Bioequivalence studies guide

JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY,
KAKINADA
SUBMITTED TO
M.VINAY KUMAR SIR SUBMITTED BY
K.PUSHPA LATHA
1ST M.PHARM
PHARMACEUTICS
BIO EQUIVALENCE STUDIES

Bioequivalence:
Denotes that the drug substance in two or more identical dosageform , reaches the
systemic circulation at the same relative extent i.e., their plasma concentration-time
profiles will be identical with out significant differences are statistically significant
differences are observed in the bioavailability of two or more drug products, bio-
equivalence is indicated.
Evaluation of bio equivalence studies:
Analytical methods:
• Accuracy, precision, and specificity.
• More than one analytical method not be valid.
• Data presented in both tabulated and graphical form.
• Plasma drug concentration versus time curve for each drug product and each subject.

Pharmacokinetic evaluation of the data:
• single dose study: AUC0-t,AUC0-∞, Tmax, Cmax, Cmin and percent
fluctuation[100*(Cmax-Cmin)/Cmin].
Statistical evaluation of the data:
• No statistical difference between the bioavailability of the test product and
the reference product.
• Bell-shaped curve
• Log values resembles more closely a normal distribution
Analysis of variance(ANOVA):
• No significance difference
• AUC0-∞, Tmax&Cmax

• Evaluate variability in subjects, treatment groups, study period , formulation &
other variables.
• Variability in the data is large then two drug products are bioequivalent.
• Statistically significant , if p≤0.05.
• P-level of stastitical significance.
• If p≥0.05 the difference between the two drug products are not statistically
significant
• To detect small differences between the test products , a power test is
performed
• Sample size, variability of the data & desired level of significance
• Power is set at 0.80 with an α=0.2 & a level of significance of 0.05.

Two one sided tests procedure:
• Confidence interval approach
• Greater 20%
• 90% confidence limts
• Students t-distribution of the data
• With in 20%
• Low 90% confidence interval for the ratio of means cannot be less than 0.8 & the
upper 90%confidence interval for the ratio of the means cannot be greater than 1.20
• Log transformed data , 90%confidence interval is set at 80 to 125%
• Confidence limits termed the bio equivalencecui interval
• No statistical difference the mean AUC & Cmax values of the test drug product
should not be less than 0.8(80%) nor greater than 1.25(125%) of the reference
product based on log transformed data.

CLINICAL SIGNIFICANCE:
 Clinical interpretation is important in evaluating the results of a
bioequivalence study
 Difference of less 20% in AUC & Cmax between the drug products are
unlikely to be clinically significant n patients
 A small , statisically significant difference if the study well controlled &the
no. of subjects is sufficient large.
 Above MEC & do not reach the MTC
 Elderly or patients
 Normal healthy volunteers
 Minimize product to product variability by different manufactures & lot to
variability with a single manufacturer .

Study design:
Steps involved in designing a study:
 outline of the study
 Title the study
 Research questions
 Research hypothesis(specific hypothetical and sequential sets of questions to be
addressed)
 Significance of the study
 Comprehensive description of the study
 Inclusion and exclusion criteria
 What out comes and other factors will be measured
 How will data be analysed

Study design conti…
 How will the data be safe guarded
 Potential problem areas_ logistics
 Limitations of study and how can these be corrected
 Is the study proposal approved(approved pending ) by IRB
POTENTIAL PROBLEMS AND SOLUTIONS IN DESIGNING
STUDIES:
 Research question is too broad/general
 Not enough subject available
 Proposal confusing (or) unclear
 Too expensive

TYPES OF STUDY DESIGNS:
1. Observational studies
2. Experimental studies
 In both, the effects of casual may be assessed. The effects are the outcomes, where
the causes are usually generically called “exposures”
 The key difference for experimental and observational study is the control of
exposures.
 In some experimental studies the investigator can control exposure and determines
who get exposure and how much exposure one gets.
 In some experimental studies, the investigator able to control allocation of exposure
groups. Such studies are generally referred as quasi-experimental studies
 Quasi-experimental studies more easily conducted and poorer internal validity when
compared to randomized studies.

• In contrast, the observational studies , the investigator does not get to control
the exposure but classifies the participants based on their pre existing
exposure status. The determinants of the exposure lie in the population, or
otherwise outside the control of the investigator.
Observational Studies Experimental studies
1. Descriptive or case-series 1. Controlled trails
2. Retrospective or case-control a)parallel designs
3. Cross-sectional (prevalence),surveys b)sequential designs
4. Prospective (cohorot) c)external controls
5. Retrospective cohort 2)studies with no control

Obeservtional studies:
1) Retrospective studies:
• Begin with disease/condition/outcome and look back for fetures (exposure) of
those with or with out outcome
• Useful for:
o Hypothesizing causes of disease
o Identifying risk factors
• Weaknesses:
o Biased case and and/ or control selection
o Biased exposure ascertainment
o Temporal sequence of exposure/outcome
• Advantages :
o Data availability (design of choice for choice for chart reviews)
o Usually expensive
o Can be performed quickly

 Matching cases and controls:
o prevents imbalance of known risk factor and potential confounding
o Can reduce variability (increase efficiency)
o Require special analysis techniqes
2)Retrospective cohort design:
 Use previously collected data on well defined cohort
 Common approach for disease or treatment registries since meticulous record
keeping is required
 All follow up took place in the past
 Subject to many of the same biases of other retrospective designs
 Allows estimation of prospective-like measures

 Cross-sectional design:
o Classifies a population or group with respect to both out come and exposure at a
single point in time
 useful for
 Disease description
 diagnosis and staging
 describing disease processes, mechanism
 weaknesses:
 Subject to sampling and recall biases
 Temporal order problem
 Can’t estimate disease incidence only prevelance
 Surveys:
 Single point in time studies; many utilizes sampling techniques to assure
generability
 Complex survey designs use probability sampling

 Target population is divided into clusters; subsets of cluster are sampled randomly
 Certain clusters may be “over sampled” to assure representation
 statistical analyses require special methods that correct variance for study designs
• Prospective design:
 Start with cohort study and follow up for occurrence of disease/ outcome
 Considered the optimal design for observational studies
 Uses for:
 Finding causes and estimating incidence of disease
 Identification of risk factors
 Following natural history , determining prognosis
• Weaknesses:
 Subject to selection bias and survillence bias
 Losses to follow up or drop outs
 Temporal changes in health habits.

 Can be expensive and always take time
 Advantages :
 Correct temporal relationship between exposure and disease/ outcome
 Allows estimation of disease incidence and relative risks
EXPERIMENTAL STUDIES:
CLINICAL TRAILS:
 Participants are assigned to an experimental treatment and followed for event
of interest
 Clinical trail may be:
a) be randomized or non- randomized
b) include a control group or have no control group
c) compare current treatment to an historical control
d) employ parallel or cross over design
e) employ blinding of investigator and/ or participant

 The randomized , double-blind,placebo- controlled parallel design is
considered to be the best determine efficasy
Randomization:
 Purpose: to balance group on both observed and unobserved factors
 No guarantees: balance occurs in expectation(i.e., there is chance that
some factors will not be balanced)
 In cross over design, it’s best to randomize treatment order(if possible)
 Stratification used to assure balance on factor of interest.

 Parallel design:
o This designs are helpful to minimize the experimental variables and to avoid a
bias
o In this design, two formulations are administered randomly to the volunteers
o Disadvantage:
o Inter subject variation is not being corrected
Parallel design is preffered in case of:
 The inter subject variability is relatively low compared with intra subject
variability
 The drug is toxic or it may have long half life examples: triptorillin, goserillin
acetate
 The population of interest consists of very ill patients
 The cost for increasing the no. of subjects is less than that of adding additional
treatment period

 Non control or non randomized designs:
 Patients not assigned to treatment (or treatment order) via
randomization; interpret with caution
 External or historical controls: compare current experiment to an
external control group (eg: from prior study or literature) interpret
with caution
 Uncontrolled trail: experimental group only (no comparsion);
interpret with caution

Cross over design:
A cross over design is a repeated measurements design such that each
experimental unit(patient) receives different treatments during the different time
periods i,e., the patients cross over from one treatment to another during the
course of trail. This is in contrast to a parallel design in which patients are
randomized to a treatment and remain on that treatment through out the duration
of the trail
 The reason to consider a cross over design when planning a clinical trail that it
could yield a more efficient comparison of treatment than parallel design
 Institutively this seems reasonable because each patient serves as his/her own
matched control
 Every patient should receive both A and B
 Cross over design are popularly used for agriculture, medicine ,
manufacturing, education and many other disciplaines

 A comparision is made of the subjects response on A vs B
 Cross over designs design of choice for bio equivalence trails
 The main objective of BE trails is to determine whether test and reference
pharmaceutical formulation yield equivalent blood concentration level
 Learning objectives and outcomes:
 Distinguish between situation where a cross over design would not be
advantageous
 Use the following terms appropriately: first order carry over test, sequence,
period, wash out, aliased effect
 State why an adequate wash out period is essential between periods of cross
over study in terms of aliased effects
 Evaluate the cross over designs as to its uniformity and balance and state the
implications of these characteristics

 Provide an approach to analysis of event time and data from a cross over study
 distinguish between population bio equivalence and individual bio equivalence
 Relate the different types of bio equivalence to prescribility and switchability
Limitations and dis advantages:
 Mainly it having 2 problems
 First is the issue of “order” effects, because it is possible that the order in
which treatments are administered may effect the outcome
for eg: might be a drug with many ADR giving first , making patient taking a
second less harmful medicine, more sensitive to any adverse effects

 Second is the issue of “carry-over” between treatments which
confunds the estimates of the treatments effect. In practice carry- over
effect can be avoided with a long sufficiently long wash out period
between treatments
Cross over study:
Usually a substantial inter subject variability exists in drug levels
achieved from any given dose of medication in panel
 The cross over design minimize the effect of inter subject variability
in the study by using each subject as his own control
It is of 2types
1.Latin square cross over design
2.Balanced incomplete block design

 Latin square cross over design:
 A standard approach for conducting a comparative bioavailability study is
randomized, balanced, cross over design called a latin square or complete cross
over design
I. Each subject receives each formulation
II. Each formulation is administered once in each study period
III. It includes two way cross over design, three way or four way cross over design
IV. In two way cross over design 12 subjects are used to study the BE for two
formulations , treatment A, treatment B
V. 1 to 6 subjects receive treatment A , 7 to 12 receives treatment B and the second
study period is started after washing period

 In second study period 1 to 6 receives treatment B, where 7 to 12 receives treatment A.
there fore each subject as his own control
 Advantages:
 It minimizes the effect of inter subject variability in the study by using each subject
as his or her own control
 It requires less no.of subjects to get meaning full results
 It minimizes the effect of time on bioavailability since each dosage form is
administered in each study period
 Dis advantages:
 It requires longer duration to complete the study
 The time to complete the trail is based on no.of formulations evaluated in the study
 Increased no. of study periods leads to high subject dropouts and the study becomes
difficult

 Balanced in complete block design:
A BBID eliminates many of the difficulties encountered
with the latin square design. Salient features of design :
 Each subject receives not more than two formulations
 Each formulation is administred te same no of times
 Each pair of formulations occurs together in the same no of subjects
 Table shows 4 formulations
 Eac subject receives 2 formulations
 Each formaulation administred 6
times and eah pair of formulation
Occur together in 2 subjects, pairs
Are AB,AC,AD,BC,BD,CD

 Replicated cross over design:
 These study deigns are critical when an individual BE approach is used to
allow estimation of with in – subject variences for T and R measures and the
subject by formulation interacton varience component
 Following is the four – period , two- sequence, two- formulation design
recommended for replicated BE studies
Period 1 2 3
Group 1 T R T
Group 2 R T R
4
R
T

 Fasting & Fed State Conditions
 Fasting Conditions:
 Single dose study:
 Overnight fast (10 hrs) and subsequent fast of 4 hrs
 Multiple dose study:
 Two hours fasting before and after the dose

 Fed State Studies
 Required when:
 Drug recommended with food
 Modified release product
 Assessment of Cmax and Tmax difficult with fasting state study
 Requires consumption of a high fat food, 15 minutes before dosing
 Provide 950-1000 kcals
 Fat- 50%, Proteins 15-20%, Carbohydrate- 30-35%
 Ethnic & cultural variation considered
 Specified in protocol

 Steady State/ Multiple Dose Studies
 Long elimination half life→ Accumulation in the body
 Toxic drugs requiring multiple dose therapy
 Some Modified-release drugs
 Combination products
 Drugs inducing own metabolism
 Drugs showing non-linear pharmacokinetics
 Disadvantages:
 Difficult to conduct
 Costly
 Longer monitoring
 Longer exposure to drug

 Evaluation of data:
 Statistical Evaluation
 Primary concern of bioequivalence is to limit Consumer’s &
 Manufacturer’s risk
 Cmax & AUC analysed using ANOVA
 Tmax analysed by non-parametric methods
 Use natural log transformation of Cmax and AUC
 Calculate Geometric means of Cmax of Test [Cmax’t]
 Calculate Geometric means of Cmax of Reference [Cmax’r]
 Calculate Geometric Mean Ratio = [Cmax’t] / [Cmax’r]
 Calculate 90% confidence interval for this GMR for Cmax
 Similarly calculate GMR for AUC

To establish BE:
 The calculated 90% CI for Cmax & AUC, should fall within range:
 80-125% (Range of Bioequivalence)
 Non-parametric data 90% CI for Tmax should lie within
 clinical acceptable range
BE Results
T/R (%)80% 125%
Demonstrate BE
Demonstrate BE
Fail to Demonstrate BE
Fail to Demonstrate BE Fail to Demonstrate BE

 Study submission and drug review process:
 IND / NDAS:
 Documentation of bioequivalence may be useful during the investigation of new
drug to find links between
(I) early and late clinical trial formulations
(II) formulations used in clinical trial and constancy studies, if different
(III) clinical trial formulations and to be marketed pharmaceutical drug product
(IV) other evaluation, as appropriate.
 In each comparison, the new formulation or new method of manufacture is the
pharmaceutical test product and the prior formulation or method of manufacture
is the reference product.

 It is suggested that the determination of re-document
bioequivalence during the new drug application period because the
pharmaceutical test product produces higher or lower measures of
rate and extent of absorption in our body or because the
performance of the pharmaceutical test or reference is more
 In some cases, BIOINEQUIVALENCE is observed because of
insufficient numbers of subjects entered into the BE study.

ANDAs
 Bioequivalence studies are a critical component of ANDA
submissions.
 The purpose of these studies is to demonstrate BE between a
pharmaceutically equivalent generic drug product and the
corresponding reference listed drug (21 CFR 314.94). Together with
the determination of pharmaceutical equivalence, establishing BE
allows a regulatory conclusion of therapeutic equivalence.
 In addition to a BE study under fasting conditions, it is recommended
a BE study under fed conditions for administered the immediate-
release drug products in orally

Bio-pharmaceutical classification system:
 Introduction
The Biopharmaceutics Classification System (BCS) is a scientific
framework for classifying drug substances based on their aqueous solubility and
intestinal permeability. This classification system was devised by Amidon et al.
This concept underlying the BCS published finally led to introducing the
possibility of waiving in vivo bioequivalence (BE) studies in favor of specific
comparative in vitro testing to conclude BE of oral immediate release (IR)
products with systemic actions.

Solubility determination
 The solubility of any substance can be defined as the amount of substance that has
passed into solution when equilibrium is attained between the solution and excess
(undissolved substance) at a given temperature and pressure.
 A drug substance or an active pharmaceutical ingredient (api) is considered highly
soluble when the highest dose strength is soluble in 250 ml or less of aqueous
medium over a specific ph range.
 The ph solubility profile of the drug substance is determined at 37 + 1°c in aqueous
medium with ph in the range of 1-7.5 as per united states food and drug
administration (USFDA) guidelines,1.2-6.8 as per world health organization (WHO)
guidelines and 1-8 as per european medicines academy (EMEA)

 . Standard buffer solutions described in pharmacopoeias are considered
appropriate for use in solubility studies. Methods other than shake-flask method
can also be used with justification to support the ability of such methods to
predict equilibrium solubility of test drug substance
 Permeability determination:
The methods that are routinely used for determination of permeability include the
following:
a. Pharmacokinetic studies in human subjects including mass balance studies
and absolute bioavailability (BA) studies or intestinal permeability methods
b. In vivo or in situ intestinal perfusion in a suitable animal model
c. In vitro permeability methods using excised intestinal tissues
d. Monolayers of suitable epithelial cells e.g. Caco-2 cells or TC-7 cells

 In mass balance studies, unlabeled, stable isotopes or
radiolabeled drug substances are used to determine the extent of
drug absorption.
 In absolute BA studies, oral BA is determined and compared
against the intravenous BA as reference.
 Intestinal perfusion models and in vitro methods are suggested
for passively transported drugs.

 It is a scientific framework for classifying drug substances based on their aqueous
solubility and intestinal permeability.
 It is a drug-development tool that allows estimation of the contributions of three
major factors, dissolution, solubility and intestinal permeability that affect oral
drug absorption from IR solid oral dosage forms.
 It was first introduced into regulatory decision-making process in the guidance
document on immediate release solid oral dosage forms: Scale-up and
postapproval changes.
 The drugs are divided into high/low-solubility and permeability classes.
Currently, BCS guidelines are provided by USFDA, WHO and EMEA

 Class boundaries:
 Solubility
 Permeability
 Disssolution
Classification
According to BCS, drug substances or APIs are divided into
high/ low solubility and permeability classes as follow:
Class I : High Solubility - High Permeability
Class II : Low Solubility - High Permeability
Class III : High Solubility - Low Permeability
Class IV : Low Solubility - Low Permeability

 In combination with the dissolution, the BCS takes into account the
three major factors governing BA, viz. dissolution, solubility and
permeability.
 The BCS in accordance with WHO guideline is shown in Figure 1.
This classification is associated with drug dissolution and absorption
model, which identifies the key parameters controlling drug
absorption as a set of dimensionless numbers.
 Absorption number, (An) = Mean residence time/mean absorption
time Dissolution number, (Dn) = Mean residence time/mean
dissolution time

Bio pharmaceutical classification system

Applications of BCS:
Class 1- high solubility & high permeability
1. Achieve a target release profile associated with a particular
pharmacokinetic and /or pharmacodynamics profile
2. Formulation approaches include both control of release rate and
certain physicochemicak properties of drug like ph- solubility profile
of drug
Class 2: high permeability& low solubility
1. Micronisation
2. Addition of surfactants
3. Formulation as emulasions micro emulsions systems
4. Use of complexing agents like cyclodextrins

Class 3- low permeability & high solubility
1. Require the technologies that address to fundamental limitations of
absolute or regional permeability
2. Peptides and proteins constitute the part of class 3 and the
technologies handling such materials are on rise now days
Class 4- low solubility & low permeability:
1. Major challenge for development of drug delivery system and choice
for administering such drugs is parenteral (solubility enhancers)
2. Fortunately , etreme examples are the exception rather than the rule
and are rarely develop and reach the market

Bioequivalence studies guide

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