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%
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
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 (t˝) 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
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 /
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
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 (
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.
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)
(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-Ą
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
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 (
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)
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
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 (
6. Approval letter from the Drug
Controller (
7. Letter from the sponsor to the
bioequivalence center along with the copy of the approval letter from the Drugs
Controller (
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 :
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 :