimen.

c.       Description of possible risks and benefits involved, if any, and the facilities available to tackle them.

d.      Right to opt out of the study without assigning any reason and without any liability .

 

4.4 DRUG INFORMATION

 

The drug information is based on the published literature.

a.        physicochemical  characteristics.

b.        Pharmacological effects ( clinical therapeutic and toxic).

c.         Pharmacokinetic characteristics.

 

4.5 PRODUCT INFORMATION

 

Product information is for reference and test formulation .

a.        Information including brand name , date of manufacture .label claim country of origin , date of expiry, batch no., name of the manufacturer for both test and reference formulation.

b.        Information regarding the test and standard product should include an analytical profile , dissolution data and batch size . the batch should be manufacturer under the same  production and GMP controls ; it should be at least 10% of the individual lot or a minimum of 10000 units .

c.         The source of the innovator ‘product should also be supplied /mentioned.

 

4.6 STUDY DESIGN

 

Appropriate study design such as :

 

a.      Single – dose, randomize , two period, two treatment, complete crossover

b.      Latin square design to be used for comparing  three formulations

c.       Balanced incomplete block (BIB) design in case of more three than formulations

d.      Wash out period between any two subsequent treatments should not be less than 5 times of the reported value of elimination half life (based on intravenous data) between the two treatments.

 

 

 

 

 

4.7 HEALTHY VOLUNTEERS

 

Number of volunteers

Should this number be changed as it does not account for drop outs?.

Twelve normal, healthy adult male volunteers.

 

Inclusion criteria

 

a.      Adult healthy male between age of 18 and 50 years.

b.      Height and weight according to the tables published by the LIC of India. A maximum variation of 10% on the either side is permissible.

c.       The following hematological and other parameters should be within normal limits for those specific age, weight and healthy combinations :

(i)                  Complete blood count

      (Total and differential count)

(ii)                ESR

(iii)               Liver function test

·          SGOT

·          SGPT

·          Serum bilirubin (direct and indirect)

·          Serum alkaline phosphates

 

(iv)              Serum creatinine

(v)                Blood sugar : fasting, postal – prandial

(vi)              Urine : routine and microscopic

(vii)             Australia antigen.

(viii)           HIV antigen

 

d.      No abnormility on clinical examination

e.      No history of any illness in past 8 weeks

 

Exclusion criteria

 

a.      Consumption of tobacco in any form

b.      Addiction to alcohol or history of any drug abuse

c.       History of kidney or liver dysfunction

d.      History of jaundice in the past 6 months

e.      History of drug allergy to the test drug or any chemically similar to the drug under investigation

f.        Administration / intake of any prescription or OTC medication for 2 weeks before the study

g.      Patient suffering from any chronic illness such as arthritis , asthma etc

h.      Subject suffering from any psychiatric (acute or chronic) illness

i.         Participation in any bioavailability / bioequivalence study in the past 12 weeks

j.        Intake of barbiturates or any enzyme – inducing drug in the past 3 months

k.       HIV – positive volunteers

 
4.8 CONDUCT OF THE STUDY

 

a.      All the volunteers should be admitted in the ward / bioavailability center atleast 12 hours prior to the administration of the study drug dosage form. They should be housed for adequate time.

b.      Time of administration of medication (test and reference) should be in the morning and the same time should strictly adhered during the entire study

c.       Tea, coffee and caffeine / xanthine -  containing beverages and food should not be allowed during each phase of the study/

d.      All volunteers should fast for 12 hours predosing and continue to fast for at least 2 hours postdosing or for adequate time after dosing.

e.      Uniform and identical meals should be provided at identical times to all volunteer from the time they are admitted and till the time they are housed.

f.        A precise work schedule should be prepared in advance and explained to the volunteers.

g.      The exact time of drug administration, sample collection and meals should be strictly adhered to and the actual time of each activity should be properly and accurately documented.

h.      Test tubes/vials required for sample collection should be elaborately labeled in advance and kept ready.

i.         Standard quantity of water (one glass : 200 ml) should be allowed for proper ingestion of the drug.

j.        The investigator will provide the medication to the volunteers and the volunteers must consume the drug in the presence of the investigator.

k.       After ingestion of the medication, the volunteers should rest in supine position at least for 2 hours to ensure proper gastric emptying. Subsequently, although the volunteers are ambulatory, any strenuous physical or mental activity should not be permitted.

l.         Blood samples should be collected by using disposable needles, syringes , etc.

m.     Multiple blood sample collection should be done by using disposable in dwelling venous canulae / catheters in order to avoid repeated venupuncture.

n.      Care should be taken not to withdraw more than 250 ml of blood per volunteer in one month.

o.      An attending physician will be present at all times to oversee the conduct of the investigation as well as to answer any queries by the volunteers.

p.      Emergency medical equipment and support services will be available and accessible throughout the investigation.

 

4.9 BLOOD SAMPLING

 

a.      adequate number (at least three) of venous blood samples should be collected during the absorption phase and should be well spread  over the expected duration of the absorption phase.

b.      At least 3 blood samples should be collected around the expected time of peak blood levels. This can be made possible by adjusting 1 to 2 points each from absorption points (last ones) and distribution points (earlier ones) as near the Cmax as possible. Its timing should be decided on the basis of the published information. In the absence of any published information a pilot study with limited volunteers may be performed earlier to the actual bioequivalence study.

c.       Sampling container should be sterilize using dry heat. While transferring the sample from the syringe , the needle should be removed and the syringe should be slowly emptied along with the sides of the container. Should it be mandatory to use presterilized instruments.?

d.      The sampling container should contain an adequate amount of anticoagulant. The anticoagulant (in case of plasma) should be chemically compatible with the drug and should not increase the volume of blood sample stored in the container.

e.      The separation of plasma or serum should be done immediately following the collection and it should be stored in two identical containers with identical labels.

f.        Once separated the plasma, serum or the biological fluid collected, should be immediately frozen to –10oc to –20oc till the time of analysis. Adequate precautions should be taken in case of light- sensitive drugs.

g.      The analysis  should be performed immediately if the substance (drug) is known to degrade on storaging.

 

4.10 ANALYTICAL METHOD

 

Details of the analytical method intended to be used for the analysis of the biological sample should be included in the protocol.

 

4.11 EVALUATION PARAMETERS

 

The following pharmacokinetic parameters from blood/serum concentration time data should be determined for each volunteer in each treatment :

a.      Cmax

b.      Tmax

c.         AUC : both AUC o-t and AUC 0-¥

 

All the above parameters should be tabulated for each volunteer for both the treatments to permit the calculation of mean, standard deviation and coefficient of variation.

 

4.12 STATISTICAL ANALYSIS

 

a.      The methods/techniques to be used should be decided and mentioned in the protocol before the study begins

b.      Whenever a user-defined software is used for analysis, it should be validated.

c.       When the analysis is performed manually, all the results of intermediate steps should be properly recorded.

 

 

5. PROTOCOL AND STUDY DESIGN FOR  EXTENDED RELEASE DOSAGE FORMS

 

 

5.1 Bioequivalence studies are required in the following two situations :

 

A.      An  E.R. product with the same label claim and dosing frequency is already available internationally or in India.

 

B.      An E.R. product is developed for the first time.

 

5.2 PROTOCOL & STUDY DESIGN

 

The study protocol shall contain the following elements.

 

5.2.1  Information on Sponsor/ Investigator

 

The names of the principal investigator(s), other persons responsible for conducting the study, and the names of the laboratory and the clinical pharmacology unit where the study will be conducted.  Complete official address of the premises of the laboratory and contact phone numbers of the person in-charge of the study, with time for contact (in case of more than one phone numbers), should also be mentioned.

 

The name of the sponsor and/or other person responsible for monitoring the study should be mentioned, along with the complete, official as well as residential addresses and phone numbers with contact times.

 

5.2.2 Objective of the Study

 

To evaluate comparative bioavailability of an ER formulation, with a reference ER formulation or a conventional dosage form given in equivalent doses at appropriate frequencies.

 

All the applicants should conduct bioequivalence by comparing the test product with the reference product with the same label claim and frequency of administration, because its clinical efficacy and safety profile is usually well documented in extensive trials.

 

The reference product can be one of the following :

 

a)      ER product of the innovator with same label claim and frequency of administration.

b)      Conventional release dosage form of the innovator or applicant given in equivalent doses at appropriate frequencies

 

5.2.3 Ethical Considerations

All studies shall be conducted in accordance with the Helsinki Declaration.

 

There should be an ethical committee to approve the study and ensure that the study is carried out as per scientific and ethical norms.  The ethical committee should consist of qualified personnel including at least a clinical pharmacologist, a consultant physician, a social worker and a person with legal background.  Sufficient and relevant data on animal studies and human studies should be made available to the ethical committee.  If for any reason, the approved protocol needs amendments, the amended protocol should be approved by the ethical committee and the Drugs Controller General (India).  Finally, the amended and approved protocol should be submitted with the report.

 

If the formulations under study, are likely to adversely affect the health of the volunteers, e.g. anticancer drugs, oral hypoglycemic drugs, etc., healthy volunteers should not be enrolled in the study.  Patients should be enrolled in such cases.  An informed and written consent form of the volunteer must be obtained and kept on record.  The participating volunteers should be provided with full information about the study in a language understandable to them.

 

The information to the volunteer should include :

Informed consent process according to GCP guidelines

a)      Description of the drug.

 

b)      Procedure of the study, i.e. mode of administration, number of blood samples, collection of any biological specimen, details of consumption of meals and fluids, physical activities and details of restriction.

 

c)       Description of possible risks and benefits involved, if any, and the facilities available to tackle them.

 

d)      Right of the volunteer to opt out of the study without assigning any reason and without any liability.

 

e)      Right of the investigator to withdraw the volunteer from the study with valid reasons.

 

 

 5.2.4 Drug Information

 

The drug information should be based on the published literature

 

a)      Physicochemical properties

b)      Pharmacodynamic effects.

c)       Pharmacokinetic properties including  active and inactive metabolites.

d)      Profile and management of adverse drug reactions

 

5.2.5  Product Information

 

Following information  for reference and test should be provided.

 

a)      Brand name, date of manufacture, label claim, country of origin, date of expiry, batch number, name of the manufacturer, analytical profile and dissolution profile.

 

b)      Additional information for the test product should include batch size. The batch should be manufactured under the same production and GMP controls; it should be at least 10% of the individual lot or minimum of 10,000 units.

 

5.2.6  Study design

 

5.2.6.1  Situation A:

 

ER product with similar dosage regimen is already marketed. The test product will be compared with reference product using one or more studies as applicable.

 

Study A-1       A single dose, randomized, two-period, two-treatment, two-sequence crossover study, under fasting conditions, comparing equal doses of the test and reference products.

 

Study A-2       For drugs whose conventional dosage form shows interference in

                       absorption with food,  a study with following design may be carried out.

 

A single dose, randomized, three-treatment, three-period, six sequence, crossover, limited food effects study, comparing equal doses of the test product administered under fasting conditions with those of the test and reference products administered immediately after a standard breakfast.

 

If the conventional release product and the innovator’s ER formulation does not show interference in absorption with food, then the food effect studies may not be required under following circumstance:

 

     ·   If there is convincing evidence that the basic dosage form itself has not shown food

        interference as documented in studies/ literature.

 

Under this condition, the permission to undertake single dose fasting study design may be granted from case to case basis.

 

Study A-3       The following study is recommended for those drugs which are likely to accumulate on multiple dosing as indicated by literature/preliminary data of

AUC(o-t) <= 0.8 for the innovator’s ER formulation.

AUC(0-t)

 

A multiple dose, steady state, randomized, two-treatment, two-period, two-sequence crossover study comparing equal doses of the test and reference formulations.  For safety reasons, this study can be performed in the non-fasting state, after providing justification in the protocol.

 

A very well planned acute-on-chronic multiple dose, steady state level study may obviate the need for an acute single dose (fasting and fed) study.  Since multiple dose, steady state level study simulates real life condition including influence of meals as well as circadian effects on the performance of the ER product.

 

For those drugs where use in healthy volunteers may be ethically questioned (e.g. anticancer, antipsychotics, hormones), multiple dose steady-state level studies can be conducted in patients.

 

5.2.6.2  Situation B.

 

ER formulation developed for the first time

 

In such a case data regarding the behaviour of the molecule in ER form is unknown.  It is advised that a pilot study in 6 volunteers is undertaken to predict adequate performance to proceed with complete study.  If same statistical design as on page number                is adopted, then the data generated in this pilot study can form part of the complete study.

 

Study B-1  A single dose randomized, three treatment, 3 period, 6 sequence, cross

                      over limited food effects study, comparing equivalent doses of the test

                      product, and the conventional formulation administered under fasting

                      condition with that  the test product administered immediately after a

                       standard breakfast. 

 

Study B-2       A multiple dose, steady state, randomized, two-treatment, two-period, two-sequence crossover study under fasting conditions (or non-fasting conditions for safety reasons providing justifications in the protocol) comparing equivalent doses of the test and reference formulations.

 

 

FLOWCHART FOR SELECTING STUDY DESIGN

NEW E.R. DOSAGE FORM

 

Is a similar product (with same label claim) marketed internationally?

 

 


YES                                                                                          NO

Situation A                                                                                 Situation B

 

 


                                                                                                            PILOT STUDY

                                                                                                            (6 VOLUNTEERS)

Does it accumulate in the body?

 


                                                                                                B1        SINGLE DOSE STUDY

YES                                                      NO

                                                                                                B2        MULTIPLE DOSE

STEADY-STATE LEVEL STUDY

A3

 


MULTIPLE DOSE

STEADY-STATE LEVEL STUDY *

 

 

Is food likely to interfere with absorption of drug/ dosage form?

 


                        NO                                                                               YES

 

 


            A1        SINGLE DOSE FASTING                         A2        SINGLE DOSE

FOOD EFFECTS STUDY

 

 

 

*   Since multiple dose study simulates real-life situations (including influence of meals) it may obviate the need of single dose food effects study.

 

 

 

 

5.2.7  Study Subjects

 

Twelve evaluable normal healthy human adult volunteers.

 

5.2.8  Inclusion Criteria

 

a)      Adult healthy males  or females (for those studies requiring female volunteers only) between the age of 18 and 45 years.

 

b)      Height and weight according to the tables published by the LIC of India.  A maximum variation of upto 10% on either side is permissible.

 

c)       The following haematological and other parameters should be within normal limits for  specific age, weight and height combinations

 

i)          Complete blood count (total and differential count)

ii)         ESR

iii)         Liver function tests

 

·          SGOT (AST)

·          SGPT (ALT)

·          Serum bilirubin (Direct & Indirect)

·          Serum alkaline phosphatase        

      iv)  Serum creatinine

       v)  Blood sugar : fasting, post-prandial

      vi)  Urine : routine and microscopic

     vii)  Australia antigen

     viii) HIV antibody

       ix) HCV antibody testing

 

d)         No relevant abnormality on clinical examination

e)         No history of any major illness in the past 8 weeks.

 


5.2.9  Exclusion Criteria

 

a)      Habituation of tobacco necessitating uninterrupted tobacco consumption

b)      Addiction to alcohol or history of any drug abuse.

c)       History of kidney or liver dysfunction.

d)      History of jaundice in the past 6 months. Female volunteers with positive pregnancy tests, or not practising physical contraception, and nursing mothers (for those studies requiring female volunteers only)

e)      History of drug allergy to the test drug or any drug chemically similar to the drug under investigation.

f)        Administration/Intake of any prescription or OTC medication for two weeks before the study.

g)      Patients suffering from any chronic illness such as arthritis, asthma, etc.

h)      Subjects suffering from any psychiatric (acute or chronic illness).

i)        Participation in any bioavailability/bioequivalence study in the past 12 weeks.

j)        Intake of barbiturates or any enzyme-inducing drug in the last 3 months.

k)      HIV positive volunteers.

l)        History of significant blood loss due to  any reasons, such as blood donation in the past 12 

      weeks.

m)    History of any bleeding disorder.

 

 

5.2.10  Conduct of Clinical study

 

a)      All the volunteers should be admitted to the ward/bioavailability centre at least 12 hours prior to the administration of the study drug dosage form.  They should be housed for adequate time.

 

b)      For multiple dose studies lasting for less than a week arrangements could be made to house the patients but for studies of longer duration for practical reasons volunteers may be allowed to go home and visit for drug dosing and collection of blood samples.

 

c)       Time of very first administration of the medication (test and reference) should be in the morning and the same time should be strictly adhered to during the entire study.

 

d)      All study subjects should fast overnight at least for 10 hours before the administration of the first study dose and at least four hours post dosing in case of morning dose.

 

The protocol should stipulate the time of meals in relation to the administration of the dosage form.

 

In case of multiple dose studies the second dose be given in the afternoon/evening.  All volunteers should fast 2 hours before dosing and continue to fast for two hours after the dose.  The same time should be strictly adhered to during the entire study.

 

e)      Tea, coffee and caffeine/xanthine-containing beverages and foods should not be allowed during each phase of the study.

 

f)        Uniform and identical meals should be provided at identical times to all volunteers from the time they are admitted and throughout the time they are housed.

 

g)      For studies under fed conditions a standard & uniform breakfast should be given prior to dosing. (Due to the variation in diets in different parts of India, the fat content should be mentioned).

 

h)      A precise work schedule should be prepared in advance and explained to the volunteers.

 

i)        The exact time of drug administration, sample collection and meals should be strictly adhered to and the actual time of each activity should be properly and accurately documented.

 

j)        Test tubes/vials required for sample collection should be elaborately labelled in advance and kept ready.

 

k)      Standard quantity of water (one glass : 200 ml) should be allowed for proper ingestion of the dosage form.

 

l)        The investigator will provide the medication to the volunteers and the volunteers must consume the drug in the presence of the investigator.

 

m)    After ingestion of the medication, the volunteers should rest in supine position at least for two hours to ensure proper gastric emptying.  Subsequently, although the volunteers are ambulatory, any strenuous physical or mental activity should not be permitted.

 

n)      Blood samples should be collected by using disposable needles, syringes, etc.

 

o)      Multiple blood sample collection should be done by using disposable in-dwelling venous cannulae/catheters in order to avoid repeated venepuncture.

 

p)      Care should be taken not to withdraw unduly large quantity of blood that can adversely affect haemoglobin concentration in the subjects.

 

q)      An attending physician will be present at all times to oversee the conduct of the investigation as well as to answer any queries by the volunteers.

 

r)       Emergency medical equipment and support services will be available and accessible throughout the investigation.

 

5.2.11  Blood sampling

 

a)      Adequate number (at least three) of venous blood samples should be collected during the absorption phase and should be well spread over the expected duration of the absorption phase.

 

b)      At least 3 blood samples should be collected around the expected time of peak blood levels.  This can be made possible by adjusting 1 to 2 points each from absorption points (last ones) and distribution points (earlier ones) as near the Cmax as possible.  Its timing should be decided on the basis of any published information.  In the absence of any published information, a pilot study with limited volunteers may be performed earlier to the actual bioequivalence study.

 

c)       For steady state levels at least 4 trough concentrations should be taken for confirmation.  Trough concentrations or Cmin is the concentration before the next dose.

 

d)      Sampling containers should be sterilized using dry heat.  While transferring the sample from the syringe, the needle should be removed and the syringe should be slowly emptied along the sides of the container.

 

e)      The sampling container should contain an adequate amount of anticoagulant for plasma.  The anticoagulant (in case of plasma) should be chemically compatible with the drug and should not increase the volume of blood sample stored in the container.

 

f)        The separation of plasma or serum should be done immediately following the collection and it should be stored in identical containers with labels.

 

g)      Once separated, the plasma, serum or the biological fluid collected, should be immediately frozen to -10o c to 20o c till the time of analysis.  Adequate precautions should be taken in case of light-sensitive drugs. 

 

h)      The analysis should be performed immediately if the substance (drug) is known to degrade on storing.

 

5.2.12  Analytical Method

 

Details of the analytical method intended to be used for the analysis of the biological sample should be included in the protocol.

 

5.2.13  Evaluation  of Parameters

 

The following Pharmacokinetic parameters from blood/serum concentration time data should be determined for each volunteer in each treatment

 

a.          Cmax

 

b.         Tmax

 

c.          AUC0-t

 

Cmax and AUC (Original and log transformed) should be tabulated for each volunteer for both the treatments to permit the calculation of mean, standard deviation, and coefficient of variation.

 

A.      The Cmax, and AUC of the test formulation should be between 80-120% of the reference formulation, for untransformed data and 80 to 125% of the reference formulation for log transformed data.

 

B. In case of single dose, fasting, and fed trial.

 

·          The Cmax of conventional formulation should be more than Cmax of ER formulation.

 

·          The AUC (log transformed) of ER formulation should be between 80 to 125% of the AUC of an equivalent dose of conventional formulation.

 

·          If AUC(o-t) / AUC(0-t) > 0.8, the drug is non accumulating where

            AUC(o-t) = AUC over the usual dose interval

            AUC(0-t) = AUC calculated upto time t

 

C.         Steady State Studies

 

·          Degree of Fluctuation (% Fluctuation) = (Cmax-Cmin/Cmin) X 100

                       

OR

 

% Fluctuation = AUC (above Cavg) + AUC (below Cavg)X 100

                                                AUCss

 

 

 

 

·          Fluctuation Index (%PTF) = (Cmax - Cmin) / Cavg) X 100

 

where C avg = AUCss/t and

t = dosing interval

 

 

 

5.2.14      Statistical Analysis

 

 

a)      The method / techniques to be used should be decided and mentioned in the protocol before the study begins.

 

b)      Whenever a user-developed software is used for analysis, it should be validated.

 

c)       When the analysis is performed manually, all the results of intermediate steps should be properly recorded.

 

 

 

 

6.  METHODOLOGY FOR CONDUCT OF A BIOEQUIVALENCE STUDY

 

Following are the important sequential steps for undertaking a bieoquivalence study :

1.      Request letter from the sponsor to the center to undertake a bieoquivalence study on a product with relevant details.

2.      Consent letter by the bioequivalence center along with the checklist for the information to be provided by the sponsor.

3.      Letter with quotations and other formalities from the center to the sponsor.

4.      Protocol of the study prepared by the clinical pharmacologist and approved by the Ethical Committee to be forwarded to the sponsor.

5.      Consent letter and the protocol to be forwarded by the sponsor to the Drug Controller (India) for approval

6.      Approval letter from the Drug Controller (India) for conducting the bioequivalence study under the supervision of the chief investigator at the bioequivalence center.

7.      Letter from the sponsor to the bioequivalence center along with the copy of  the approval letter from the Drugs Controller (India) requesting commencement of the study. The sponsor should provide all the information as per the checklist in item 2.

8.      The study is planned by the center on screened  healthy male volunteers whose laboratory investigations should be carried out within one month prior to the commencement of the study.

9.      Volunteers should be requested to report at the center at least 12 hrs befopre the study commences and should not be allowed to smoke or to take tea, beverages, alcohol, etc. during the study period.

10.  Informed written consent should be obtained from volunteers and should be countersigned by witness.

11.  The study should commence at a predetermined time and must be conducted strictly according to the approved protocol. Activity record should be filled in during the study.

12.  The volunteers participating in the study should be asked personally for any symptoms or side effects which should then be recorded in the record sheet. “Do you have any complaints?”

13.  Plasma/Serum samples should be frozen and stored at 20oC. For light sensitive drugs, vials should be covered. The plasma/serum samples should be properly coded and labelled.

14.  Drug analysis from the plasma/serum samples should be carried out by a previously validated analytical method, by an assayist from whom the name the product related to any sample is concealed by means of a suitable coding method.

 

After an appropriate washout period mentioned in the protocol, the crossover study should be carried out in a similar fashion. The bioavailability of the test formulation should be compared with that of the standard formulation and the data on Cmax, Tmax, AUC should be statistically analyzed.

 

 

 

Dissolution data should be supplied by the sponsor on both the test and the standard preparations to the bioequivalence center.

 

In-vitro dissolution studies are required where bioequivalence is not applicable e.g. anti-cancer drugs, drugs which are systemically not absorbed, etc.

 

The report should be certificed by the chief investigator, medical supervisor and the clinical pharmacologist and then submitted to the sponsor.
An office copy of the report along with the  raw data should be preserved up to 3 years from the date of completion of the study at the bieoquivalence center.

Does the time frame need modification

 

 

 

 

7.  ANALYTICAL METHODOLOGY AND VALIDATION

 

7.1 INTRODUCTION

 

Analytical methods that are used for the quantitative determination of drugs and their metabolites evalutation and interpretation  of bioavailability , bioequivalence and pharmacokinetic data. It is essential to use well chararcterized and fully validated analytical methods to yield reliable results that can be satisfactorily interpreted.

 

7.2 OBJECTIVE

 

The objective of analytical validation on sample of biological origin (plasma, urine, etc.) is to demonstrate the reliability of results for active ingredients and/or metabolites obtained from bioavailability studies.

Although there are various stages in the development and validation of an analytical procedure, the validation of the analytical method can be envisaged to consist of two distinct phases :

1.      The development phase, comes before the actual start of the study and involves the validation of the method on human plasma samples and spiked plasma samples.

2.      The study phase in which the method is applied to the actual analysis of samples from pharmacokinetic, bioavailability and bioequivalence studies.

 

7.3 DEVELOPMENT PHASE

 

The validation criteria should be those currently used in analytical chemistry (Good Laboratory Practice) and consist of  :

 

1.      Stability of stored samples

2.      Specificity and selectivity

3.      Accuracy (relative recovery)

4.      Precision (repeatability and reproducibility)

5.      Range and linearity

6.      Sensitivity

(Limit of detection & limit of quanitfication)

7.      Calibration of instruments

8.      Documentation of results

 

Each test procedure should be validated for each type of biological sample.

Stability of stored samples

A significant time lapse can be there between the time of sampling and the time of analysis. For this reason it is necessary to know :

i.The stability of the substance been examined in the biological fluid in the precise   storage conditions.

ii.The absorption of the substance by the sampling container and the stopper (especially in the case c of plastic and rubber closures).

 

The stability data should include freezing and thawing cycles representative of actual sample handling.

 

Specificity/Selectivity

This is to ensure that the signal measured with the test procedure comes only from the substance being analyzed with no interference from endogenous sompounds, drug metabolites or co-administered drugs.

 

Accuracy (Relative Recovery)

Accuracy expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and  the observed value found by using the method for quantitation. Accuracy provides the indication of systematic errors.

Accuracy can also be established by doing the recovery experiment by the method of standard addition. In this method, to a pre-analyzed sample, known levels of the standard are added and the quantitation of the target compound is done by the proposed assay method. In this, there is a zero level (assay level) and three more levels. To these three levels, a known  amount of the standard drug is added. After a quantitative analysis of all these four solutions, a graph of the amount of the drug found by the proposed method on the X-axis and the amount of the standard drug added on the Y-axis is plotted. The intercept on the Y-axis should correspond to the zero assay level.

The analysis of quality control samples (QC samples) should be done  on every analytical day, along with the linearity range levels. The QC samples should essentially envisage low, medium and high levels of the samples to be analyzed. The inter-day variation and intra-day variation should also serve as an important measure of the accuracy of the method being used for that particular quantitation.

 

Precision (Repeatability and reproducibility)

The precision of a test procedure expresses the closeness of agreement obtained from the multiple sampling of the same homogeneous samples under certain prescribed conditions. Precision provides an indication of random errors.

 

(i)                  Repeatability

        

Repeatability expresses the

 

·           same analyst

·          same apparatus

·          short interval of time

·          identical reagents.

 

(ii)                Reproducibilility

Reproducibility expresses the precision under different  conditions for e.g.,

 

·          different analysts

·          apparatus from different manufacturers

·          different days

·          reagents from different sources.

 

 

Range and Linearity

 

The range of the test procedure is the interval between the upper and the lower levels of analysis (including these levels) for which the procedure has been demonstrated as suitable with precision, accuracy and linearity using the specific method.

The range of the standard solution should extend from the lowest to the highest concentration samples that are anticipated in the study. There should be a minimum of six standards over the range.

The linearity of a test procedure is its ability to obtain test results directly proportional to the concentration of the analyte in the sample over a given range.

 

Sensitivity

 

Sensitivity is the capacity of the test procedure to record small variations in concentration.

(a)   Limit of detection (LOD) :

     The lowest concentration of drug that will yield an assay response significantly

      different from that of a sample blank.

 

(b)   Limit of quantitation (LOQ, sensitivity limit) :

The lowest concentration of drug that can be determined with acceptable precision and accuracy under the stated experimental conditions. This should be lowest point in the standard calibration curve.

 

Calibration of Instruments

 

The calibration of instruments to be used should be done regularly and as per standard procedures in USP/BP/IP.

The calibration should be done before starting the analysis at the development phase and thestudy phase.

 

Documentation of Results

The results will be documented in the following sequence :

 

·          Protocol describing method adapted.

·          Validation and summary (developmental phase)

·          Data on linearity of standard in plasma

·          Data on intra-day precision and accuracy

·          Data on inter-day precision and accuracy

·          Standard curves

·          Calculation and formula

·          Certificate of calibration of instruments

·          Certificate of analysis on working standard (pure ingredient) demonstrating identity and purity, provided by the sponsor.

 

7.4 STUDY PHASE

 

Many of the principles for establishing a valid method are relevant to the development phase work on validation. In general with acceptable variability as defined by validation data, the analysis of biological sample can be done by single determination without a need for a duplicate or replicate analysis. The need for duplicate analysis should be assesesed on a case-by-case basis. For example,  for a robust procedure of low variability, with accuracy and precision routinely well within tolerance , single analysis would suffice. For a difficult procedure with a labile analyte , when the precision and accuracy tolerances are difficult to achieve duplicates may be essential. A procedure should be developed that documents the reason for re-analysis.

 

A standard curve should be generated for each analytical run for each analyte and should be used to calculate the concentration of the analyte in the unknown samples assayed with that run. It is important to use a standard curve that will cover the entire range of concentrations in the unknown samples. Estimation of unkowns by extrapolations  of standard curves below the low standard or above the high standard is not recommended. Instead, it is suggested that standard curve should be redetermined or sample should be reassayed after dilution. The quality control (QC) sample should be used to accept or reject the run. This QC samples are matrix spiked with analyte.

 

In Summary

 

1.      A standard curve should consist of five to eight standard points, excluding blanks (either single or replicate) covering the entire range;

2.      The response function is determined by appropriate statistical tests based on the actual standard points during each run in the validation; and

3.      The system suitability is based on the analyte and technique (a specific procedure or sample can be identified to assure the optimum operation of the system employed).

 

 

7.5 ACCEPTANCE CRITERIA FOR THE RUN

 

Accuracy and Precision

 

The acceptance criteria are not more than 15% CV for precision and not more than ± 15% deviation from the nominal value for accuracy. However , at the lower limit of quanittation (LOG) , ±20% is acceptable for both precision and accuracy. It is desirable that this tolerances be provided both for intraday and interday or interrun experiments.

 

QC Samples

 

During each assay, the quality control (QC) sample should be run. The QC samples are samples known concentration prepared by spiking drug-free biological fluid with drug. These samples should be prepared in low, medium and high concentration. To avoid possible  confusion between QC samples and standard solutions during the review process, a preparation of QC samples at concentrations different from those used for the calibration is recommended. The desirable concentrations of the QC samples are :

 

 

 

 

Low QC sample :           two to three time the sensitivity limit

 

Medium QC sample :      25 – 50% of the highest standard

 

High QC sample :            about 80% of the highest standard

 

A QC sample should be assayed following the assay of every eight to ten clinical samples. Whereas a standard curve is often determined only at the beginning of each assay date, each of the three QC samples is assayed several times during the day.

The QC sample provide the following benefits :

 

The intraday accuracy and the precision of the analytical system may be estimated. Unlike standards used for calibration, QC samples  are not used in the determination of the standard curve. Therefore, their accuracy and precision should be more representative of those of the clinical samples.

The results of the QC samples assay should be plotted on a quality control chart showing the acceptability band (mean ± SD), and the  method and/or reagents should be standardized whenever any QC ssmple assay result crosses the boundaries of this band.

 

Repeat Analysis

The protocol for repeat analysis should be established a priori. Some aberrant values can be identified; they can be attributed to processing errors, equipment failure, poor chromatography or QC sampels outside the pre-defined tolerance. Cautious use of ‘pharmacokinetic file’, such as a double peak may call for repeat analysis of some samples  in the study, but the reasoning should be clearly documented.

 

 


 

8. VALIDATION OF ANALYTICAL METHOD : IMMUNO AND

   MICROBIOLOGICAL ASSAYS

 

Many of the parameters for and principle of analytical validation for chemical methods are also applicable to immuno-and microbiological methods, but there are some specific differences. In immuno – and microbiological assays, the response must be shown to be related to the concentration of the analyte in question.

 

Selectivity Issues : As with chromatographic methods, it must demonstrated that the bioassay is selective for the analyte. An alternative method, if rigorously established, may be used to compare the results of the bioassay. For bioassay, an appropriate combination of other techniques may be used to show selectivity, including the following :

1.      Comparison of standards in biological fluids with standards in buffer to detect matrix effects.

2.      Parallelism of diluted clinical samples with diluted standards to detect presence of closely related compounds.

3.      Serial separation techniques (e.g. extraction) and chromatograohy, with the bioassay as detector, to demonstrate that the response is due only to the analyte in question.

4.      Metabolite (or endogenous compound) cross-reaction assessed initially by comparison of displacement curves (in critical cases, metabolite cross reaction should also be assessed by addition of a metabolite to an analyte). Similar criteria will be applicable when drug is concomitantly administered with other drugs.

 

8.1 QUANTITATION ISSUE

 

These issues are summarized as follows :

 

1.      The criteria for precision and accuracy of immuno- and microbiological assays should be based on the requirements of the study and should match those of chromatographci methods. Any decision to run the sample analysis in single, duplicate or triplicate should be based on variability.

2.      Immunoassay standard curves are essentially non-linear and in general require more concentration points to define the fit over the range claimed.

3.      It should be established that an acceptable curve fitting model is being used by examining the statistics for the goodness of the fit and by back – calculating results of standards and control samples.

4.      Both upper and lower LOQ values must be defined by acceptable accuracy, precision or confidence- interval criteria based on the study requirements.

5.      For all assays, the key factor is the accuracy of the reported results. This accuracy may be improved by the use of replicate samples. When replicate samples need to be measure during validation to improve accuracy, the same procedure must be followed for unknown samples.

6.      If there are intermediate steps between the plasma ( or other biological matrices) and the final assay (such as extraction of biological sample followed by immunoassay) and if parallel processed standards in the biological matrix are not being used,it is necessary to establish recovery and use it in determining results. The possible approaches to assess efficiency and the reproducibility of recovery are the use of radiolabelled tracer analyte (quantity too small to affect the assay), the advance establishment of reproducible recovery and the use of an internals tandard that is recognized by the antibody but can be measured by the another technique.

7.      The correction for nonspecific matrix effects can be accomplished with separation techniques may be used in defining the standard curve for both controls and samples. The use of standards in the matrix is recommended. This approach will obviate many of the earlier mentioned concerns.

 

8.2 OTHER ISSUES

 

Commercial Kits

 

Commercial kits are available for both immuno – and microbiolgical assays and the analytical methods based on such kits should be validated. The validation assures thath the bioassay kit is applicable to the study problem and that subsequent batches or lots of kits have performance characteristics similar to the original validated kit or test. Any modifications and extentions of assay from one kit (or test) to another must be validated.

 

Some situations exit in studies in bioavailability and bioequivalence in which :

1.      the parent drug cannot be measured in biological samples and only the metabolite can be measure.

2.      The parent drug and active and/or inactive major metabolite (s) can be measure,

3.      More than one metabolite is present or

4.      The accumulation of metabolite is augmented (e.g. in the case of renal impairment).

 

The following suggestions are made :

1.      All methods applied for measuring drug and metabolite(s) should be validated for that particular study matrix with the same general parameters mentioned earlier (accuracy, precisio, specificity, recovery and reproducibility).

2.      Pharmacokineitc, bioavailability and bioequivalence studies should be based on the moieties that contribute significantly to the pharmacologic or therapeutic effect.

 

8.3 PROCEDURES

 

I.                    A short description of the main principle of the test procedure should be indicated.

II.                 Test procedure including the conditions of sampling must be described precisely, preferably in a standard format as given below :

(a)   The mode of sampling

(type of container, anticoagulant, etc.)

(b)   The conditions of storage before analysis.

(c)    Exact description of the test conditions including precautions, method of extraction, reagents, reference material etc.

(d)   The exact description of the apparatus used.

(e)   The verification of test procedure under defined operating conditions

(system suitability)

(f)     Details of the calculations of the analyte from the biological sample.

(g)   Statistical evaluation of analyzed data.

 

III.               In  case a reference substance (active ingredient) is used for the test (if pharmacopoeial or other official standards are not used), then its identity and purity must be fully established. The specification and method of analysis should be provided by the sponsor.

IV.                If a prodrug is under study, then the drug into which it gets transformed should be quantified.

V.                  If the metabolites are active and if a large amount of variability is observed in the analysis of the drug, then the quantity of metabolites must be examined.

VI.                The sponsor should be justify the rejection of any analytical data and provide a rationale for selection of reported values. This data should be kept with the sponsor.

VII.             Outliers may also be observed in AUC or Cmax parameters. For reasons other than a documented clinical problem or analytical error, outliers should not be discarded.

VIII.           The center should store the raw data and the reference samples of the brand leader (standard preparation) and the sponsors sample for a period of 3 years after completion of the study. After submission of the study report to the sponsor, the biological samples should be stored for reference at least for one month.

 

 

 

 

 

 

9.  IN VITRO DISSOLUTION

 

Should this be a part of the guideline?

Dissolution testing is required for all dosage forms in which the dissolution of the drug is necessary for the product to exert the desired therapeutic effect. This chapter sets forth the guidelines for the dissolution testing of modified release dosage forms.

 

It is necessary to demonstrate  the controlled release nature of a drug from a controlled release formulation  by both in-vivo - in-vitro methods. The manufacturers of controlled release drug products are urged to develop reproducible and sensitive in-vitro methods to characterize the release mechanisms of the controlled release  drug products.

 

 

9.1  General guidelines for dissolution testing of modified   release dosage    

      forms :

 

 

9.1.1  Dissolution Testing

 

 

Dissolution testing should be conducted on 12 individual dosage units of the test and reference products used in the bioequivalence studies.  The  potential  for  pH dependence of drug release from an extended release product is well recognized. Dissolution profiles should therefore be generated in aqueous media (preferably deaerated water) or dilute acid  or buffered aqueous solution of the following pH ranges : 1-1.5, 4-4.5,  6-6.5 and 7-7.5. Early sampling times of 1,2 and 4 hours should be included in the sampling schedules to provide assurance against premature release of the drug (dose dumping) from the formulation. The usual volume of the medium is 500 to 1000 ml, with the use of greater volumes (upto 2000 ml) allowed for drug having limited solubility. The quantity of medium used should be not less than 3 times that required to form a saturated solution  of the drug substance. Addition of solutes (i.e. surfactants)  and electrolytes to aid in solublization of the drug must be balanced against the loss of discriminatory power of the test. The use of hydroalcoholic media is generally not favoured. The use of such media if warranted should be supported by a documented in-vitro and in-vivo correlation.

 

 

 

The general dissolution conditions to be followed are shown below :

 

1

Apparatus

USP 23 Apparatus 1 (rotating basket)

USP 23 Apparatus 2 (paddle)

2

Rotation speed

100 rpm(basket)

50 and 75 rpm (paddle)

3

Temperature

37± 0.5 0 C

4

Units to be tested

12

5

Dissolution medium

900 ml of aqueous media of various pH.

Appropriate surfactants may be used for water  insoluble drugs.

6

Sampling schedules

1,2 ,4 hours and every two hours thereafter, until 80 % of the drug is released.

7

Tolerance

To be established on data generated based on data generated on the first 3 batches.

8

Sampling time

Specimen have to withdrawn within a tolerance of  ±  2 % of the stated time in hours.

9

Content uniformity

Content uniformity testing of the test product lot  should be performed as described in the USP 23.

 

 

 

9.1.2   Specifications

 

The purpose  of establishing dissolution specifications is to ensure batch -to -batch consistency  within a range which guarantees acceptable  biopharmaceutical performance in-vivo. Specification limits therefore have to be defined based on experience gained during the drug development stage and bioequivalence studies. In most cases arriving at specifications limits requires thorough in-vitro - in-vivo comparison studies.

 

Dissolution specification should consist of atleast three points. The first specification is intended to prevent "dose dumping" and therefore should be set after a testing interval of one to two hours or corresponding to a dissolved amount of  20 - 30% of labelled drug substance. The second specification point should define the dissolution pattern and thus be set around 50 % release of labelled drug substance.

 

The final specification point should ensure (almost) quantitative drug release, which is generally understood as  ³ 80%. The dissolution run in quality control therefore should be extended for the time interval until at least 80% of drug substance is dissolved. Shorter test intervals can be acceptable  in special cases but require justification on the basis of an in-vitro - in-vivo comparison  study and should be upto  24 hours.

 

The acceptance range for the dissolution pattern at the time intervals specified should be defined case-by-case  on the basis of the in-vitro - in-vivo comparison study and taking into consideration the capability of the manufacturing process and the commonly accepted range of 95 - 105% of stated amount for the average content of drug substance. Where both upper and lower limits are specified at any time point, the difference between them should usually not exceed 20%  of the labelled content of drug substance in the formulation unless limits have been shown to provide reproducible and acceptable in-vivo performance.

 

Specifications for the dissolution procedure to assure quality control will be determine on a case by case basis. In general future validation will be required to expand dissolution specifications beyond those established for the biobatch.

 

 

9.2  Key validation points for dissolution testing are :

 

1.      Reproducibility of the method.

 

2.      Proper choice of medium.

 

3.      Maintenance of sink conditions.

 

4.      Control of solution hydrodynamics.

 

5.      Dissolution rate as a function of pH

 

9.3 The validated dissolution test method should 

       establish:

 

1.      Lack of dose dumping-indicated by a narrow limit on the one hour dissolution specification.

2.      Controlled release characteristics- by employing additional sampling windows overtime.

(Narrow limits with an appropriate Q value system will control the degree of  first order release).

3.      Complete drug release - indicated by a 75 - 80% minimum release specification at the last sampling interval.

4.      Dosage form pH dependence  / independence indicated by percent dissolution in water or appropriate buffer , simulated gastric * and simulated intestinal  * fluid.

 

(*) - minus enzymes

 

 

 

The dissolution range at each time point should be subjected to the following acceptance table.

 

Dissolution Range (window) : Q 1 - Q2 for each time point

 

Q1 and Q2 are the lower and upper limits set for percent release at each time point

 

S1: Each of six units within Q1 + 5 to Q2 - 5 range

 

S2 : Each of twelve units within Q1 to Q2 range

 

S3 : NLT 22 units within Q1 and Q2 range.

       NMT 2 units within Q1 - 10 to Q2 + 10 range or  Q1-5 to Q2+5 range, depending on the nature of the product..

 

The dissolution limits at each point are set after reviewing the dissolution profile of the lot on which acceptable bioavailability data  has been obtained. The standard is set on the basis of 12 tablet dissolution for the S2 range in the acceptance table. For the S1 stage (6 units) , the dissolution range is made tighter. For the S3 stage (24 units) the dissolution limits are slightly widen to allow for outliers. The range nevertheless should be sufficiently constrained so as to prevent dose dumping.

 

 

9.4  Equipment selection criteria :

 

9.4.1 Apparatus Suitability Test -

 

Individually test 1 tablet of the USP Dissolution Calibrator, Disintegrating Type and 1 tablet of USP Dissolution Calibrator, Nondisintegrating Type, according to the operating conditions specified. The apparatus is suitable if the results obtained are within acceptable range stated in the certificate for that calibrator in the apparatus tested.

 

 

9.4.2  Dissolution Medium -

 

Use the solvent specified in the individual monograph. If the Dissolution Medium is a buffered  solution, adjust the solution so that its pH is within 0.05 unit of the pH specified in the individual monograph.[ NOTE - Dissolved gases can cause bubbles to form, which may change the results of the test. In such cases , dissolved gas should be removed prior to the testing]

 

9.4.3  Time -

 

Where a single time specification is given, the test may be concluded in a shorter period if the requirement for the minimum amount dissolved is met. If  two or more times are specified , specimens are to be withdrawn only at the stated times, within a tolerance of ± 2%.

 

The suitability test has to cover each individual apparatus and to consist of the full programme, meaning both calibrator types. Both paddle and basket equipment have to be qualified.

 

 

The automated systems should be validated with respect to all parameters and there shall be evidence of no significant differences between data obtained with the manual dissolution equipment and the automated systems.

 

The above discussed information should be used for setting dissolution specifications for all modified release dosage forms.

 

Deviations from the key elements , dissolution procedure or acceptance table must be approved by the Drug Controller , India.

 

These Guidelines should be helpful and   applicable for all involved in in-vitro dissolution test. However, there was special emphasis on providing reliable guidance for industrial research and development, process validation and quality control, making the Guidelines especially applicable for industry, drug authorities and control laboratories but also for universities , hospitals, pharmacies or others , when involved in  (bio) pharmaceutical quality evaluation.

 

In general this Guidelines should be understood as recommendations based on scientific knowledge and experience. They should be helpful in the dialogue with drug regulatory authorities; however , they are not intended to represent any official requirements in this field.

 

 

 

9.5  Guidelines for oral extended (Controlled) release  dosage forms

Should this be referred to as per the pharmacopeia?

1. Apparatus

USP Type  I (Basket) for capsules

 

USP Type II (Paddle) for tablets

 

Impact of sinker which is recommended when specimen tends to float.

2. Impact of rotation speed

50,75 or 100 rpm

3. Temperature

37o ± 0.5 o C

4. Impact of dissolution medium

Aqueous medium of various pH

 

Volume - 500 ml, 750 ml, 900 ml or 1000 ml

5.  Sampling schedule

1,2,4, hours and every two hours     thereafter, until 80% of the drug is release

6. Sink condition

Solubility of the compound

7. Property of the dosage form

Sinking , floating, Oros

 

 

 

 

 

 

9.6  Dissolution specification :

 

The purpose of establishing dissolution specification is to ensure batch to batch consistency within a range which guarantees acceptable biopharmaceutical performance in vivo .

 

1.      The first specifications is intended to prevent dose dumping and therefore should be set after a testing interval of one or two hours or corresponding to a dissolved amount of  20 to 30 % of the labelled drug substances

2.      The second  specification point should defined the dissolution pattern and thus be set around 50% release of labelled drug substance.

3.      The final specification point should ensure (almost ) quantitative drug release, which is generally understood as > 80%. The dissolution run in quality control therefore  should be extended for the time interval until atleast 80 % of drug substance is dissolved and should atleast cover 24 hours.

 

 

 

9.7  Method Validation :

 

 

Validation of an analytical method is the process by which it is established , by laboratory studies, that the performance  characteristics of the method meet the requirments for the intended analytical applications. Performance characteristics are expressed in terms of analytical parameters. Typical analytical parameters that should be considered in the validation of the types of assays described in this document are listed in Table 1

 

 

Table 1 : Typical Analytical Parameters used in Validation

 

                                          Accuracy

                                          Precision

                                          Specificity

                                          Linearity

                                          Limit of quantification

                                          Limit of detection

                                          Range

                                          Ruggedness

                               

 

Data elements recommended for Analytical Method used for Dissolution Testing

   

                                   Accuracy

                                   Precision

                                   Specificity

                                   Linearity

                                   Range

                                   Ruggedness

 

 

10. STATISTICAL  EVALUATION FOR CONVENTIONAL DOSAGE FORMS

 

 

10.1 GENERAL

 

Statistical methods are used to estimate the certanity of statement and precision of measurement  about the population (larger group) after observing a random sample (smaller group) of  its members.

It is therefore absoulutely essential to ensue that the following points are duly taken  into consideration in order to improve the quality after study :

i. The services of a statistician should be enlisted right from very beginning of the study.

ii. All raw data must be edited to ensure quality of data from time to time. Case record forms must be checked for :

(a)   completeness

(b)   internal consistency

(c)    consistency of others

(d)   missing data

(e)   abnormal findings

(f)     use of uniform laboratory and other methods

 

 
10.2 STUDY DESIGN

 

The choice of design should be based on many factors such as background information about formulation , variability with the laboratory, variability between volunteers, etc.

However, following are the broad guideliness for the selection of design :

Good experimental design enhance the power of the study. The validity of results does not necessarily increase with the number of subjects unless statistical aspects have been carefully considered.

Early and continuing consultation with statistician is recommended. As far as possible , thestudy could be of crossover designs and suitably randomized. Some of the designs are given below :

Two-Period Crossover Design :

In case of 2 formulations, an even number of subjects should be randomely divided into two equal groups. In the first period , each member of one group will receive a single dose of the test formulation and each member of the other group will receive the standard formulation. After a suitable wash period (generally 5 half lives), in the second period , each member of the respective groups will receive a dose of an alternative formulation and the experiment will be repeated.

 

 

 

 

 

 

The design can be depicted as follows :

 

       Vol.No                   Period 1                  Period 2

           1                            A                              B

           2                            B                              A

           3                            A                              B

     4                            A                              B

     5                            B                              A

     6                            B                              A

 

Latine Square Design :

In case of more than two formulations , a Latine Square Design should be used. For example, in a bioequivalence study of 3 formulations, a group of volunteers will receive formulations in the sequence shown below :

 

       Vol.No.                Period 1               Period 2               Period 3

           1                          A                          B                          C

           2                          B                          C                          A

           3                          C                          A                          B

 

The next group of 3 volunteers will receive formulations in the same sequence as shown above.

 

Balance Incomplete Block Design (BIBD) :

In case there are more than 3 formulations, the latin square design will not be ethically advisable, mainly because each volunteer may require the drawing of too many blood samples.

However, if each volunteer expected to receive at least two formulation, then such a study can be carried out using Balance Incomplete Block Design (B.I.B.D.). As per this design, if there are four formulations, six possible pairs or formulations can be chosen from four formulations. Then, the first 6 volunteers will receive these six pairs formulations and the next six volunteers will receive the same six pairs in reverse order.

 

Vol. No.           Period 1           Period 2

    1                       A                     B

    2                       A                     C

    3                       A                     D

    4                       B                     C

    5                       B                     D

    6                       C                     D

    7                       B                     A

    8                       C                     A

    9                       D                     A

   10                      C                      B

   11                      D                     B

   12                      D                     C

 

10.3 NUMBER OF VOLUNTEERS

 

The minimum acceptable number will be 12.

 

n ³  {(s)2}        (ta+ tb)2 + 0.25 ta2

           -----------

        2D2

Where ,

a = Required level of significance (0.05)

b = Required power of test (0.80)

s2 = Error mean sum of squares from ANOVA (estimated / guess)

D = Minimum difference between the means which if present, ought to be detected

 

In disease states, where combination products are prescribed, a three-way cross over study should be conducted on 18 (eighteen) volunteers.

 

10.4 PHARMACOKINETIC ANALYSIS

 

In single dose studies, the following pharmacokinetic parameters for the reference and trial substances should be measure :

(a)   Area under the plasma / blood concentration time curve from time zero to time t (AUC 0-t) calculated using trapezoidal rule.

 

(b)   Area under the plasma/ blood concentration time curve from time zero to infinity (AUC 0-a) calculated using the following expression :

 

                        AUC0-¥ = AUCa-t +     Ct

                                                         --------

                                                             Kel

 

Where Ct = least measurable drug concentration and Kel = terminal elimination rate constant

(c)    terminal elimination half-life of drug (t½)

(d)   peak drug concentration (Cmax) obtained directly from the data.

(e)   Time to peak drug concentration (tmax) obtained directly from the data.

 

10.5 STATISTICAL ANALYSIS

 

The pharmacokinetic paramaters, Cmax, Tmax and AUC should be subjected to a three-way analysis of variance, (3-way ANOVA) in order to test differences due to formulations, period and subjects. A more complex ANOVA may be appropriate in some circumstances; for example, if treatments are replicated. The standard Parametric ANOVA assumes homogeneity of variances, normality and additivity of independent variables.

In order to ensure homogeneity of variances between treatments, Barttlet’s test or a similar test should be carried out prior to performing the ANOVA. Barttlet’s test being sensitive to departures from normality,the test for normality is not performed routinely.

If Barttlet’s test indicates a significant difference in variances amongst the treatments, then a Parametric ANOVA should not be performed in the untransformed data. The data may be log-transformed  and Barttlet’s test should again be conducted on the log-transformed data prior to an ANOVA.

The primary comparison of interest in a bioequivalence study is the ratio of average parameter data (AUC or Cmax) from the test and reference formulations rather than the difference between them. Log transformation of data allows the General Linear statistical model to draw inferences about the ratio of the two averages on the original scale. Log transformation thus achieves the general comparison based on the ratio rather than on the difference.

Moreover, plasma concentration data, including AUC anc Cmax, tend to be skewed and their variances tend to increase with the means. Log transformation corrects this situation and makes the variances independent of the mean.

Further, the frequency distribution skewed to the left, i.e., those with a log tail to the right are made symmetrical by log transformation.

In case no suitable trannsformation is available, the non parametric method should be used. T-max values being discrete, data on Tmax should be analyzed using non-parametric methods.

In respect of each pharmacokinetic parameter, a 95% confidence interval using Westlake’s method should be presented.

 

10.6 PRESENTATION OF DATA

 

Data presentation should be done by attaching copies of tables listed on page 34.

 

10.7 OUTLIER CONSIDERATIONS

 

In bioequivalence studies, outliers are defined as subjects having discordant values of one or more pharmacokinetic parameters in comparison to other values in a study.

The existence of an outlier is indicative of either product failure or a type of a inclusion of a subject from a sub population which is relatively rare.

Outliers should be detected using appropriate statistical tests. However, outliers should not be dropped from the analysis purely on the basis of a statistical test. A scientific explanation should be provided to justify the exclusion of a subject from statistical analysis.

 

10.8 RESULTS AND CONCLUSION

 

First , the ratio of the reference product mean to the test product mean should be examined to see whether it lies within or outside the acceptable Westlake interval, example 0.8 – 1.2 or 0.9 – 1.1 as the case may be , for this determines the clinical significance of the difference.

If the ratio is within the acceptable limit, then the statistical significance should be considered. If the difference  is statistically significant , it would mean that although the test product is acceptable , it is really different from the reference product; otherwise, it would mean that the observed  difference could be due to chance and the products are unlikely to be really different.

If the ratio is outside the acceptable limit, and also statistically significant, then it would mean that the test product is really and unacceptably different from the reference product. Otherwise, it would mean that the difference, though unacceptable, could be due to chance.

In either case, the power of the experiment to detect the minimum unacceptable difference at the chosen significance level should be calculated and stated, for it would indicate whether the experiment was sensitive enough to detect what is clinically important.

 

10.9 STATISTICAL ANALYSIS

 

Summary

No. of Volunteers       : Twelve

Study Design              : Two-period crossover, Latin square (for 3 formulations including

                                      standard formulation), Balanced Incomplete Block Design 

                                      (BIBD) for more than 3 formulations and when each volunteer is

                                      expected to receive at least 2 formulations.

 

Statistical Analysis    :  Each pharmacokinetic parameter should be subjected to a 3 way

                                      Analysis of Variance (3-way ANOVA) to test differences due to

                                      fornulation, period and subject.

 

                                      95% confidence intervals using Westlake’s method should be

                                      presented for all pharmacokinetic parameters.

 

Documentation           : Copies of tables listed on the next page in the formats specified

                                      on subsequent pages should be enclosed.

 

 

 

11. STATISTICAL evaluation for extended dosage forms

 

 

 

The statistical methods applicable to the conventional formulation are not considered to be adequate for ER formulations because of relatively high probability of increased intersubject variability in bioavailability including dose/dumping.

 

11.1 CONSIDERATIONS  IN STUDY DESIGNS :

 

The decision regarding the types of studies and the appropriate study designs to  be used depends on the available information about the active drug entity, its clinical pharmacokinetic and biopharmaceutical properties. Also , while designing bioequivalence studies of ER formulations the following factors need special considerations :

 

a)      Does an ER formulation contain a totally new chemical entity ?

 

b)      Is it the first or subsequent  market entry of an ER formulation?

 

c)       What is the extent of drug accumulation after repeated dosing ?

 

d)      What is the potential for adverse drug reaction ?

 

e)      What are the  claims for safety and efficacy of the ER formulation ?

 

 

 11.2  proposed study designs :

 

The proposed  studies to be conducted under various situations are listed below :

 

11.2.1  Situation A :

 

As defined in the protocol section 3.2.6.1 (page no        )

 

11.2.1.1 STUDY A-1 :

 

A single dose, randomized, two period, two treatment, two sequence cross over study under fasting conditions, comparing equal doses of the test and reference products.

 

Objective :

 

To compare rate and extent of absorption of test product with that of a reference product when administered in equal labelled doses.

 

Design :

 

Single dose, two treatment, two period, two sequence cross over.

 

Equal number of subjects should be randomly assigned to two dosing sequences as follows :

 

--------------------------------------------------------

Vol.           Period 1                       Period 2

--------------------------------------------------------

1.                       A                                 B

2.                       B                                 A

3.                       B                                 A

4.                       A                                 B

5.                       B                                 A

6.                       A                                 B

.

.

.

.

.

12

---------------------------------------------------------

 

Washout  Period :

 

The minimum washout  period between two phases of the study should not be less than ten elimination half -lives of the drug.

 

No. of  subjects :

 

Minimum twelve; however it is desirable that sample size be estimated on the basis of intra/inter subject variability, level of significance (a), power of test (1 - b) and difference to be detected.

 

Pharmacokinetic Data :

 

AUC0-t , Cmax, Tmax

 

 

Statistical Methods :

 

Data on AUC and Cmax in absolute forms as well as in logarithmic form should be analyzed statistically using Analysis of Variance (ANOVA).

 

Data on Tmax should be analyzed using a non parametric test.

 

 

The 90% confidence intervals:

 

a) Conventional confidence interval and

 

b) Westlake confidence interval.

 

 

Statistical Tables to be presented :

 

 

Table no.

 

topic

     1

Demographic profile of volunteer

     2

Randomization schedule

     3

Volunteer wise plasma-concentration profile for test product

     4        

Volunteer wise plasma – concentration profile for reference product

     5

Volunteer wise AUC0-t, Cmax ,Tmax, log AUC, log Cmax for test  product

     6

Volunteer wise AUC0-t, Cmax Tmax, log AUC, log Cmax for reference           product

     7

Summary pharmacokinetic parameters for test and reference product

     8

Volunteer wise values of AUC for test, reference, difference, ratio and log of ratio.

      9

Volunteer wise values of Cmax  for test, reference, difference, ratio and log of ratio.

      10

Summary ANOVA for AUC

      11

Summary ANOVA for log (AUC)

      12

Summary ANOVA for Cmax

      13

Summary ANOVA for log (Cmax)

      14

Conventional  and  Westlake 90 % Confidence Interval for AUC, log AUC, Cmax , log Cmax of test product in terms of percentage of respective values reference product

       15

Power of a test

       16

Result of non-parametric test on Tmax

 

 

Conclusion :    

 

Confidence  intervals for AUC and Cmax within 80-120% for untransformed and 80-125% for log transformed indicate existence of bioequivalence of test and reference products. For Cmax this limit may be stretched to 70 – 130% for safe and variable drugs.

 

11.2.1.2 STUDY A-2 :   

 

Single dose randomized, three treatment, three period, six sequence  cross over design.

 

 

Objectives :          

 

1)       To determine labeling  instructions describing special conditions for administration with respect to meals.

 

2)      To provide information on pattern  of absorption of the E-R dosage form compared to that of I-R dosage form.

 

Study Design :               

 

Single - dose,  randomized, 3-treatment 3-period, 6-sequence crossover study design is depicted below :

 

Volunteer No.

Period 1        

Period 2

Period 3

1

A                          

B

C

2

B

C

A

3

C 

A

B

4

A

C

B

5

B

A

C

6

C

B

A

 

 

Note : No. of subjects should be a multiple of 6 and same sequence can be used for treating subsequent groups of 6 subjects  each.

 

Treatments :

 

A)      E-R dosage form administered under fasting condition

 

B)      E-R dosage form administered at the same time as standardized meal.

 

C)     Conventional dosage form administered under fasting conditions.

 

 

No. of Subjects :

 

12 or above (must be a multiple of 6)

 

Pharmacokinetic Data :

 

AUC0-t , Cmax, Tmax

 

Statistical Methods :

 

Data on AUC ,Log AUC,  Cmax   and log Cmax  should be analyzed using three way Analysis of  Variance (ANOVA).

 

Data on Tmax should be analyzed using a non-parametric test.

 

 Interpretation / Conclusion :

 

1.      A comparable food effect will be assumed if the mean values of AUC0-t and  Cmax   for the test product administered with food differ by no more than 20% of the respective mean values for the reference product.

 

2.      A difference of more than 20% suggest need for conducting additional studies .

 

If  the results of above mentioned studies indicate that the test product is equivalent to reference product in terms of

 

a)      Extent of rate and absorption in equal doses (Study A-1)

 

b)      Comparability of food effects (Study A-2) , one more study should be conducted.

 

 

11.2.1.3 Study A-3

 

A multiple dose, steady-state, , two-treatment, two-period, two-sequence crossover study

 

Objectives

 

To compare the steady state rate and extent of absorption of test extended release product with that of reference extended release product.

 

Design :

 

Multiple dose, two treatment ,two period, two sequence, steady state crossover study  with random allocation of two sequences as depicted below :

 

--------------------------------------------------------

Vol.           Period 1                       Period 2

--------------------------------------------------------

1.                       A                                 B

2.                       B                                 A

3.                       B                                 A

4.                       A                                 B

5.                       B                                 A

6.                       A                                 B

..

12

---------------------------------------------------------

 

Number of subject :   

 

Minimum 12

 

Pharmacokinetic data

 

i.         Mean trough levels on 4 consecutive days to establish evidence  of reaching steady state.

 

ii.        (Cmin)ss, (Cmax)ss, AUCss, % Fluctuation, %PTF.

 

Statistical Methods :

 

Data on AUCo-t and Cmax in absolute as well as log transformed form should be analyzed statistically using Analysis of Variance (ANOVA).

 

Data on Tmax should be analyzed using non-parametric test.

 

Data on percent fluctuation and Fluctuation Index (% PTF) should be compared statistically using ANOVA.

 

90% Confidence Intervals

 

a)      Conventional  confidence intervals and 

 

b)      Westlake confidence intervals for AUC, log AUC , Cmax, log Cmax.

 

 

 

Statistical Tables to be presented :

 

 

Table NO.

                                         Topic

 

1.       

 Demographic profile of volunteer

 

2.       

 Randomization schedule

 

3.       

 Volunteer wise plasma-concentration time profile (for day 1)for test product

 

4.       

 Volunteer wise plasma - concentration time profile (for day 1) for reference product

 

5.       

 Volunteer wise plasma - concentration time profile (for day 7 after reaching steady ) for test product

 

6.       

 Volunteer wise plasma - concentration time profile (for day 7 after reaching steady state) for reference product

 

7.       

Volunteerwise drug / metabolite concentration  in (trough / min levels) plasma just prior to the administration of next dose on days 3,4,5 and 6 prior to reaching steady state, for test product.

 

8.       

Volunteerwise drug / metabolite concentration  in (trough / min levels) plasma just prior to the administration of next dose on days 3,4,5 and 6 prior to reaching steady state, for reference product.

 

9.       

Mean values of trough levels  (Cmin) for both test and reference formulation for  four consecutive days.

 

10.   

Results of regression analysis of four (Cmin) values for both test and reference products for showing evidence of steady state.

 

11.   

Volunteerwise Cmin (SS), Cmax (SS), AUCo-t, (SS), percent fluctuation and Fluctuation Index (%PTF) for test product.

 

12.   

Volunteerwise Cmin (SS), Cmax (SS), AUCo-t, (SS), percent fluctuation and Fluctuation Index (%PTF) for reference product.

 

13.   

Summary pharmacokinetic parameters for test and reference product.

 

14.   

Summary ANOVA for AUC

 

15.   

Summary ANOVA for log (AUC)

 

16.   

Summary ANOVA for Cmax

 

17.   

Summary ANOVA for log Cmax

 

18.   

Summary ANOVA for percent fluctuation

 

19.   

Summary ANOVA for fluctuation index ( % PTF)

 

20.   

Conventional and Westlake 90% confidence intervals for AUC, Cmax, % Fluctuation & % PTF

 

21.   

Power of a test

 

 

11.2.2  Situation B

 

As defined in the protocol section 3.2.6.2 (page no.       )

 

 

11.2.2.1  Study B-1 :

 

Single dose , randomized , two treatment, two period cross over study.

 

Objective: 

 

To compare the effect of concomitantly administered standardized meal on test extended release product with that on reference extended release product administered concomitantly with standardized meal.

 

Design:

 

Single dose , two treatment, two period, two sequence crossover.

 

 

Equal number of subjects should be randomly assigned to two dosing sequences as follows :

 

--------------------------------------------------------

Vol            Period 1                       Period 2

--------------------------------------------------------

1.                       A                                 B

2.                       B                                 A

3.                       B                                 A

4.                       A                                 B

5.                       B                                 A

6.                       A                                 B

.

.

.

.

.

12

---------------------------------------------------------

 

Washout period:

 

The minimum washout period between two phases of the study should not be less than 10 elimination half lives of the drug.

 

 

No. of subjects :         Minimum 12.

 

Pharmacokinetic parameters : 

 

AUC0-t, Cmax , Tmax

 

Statistical Methods :

 

Data on AUC0-t, and Cmax   in absolute form and also in logarithmic form  should be analyzed using Analysis of  Variance (ANOVA).

 

Data on Tmax should be analyzed using a non parametric test.

 

Table no

 

topic

1.       

 Demographic profile of volunteer

2.       

 Randomization schedule

3.       

 Volunteerwise plasma-concentration  time profile for test ER product

4.       

 Volunteerwise plasma - concentration time profile for reference ER  product

5.       

 Volunteerwise AUC0-t Cmax Tmax, log AUC, log Cmax for test  ER product

6.       

 Volunteerwise AUC0-t, Cmax Tmax, log AUC, log Cmax for reference      ER product

7.       

Summary  of pharmacokinetic parameters for test and reference ER product

8.       

Volunteerwise values of AUC for test, reference ,difference, ratio and log of ratio.

9.       

Volunteerwise values of Cmax  for test, reference ,difference, ratio and log of ratio.

10.   

Summary ANOVA for AUC

11.   

Summary ANOVA for log AUC

12.   

Summary ANOVA for Cmax

13.   

Summary ANOVA for log Cmax

14.   

Conventional and Westlake 90 % confidence interval for AUC, log AUC, Cmax , log Cmax of test ER product in terms of percentage of respective values of reference product

15.   

Power of a test

 

Conclusion :

 

Confidence  intervals for AUC and Cmax within 80-120% for untransformed and 80-125% for log transformed indicate existence of bioequivalence of test and reference products. For Cmax this limit may be stretched to 70 – 130% for safe and variable drugs.

 

 

11.2.2.2  Study B-2 :

 

If the results of the study B-1 demonstrate that the test ER products has a food effect then a multiple dose, steady state, two treatment, two period, two sequence crossover should be carried out  as described in study A-3

 

12.  REPORT FOR BIOEQUIVALENCE STUDIES

 

 

The report for a bioequivalence study should truly reflect the data generated from all  components of a study. The report should be arranged in discrete sections in a sequence that is intended to provide the  reviewer a clearer and comprehensive picture of a study. Each section is intended to be complete and self-sufficient, thus allowing the reviewer a relatively easy and quick access to the required information for a speedy and comprehensive review.

 

Following is a suggested format of the study report.

 

12.1  SUMMARY REPORT :

  

1       Detailed study title

1       Name of sponsor

1       Name and address of clinical analytical laboratory

1       Signature of investigator(s)

1       Study resume

1       Product information

1       Brief description of procedures

1       Results and discussions

1       Conclusion

1       Dissolution profile of test and reference products

1       Summary tables of pharmacokinetic data with statistics of untransformed and log transformed data

1       Individual serum /plasma concentration profile of test and reference product.

1       Comparative evaluation of all pharmacokinetic parameters

1       Mean plots of product profile (linear and semilog)

1       Semi-log plots product profile in individual subjects

1       Protocol and amendments

1       Sample copy of informed consent form

1       Approval letter from ethics committee

1       Curriculum vitae of investigators

 

12.2  CLINICAL REPORT :

 

1       Summary of study

1       Description of events during clinical operations e.g. dates of clinical study periods, subjects  dropouts ,enrolled subjects ,adverse events (provide data)

1       Demographic data of subjects

1       Randomization scheme with description of procedure

1       Protocol deviations

1       Results and discussion on clinical events

 

 

12.3  ANALYTICAL  REPORT :

 

 

1       Summary of analytical study

1       Description of  events during analysis of clinical samples e.g. total number of clinical samples, number of samples analyzed, dates of analysis, missing samples and procedure for calculation of concentration.

1       Results of analysis e.g. incidence of chromatographic  interferences, repeat analysis

1       Impact of problems encountered during analysis on the results and conclusion of the study

1       Results and discussion on analytical procedures and events

1       Data of all back calculated calibration curves with regression parameters reflecting accuracy and precision of analytical method

1       Data of all back calculated quality control samples reflecting precision and accuracy of analytical method

1       Serum concentration profiles of test and reference products

1       Method validation report

1       Analytical test procedure

 

 

12.4  STATISTICAL REPORT:

 

1       Summary of statistical analysis

1       Description of pharmacokinetic parameters subjected to statistical analysis

1       Description of statistical tests and procedures

1       Results and discussion

1       Computer outputs of statistical tests

 

12.5  RAW DATA:

 

 

Provide analytical raw data :

Chromatograms of atleast 20% subjects of all periods and all sampling time intervals  along with their calibration curves and quality control samples.

 

 

 

13.  WAIVER REQUIREMENTS

 

 

13.1 Waiver required for modified release dosage forms

 

 

A single dose two-way crossover study under fasting conditions is required for each strength of a generic extended release tablet formulation with multiple strengths. The multiple dose steady state study and the food / fasting single dose three-way crossover study are to be conducted with the highest strength only.

 

For extended release capsule formulation marketed in multiple strengths, a single dose bioequivalence study under fasting conditions is required only on the highest strength, provided  that the compositions of the lower strengths are proportional to that of the  highest strength, and the capsules contain identical beads or pellets. Single dose in vivo bioequivalence studies may be waived for the lower strengths on the basis of the acceptable dissolution profiles. Multiple dose steady state and single dose food / fasting studies are to be conducted  on the  highest strength  of ther capsule formulation. 

 

 

 

 

 

 

14. DOCUMENTATION

 

It is one  of the most important aspects of any experimental work, be it the conduct of bioequivalence/ bioavailability study or any other quantitation work. Good documentation ensures quick retrieval of any information pertaining to any important activity at any given time. It can be resorted to as an ideal help in case of situation of technical or non-technical problems/disputes

A good laboratory should have a good documentation system, a good documentation room and a good documentation officer.
With respect to the conduct of bioequivalence/bioavailability studies following are the important documents that any laboratory should maintain.

 

·           Company correspondence pertaining to initiation of any particular bioequivalence/ bioavailability study.

·          Available literature on the pharmacological data on the drug which will include data studies on animals or human subjects

·          Protocol of the study

·          Ethical committee review of that particular protocol and minutes of the meetings of the board members of the ethical review committee for that particular study.

·          Violation of the protocol, if any.

·          Detailes product/s information.

·          Volunteer records includiing the following

-          Volunteer consent form

-          Symptom check list

-          Activity record sheet

-           Clinical examination form

-          Volunteer habit form

 

• Any dropouts from the study Should the reasons be recorded?

• Any adverse reaction experienced by the volunteers on the day of the study. In such   

   cases, the medical aid given in the form of treatment to such a volunteer should also be 

   maintained. A necessary mention of such incidence/s with appropriate details should be  

   made in  the study report. Such reports should be duly signed by the authorized 

    personnel comprising the chief investigator, clinical pharmacologist and medical 

    officer in-charge of that particular trial.

 

• Details of  the analytical method validation including the following :

-          System suitability test

-          Linearity range

-          Lowest limit of quantitation

-          QC sample analysis

-          Stability sample analysis

-          Recovery experiment result

 

 

 

actual analytical data of volunteer plasma samples which should include the following :

 

-          All the volunteer plasma chromatograms

-          Linearity results done on every analytical day

-          Inter-day and intra – day variation of assay results

-          Any aberrant chromatograms

-          Any repeat analysis

-          Calibration status of the instrument used for that particular analysis

-          Any other technical complications

 

     Compilation of  raw data

    All the comments of the pharmacologist regarding the data of the study submitted for 

     review.

    A copy of the final report submitted to the sponsor

 

All these documents should be maintained by the concerned bioequivalence/ bioavailability study center for a period of 3 (three) years from the date of submission of the final report to the sponsor.

 

 

 

 

 

 

 

APPENDICES

 

 

 

 

15.appendix - 1

 

IN VITRO IN VIVO CORRELATION

 

 

Should this be a part of the guideline?

15.1  Introduction :

 

 

The term in vitro - in vivo correlation refers to the establishment of a relationship between a biological parameter or a parameter derived from biological property produced by a dosage form and physico chemical property or characteristic of the same dosage form.

 

The biological properties used are the plasma concentrations form or AUC obtained following drug administration of the dosage form. The physico chemical properties are characterized by dosage form's invitro dissolution behaviour viz., percentage drug release under a given set of conditions.

 

The simplest way to demonstrate a correlation is to plot the fraction absorbed invivo versus the fraction released invitro. This relationship is often linear with a slope of 1. The intercept may or may be 0 depending upon whether there is a lag time before the system begins to release drug invivo, or the absorption rate is not instantaneous resulting in the presence of some finite quantity of dissolved but unabsorbed drug. In either case, it is a point - to-point or level A correlation when the relationship is linear with  slope of 1.

 

 

15.2  Parameters considered :

 

Recently, dissolution rate has been used as a manufacturing process standard and  is generally considered to be the in vitro parameter most likely to correlate with in vivo bioavailability.

 

In vivo studies are described in terms of the rate and extent of drug absorption. Rate of absorption is reflected by

 

1.      Peak drug concentration in plasma i.e. Cmax.

2.      Time to reach the peak i.e. Tmax

 

Extent of absorption is reflected by

 

1.      Cmax

2.      Area under the plasma drug concentration curve i.e. AUC. Usually, the AUC and  Cmax  are compared with the in vitro data

 

 

 

 

 

15.3 LEVELS OF CORRELATION  FOR EXTENDED  RELEASE ORAL   dosage forms:

 

 

For  ER dosage forms; three correlation levels have been defined in descending order of usefulness.

 

 

15.3.1  Level A :

 

It represents a point to point relationship between in vitro data and in vivo input rate of drug form the dosage form.

 

Sometimes the % of drug dissolved at a given time  is correlated to a certain parameter of the bioavailability of drug product.

 

 

15.3.1.1  Advantages :

 

1)      A point to point correlation is obtained. In vitro curve  can serve  as a surrogate for in vivo performance.

 

 

15.3.2  Level B :

 

This utilizes the principle of Statistical Moment Analysis.  According to this :

 

MDT = MDTtest - MRTsolution

 

where,

 

MDT :  Mean in vitro dissolution time.

 

MRT :  Mean residence time after the ingestion of an aqueous solution.

 

This equation is based on the fact that mean absorption time (MAT) is equivalent to the difference between MRToral & MRTiv

 

 

15.3.3  Level C :

 

This relates one dissolution time point t50% ; t90% to one pharmacokinetic parameter. Such as AUC, Cmax or Tmax  . It reflects a single point correlation. It does not reflect the complete shape of the plasma level, which is the critical factor that defines the performance of CR / SR products.

 

15.4  DEVELOPING  A  CORRELATION :

 

Conceptually, the relationship between the entire in-vitro dissolution curve and the entire plasma level curve defines correlation. Hence, the method discussed below is mainly applicable to Level A correlations, which uses the entire plasma drug concentration-time curve.

 

For establishing in-vitro in-vivo correlation, the following information should be presented :

 

i.         A table giving the dissolution profile of dosage form using standard dissolution technique (Format 1).

 

ii.       A table giving AUC as per Nelson-Wagner model (Format 2)

 

 

Although this model  is best suited for drugs following single compartment distribution, it can also be extended to drugs following multi-compartment distribution; as it is expected to behave in similar fashion in both test and reference formulations. Thus , this parameter for test and reference product can be compared statistically.

 

15.4 In-vitro correlation should be established using  the method given 

       below :

 

 

i.         Using the dissolution profile graph the time required for 50% dissolution should be estimated (Format 3).

 

ii.       Using the Nelson -Wagner model  the fraction absorbed should be estimated as shown in format 2.

 

iii.      A graph showing the percentage fraction absorbed (y-axis) against time

(x axis) should be presented  (Format 4).

 

iv.     Using this graph the time for 50% of absorption is to be estimated.

 

v.       The intensity factor is to be calculated as

 

                                     Time for 50% absorption

Intensity Factor =          ------------------------------

                                     Time for 50% dissolution

 

vi.     Transform  T (in vivo time point) to the corresponding in vitro time point applying to the equation T = In vivo/Intensity Factor

 

vii.    The percentage absorption at Tinvivo and percentage dissolution at (Tinvivo/intensity factor) should be plotted on y and x-axis respectively (Format 6).

 

      A slope = 1 of this line indicates a good in vitro-in vivo correlation.

 

The value of the positive intercept on x-axis gives the percentage drug dissolved before getting absorbed or percentage drug dissolved during Lag-time (Format 6).

 

Lag time can be estimated as the time corresponding to this intercept on dissolution profile (Format 3).

 

If from the studies indicated in the in vitro dissolution evaluation above the modified-release dosage form exhibits dissolution behavior that is independent of the variables studied, and a Level A correlation is demonstrated when the in vitro dissolution curve is compared to the drug input rate curve, it is likely that the correlation is general and can be extrapolated within a reasonable range for that formulation of the active drug entity. If, however, the dosage form exhibits dissolution behavior that varies with the in vitro conditions, it must be determined which set of dissolution conditions best correlates with in vivo performance. One can then establish whether the correlation is real or an artifact. This is achieved by preparing at least two formulations having significantly different in vitro behavior. One should demonstrate a more rapid release and the other a slower release than the biobatch.

 

A pilot BA-BE study should be performed with these formulations , and the previously established correlation demonstrated for both. The formulation modifications of these batches should be based upon formulation factors that would be expected to influence the product's modified - release mechanism and modification of these formulation factors are expected to influence the dosage form's release rate.

 

Once a level A correlation is established , it is possible that invitro testing may be utilized for establishing the effects of manufacturing modifications such as minor formulation changes, manufacturing site and equipment change, alternative excipient suppliers and a change in dosage form strength in the same formulation.

 

 

15.5  BIBLIOGRAPHY

 

1.      Food & Drug Administration. Bioavailability and bioequivalence requirements.                 Fed. Reg. 42; 1977.

 

2.      Dighe SV and William RL. Guidance for oral extended (Controlled) release dosage forms; in vivo bioequivalence and in vitro dissolution testing : Division of Bioequivalence / Office of Generic Drugs, Centre for Drug Evaluation & Research, Food and Drug Administration (9/9/93).

 

3.      Dighe SV and Adams WP. Bioavailability and bioequivalence of oral controlled release products : a regulatory perspective. In Pharmacokinetics: Regulatory. Industrial, Academic Perspectives, Ed. P G Welling & F L T S Tse, Mercel Dekker, New York.

 

4.      Rodda BE. In Bioavailability: Design and Analysis in statistical Methodology in pharmaceutical sciences, Ed. D A Berry, Marcel Dekker, New York, 1989.

 

5.      Chen ML, Patnaik RN, Dighe SV and Williams RL, Guidelines for statistical procedures for bioequivalence studies using a standard two-treatment crossover design. Division of bioequivalence/Office of Generic Drugs, Centre for Drug Evaluation & Research, Food and Drug Administration, July, 1992.

 

6.      Bowalekar SK. Statistical Aspects of Bioavailability Studies: in Bioavailability and Bioequivalence - An update ; Ed H P Tipnis New Age International Publishers 1996.

 

7.      Pidgen AW Statistical Aspects of Bioequivalence - A Review, Xenobiotica 22 (7). 1992.

 

8.      Pharmacopeial Forum 19(3)  The United States Pharmacopeial Convention Inc. 1993.           

 

 

 

FORMAT 1 : INVITRO DATA : DISSOLUTION PROFILE

 

TIME
(MIN)
TABLET  NUMBER

 

MEAN
SD
SEM
CV%
1
2
3
4
5
6

 

0

 

 
 
 
 
 
 
 
 
 
 
10

 

 
 
 
 
 
 
 
 
 
 
**

 

 
 
 
 
 
 
 
 
 
 
**

 

 
 
 
 
 
 
 
 
 
 
**

 

 
 
 
 
 
 
 
 
 
 
**

 

 
 
 
 
 
 
 
 
 
 
**

 

 
 
 
 
 
 
 
 
 
 
 

 

 

 

 

 

FORMAT 2 : INVITRO DATA : PERCENTAGE FRACTION ABSORBED : NELSON – VAGNER MODEL

 

 

 

 

 

 

FORMAT 3: DISSOLUTION PROFILE

 

 

 

FORMAT 4 : PERCENTAGE FRACTION ABSORBED v/s TIME

 

 

 

 

 

FORMAT 5 : INVITRO : DISSOLUTION PROFILE FOR TRANSFORMED TIME

 

 

In-vivo

(Hrs)

Transformed

t = In-vivo/Int. Factor

(Hrs)

Transformed

Time

(Min)

% Dissolution

 

 

 

 

 

 

 

 

 

 

 

 

FORMAT 6 : PERCENTAGE FRACTION ABSORBED V/S PERCENTAGE DISSOLVED

 

 

 

* lag Time is estimated using x and dissolution profile (Format 3)

 

 

 

16 APPENDIX – 2

 

ADVERSE DRUG REACTION MONITORING

 

16.1 PREFACE

 

Principles of Adverse drug reaction monitoring in bioavailability/bioequivalence studies for extended release formulations are essentially same as for bioavailability / bioequivalence studies of conventional release formulations. However, special attention should be paid to ,

 

·          possible adverse events due to dose dumping,

·          delayed appearance of adverse events due to extended release of the drug from the formulation

 

16.2 DEFINITIONS OF TERMS  :

 

Adverse Drug Reaction (ADR) is considered to be any undesirable reaction which occurs while a subject is receiving either test formulation or reference formulation in bioavailability / bioequivalence study.

 

Such adverse events shall include all manifestations of toxicity, hypersensitivity, overdose, dependence and drug reactions, as well as symptoms and signs.

 

16.3 SERIOUS ADVERSE DRUG REACTIONS (ADR)  :

 

Include any experience which

 

:        are fatal

:        are life threatening

:        results in permanent disability

:        require impatient hospitalization or prolongation of hospital stay

:        involve cancer, congenital anomaly or are the result of drug over dosage

 

Regardless of the above criteria, any additional adverse experiences which investigator / attending physician of the study considers serious should be immediately reported.

 

16.3.1 EXPECTED ADVERSE DRUG REACTIONS :

 

are events which, in terms of their nature, severity and frequency are included in the investigator’s brochure for research drugs or in the approved prescribing information for marketed drug.

 

 

 

16.3.2 UNEXPECTED ADVERSE EVENTS :

 

are events which are not found in the investigation’s brochure for research drug or in the approved prescribing information for marketed drugs.

 

 

16.4  RESPONSIBILITIES

 

1.      A standard statement of the Investigator’s/Physician’s obligations to record and report all adverse events during the specified period should be included in every protocol.

2.      All observed or volunteered adverse events regardless of treatment group or suspected casual relationship will be recorded on the Adverse Event page of the case report form by the Investigator/Physician responsible for the conduct of the study.

 

      Adverse DR occurring during clinical studies include those that occur :

 

·          After randomization to treatment groups ;

·          Till a period to ten half lives of the drug

 

3.      There should be provision to break the randomization code in the event of occurrence serious AE.

4.      If a serious adverse event occurs during first phase of the cross over study, subject should be withdrawn from the study.

5.      If a non-serious adverse event occurs during first phase of crossover study, fails to resolve during the washout period, the subject should be withdrawn from the study.

6.      For all adverse events, the investigator must be pursue and obtain information adequate both to determine the outcome of the adverse event and to access whether it meets the criteria for classification as a serious adverse event , which should be immediately notified to the sponsor who should notify the regulatory authorities.

7.      Study centre must have adequate resuscitation facilities to treat life threatening adverse events.

8.      Follow – up of the adverse event , after the date of therapy discontinuation, is required if the adverse event or its sequelae persist. Follow-up is required until the event or its sequelae resolve or stabilize at the level acceptable to the investigator and sponsor.

9.      Study report should have a section to report adverse events observed with treatment groups.                                              

 

 

 

 

 

 

 

 

 

 

17. APPENDIX – 3

 

GOOD LABORATORY PRACTICE

 

Good Laboratory Practice (GLP) is intended to promote the quality and validity of test data. It is a managerial concept covering the organizational process and the conditions under which laboratory studies are planned, performed, monitored, recorded and reported. Comparable quality of test data forms the basis for the mutual acceptance of the test data.

 

The application of GLP is of crucial importance to authorities entrusted with the responsibility of accessing test data and evaluating chemical and biological hazards. The issue of data quality has national and international dimension. If countries can rely on test data developed in other countries, duplicate testing can be avoided and cost to government and industry saved. Moreover, common principles and procedures for GLP facilitate the exchange of information and prevent the emergence non-tariff barriers to trade while contributing to environmental and health protection.

 

17.1 DEFINITIONS OF TERMS

 

LABORATORY

 

Body that calibrates and / or tests.

 

CALIBRATION

 

The set of operations which establish, under specified conditions, the relationship between values indicated by a measuring system, or values represented by a material measure, and the corresponding known values of a measurand.

 

TEST

 

A technical operation that consists of the determination of one or more characteristics or performance of a given product, material, equipment, organism, physical phenomenon, process or service according to a specified procedure.

 

CALIBRATION METHOD

 

Define technical procedure for performing a calibration.

 

TEST METHOD

 

Defined technical procedure for performing a test.

 

 

VERIFICATION

 

Confirmation by examination and provision of evidence that specified requirements have been met.

 

QUALITY SYSTEM

 

The organizational structure, responsibilities, procedures, processes and resources for implementing quality management.

 

RAW DATA

 

Data that cannot be easily derived or recalculated from other information.

 

REFERENCE STANDARD

 

A standard ,generally of the highest metrological quality available at a given location, from which measurements made at that location are derived.

 

REFERENCE MATERIAL

 

A material or substance, one or more properties of which are sufficiently well established , to be used for the calibration of an apparatus, the assessment of measurement method, or for assigning values to materials.

 

TRACEABILITY

 

The property of a result of a measurement whereby it can be related to appropriate standards, generally international or national standards, through an unbroken chain of comparison.

 

SOFTWARE

 

Computer code which, when implemented cause the computer to perform the described task.

 

17.2  LEGAL IDENTITY

 

Legal identity implies that the body , conducting the bioequivalence/bioavailability “study” or the parent organization to which it belongs shall be registered with an appropriate statutory body.

 

17.3  ORGANIZATION AND MANAGEMENT

 

17.3.1

 

The laboratory shall specify and document the responsibility, authority an interrelation of an personnel who manage, perform or verify work affecting the quality of analytical methods.

 

17.3.2

 

The laboratory shall have provision for supervision by persons familiar with the analytical methods and procedures, calibration, test and assessment of the results.

 

17.4  PERSONNEL

 

The laboratory shall have sufficient personnel having the necessary education, trainee, technical knowledge and experience for their assigned function.

 

17.5  ACCOMODATION AND ENVIRONMENT

 

17.5.1

 

Laboratory accommodation , calibration, and test areas, energy sources, lighting, heating and ventilation shall be such as to facilitate proper performance of calibration and tests.

 

17.5.2

 

The laboratory shall provide facilities for the effective monitoring , control and recording of environmental conditions in appropriate calibration and test areas.

 

17.5.3

 

Adequate measures shall be taken to ensure good house keeping in the laboratory.

 

17.6  EQUIPMENT AND REFERENCE MATERIAL

 

17.6.1

 

Equipment shall be adequately inspected , cleaned ,and maintained. Equipment used for the generation measurement or assessment of data shall adequately tested , calibrated and / or standardized.

 

17.6.2

 

The laboratory shall have adequate reference material required for the correct performance of calibration and test.

 

 

17.7  MEASUREMENT, TRACEABILITY AND CALIBRATION :

 

 

17.7.1

 

All measuring and / or testing equipment having an effect on the accuracy or validity of calibrations or test should be calibrated and / or verified before being put into service. The laboratory shall have an established programme for the calibration and verification of its measuring and test equipment.

 

 

17.7.2.

 

The overall programme of calibration and / or verification and validation of equipment shall be designed and operated so as to ensure that, whenever applicable, measurements made by the laboratory are traceable to national and international standards of measurement where available.

 

17.7.3

 

Where traceability to national or international standards of measurements is not applicable, the laboratory shall provide satisfactory evidence of correlation of results.

 

17.8  WORKING PROCEDURES

 

The laboratory shall have documented standard operating procedure which set forth in sufficient detail the methods, materials, and schedules to be used in the routine inspection cleaning, maintenance, testing, calibration and/or standardization of equipment, and shall specify when appropriate, remedial action to be taken in the event of failure or malfunction of equipment. The written standard operating procedures shall designate the person responsible for the performance of each operation.

 

17.9  VALIDATION OF COMPUTER SYSTMES

 

Where computers or automated equipment are used for the capture, processing, manipulation, recording, reporting,storage or retrieval of analysis data, the laboratory shall ensure that :

 

17.9.1

 

The computer software is documented and adequate for use.

 

17.9.2

Procedures shall be established and implemented for protecting the integrity of data ; such procedures shall include but not be limited to integrity of data entry or capture, data storage, data transmission and data processing.

 

17.9.3

 

It shall establish and implement appropriate procedures for the maintenance of security of data including the prevention of unauthorized access and unauthorized amendments of computer records.

 

17.10  RECORDS

 

The laboratory shall maintain a record system to suit its particular circumstances and comply with any existing regulations. Its retain on record all original observations calculation and derive data, calibration records and copy of the calibration certificate, or test report for an appropriate period. The records for each calibration and test contain sufficient information to permit their repetition.

 

The records shall include identity of personnel involved in sampling, preparation, calibration or testing.

 

17.11  OUTSIDE SUPPORT AND SERVICES

 

The laboratory shall have only those outside support services and supplies that are of adequate quality to sustain confidence in laboratory calibration and tests.

 

 

17.12  REFERENCES

 

1.      NABL Criteria for laboratory accreditation – second edition, 1994

 

2.      Good Laboratory Practices Regulations – Edited by Allen F. Hirsch

 

3.      Final Draft Guidelines for Bioavailability / Bioequivalence studies.

 

4.      Quality Manual of TDML dated 07/02/95.

 

5.   Guidelines for Laboratory Quality Auditing – Donald C. Singer and Ronald P. Upton (1993).