1

:Bioequivalence evaluation
Parallel design, replicate design, two-stage
design

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:Basic design considerations

minimize variability not

attributable to formulations

minimize bias

goal: compare performance

2 formulations
 2 pharmaceutical products
Reference Test

Bioequivalent??
How similar is similar?
Bioequivalence

The primary concern in bioequivalence assessment

is to limit the risk of a false declaration of equivalence.
Statistical analysis of the bioequivalence trial should
demonstrate that the clinically significant difference
in bioavailability is unlikely…..
 Consumer Risk

 The risk of declaring two product BE when

they’re not is called the ‘consumer risk’
 In statistical terms, this is a Type I error
– The risk of rejecting the null hypothesis when it’s true

 The consumer risk is set at 5%

 Producer Risk

 The risk of declaring two products NOT BE

when they truly are BE is called the
‘producer risk’

 In statistical terms, this is a Type II error

– The risk of accepting the null hypothesis when
it’s false
 The risks are related

 If the consumer risk is reduced, the

producer risk increases

 In statistical terms, if you lower the

acceptable risk of making a Type I error, the
risk of making a Type II error increases
…Statistical test should take into account
 The consumer (patient) risk of erroneously accepting
bioequivalence (primary concern health authorities)

 Minimize the producer (pharmaceutical company)

risk of erroneously rejecting bioequivalence

Choice:
- two one-side test procedure
- confidence interval ratio T/R 100 (1-2)
-  set at 5% (90% CI)
 Choice:
 Superiority studies - two one-side test procedure
A is better than B
 Conventional one-sided hypothesis test

 Equivalence studies
A is more or less like B (A = active y B = standard)
 Two-sided interval hypothesis

 Non-inferiority studies
A is not worse than B
 One-sided interval hypothesis
 :Average Bioequivalence

two drug products are bioequivalent

‘on the average’ when the (1-2α)
confidence interval around the
Geometric Mean Ratio falls
entirely within 80-125%
(regulatory control of specified limit)
 BE confidence interval
 The concept of the 20% difference is the basis
of BE limits (goal posts)

 If the concentration dependent data were linear,

the BE limits would be 80-120%
 On the log scale, the BE limits are 80-125%

 The 90%CI must fit entirely within specified BE

limits e.g. 80-125%
:..Variables
 Log transformation:
– For all concentration dependent pharmacokinetic
variables (AUC and Cmax)

 Analysis of log-transformed data by means of ANOVA (analysis of

variance)
– includes usually formulation (treatment), period, sequence or carry-over, and
subject factors (subjects and subjects nested within sequence)
– parametric test (normal theory)
:why log-transformation
:Why parametric testing and not non-parametric

Applicant excuse: after log transformation not normal

!distributed
 based upon test for normality, however these are
insensitive and it concerns a small study
 usually after log transformation AUC and Cmax
are normal distributed
 reason for non-normality should be explained

 non-parametric testing gives a different weighing of the values, resulting in a less pronounced
effect of the outlier and a higher probability to show BE
 ( LSM A  LSM b  t df , 0.05 SEab )
Geometric 90%CI  100. exp

Least Square
Means from ANOVA

t-statistic with
0.05 in one tail

Standard Error
• The sources of variance in the model are
– Product
– Period
– Sequence
– Subject (Sequence)

This estimates
– Residual variance
Intra-subject
variability

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 The Residual Variance (SW2)

• Sources of Variability
– Intra-subject variance in pharmacokinetics
– Analytical variability
– Subject by formulation interaction
– Unexplained random variation
 The width of the 90%CI depends on
– The magnitude of the WSV (ANOVA-CV (residual variance))
– The number of subjects in the BE study

 The bigger the WSV (within- or intra-subject variability), the wider the CI

 If the WSV is high, more subjects are needed to give

statistical power compared with when the WSV is low
80 100 125
26
 Comparison of mean ISCV observed with and
without inclusion of non caucasian subjects
30 NS
NS
25

20 NS
ISCV%

15

10

0
Mixed ethnicity Caucasians only
Anafarm, Canada
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 Comparison of mean ISCV observed with and
without inclusion of female subjects
30 p = 0.0004

25 p = 0.0009

20
NS
ISCV%

15

10

Mixed gender Male only

Anafarm, Canada
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 Comparison of mean ISCV observed with and
without inclusion of subjects with BMI > 25
30
NS
25
NS

20 NS
ISCV%

15

10

≥19.0 and <30.0 ≥19.0 and <24.9

Anafarm, Canada
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 Comparison of mean ISCV observed with and
without inclusion of subjects > 55 years of age
p = 0.0038
30

25 p = 0.0273

20 NS
ISCV%

15

10

≥18 years ≥18 and ≤ 55 years

Anafarm, Canada
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Parallel study design:

• Standard design
– Single dose cross-over study

• Alternative design
– parallel group long half-life
:Standard study design

healthy volunteers

Reference (comparator)/
Test (generic)

90% CI AUC and Cmax: Test Reference

80 – 125%

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 Inter-subject variance in
pharmacokinetics.
Analytical variability.
Subject by formulation interaction.
Unexplained random variation.

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 The width of the 90%CI depends on
– The magnitude of the WSV (ANOVA-CV (residual variance)) WSV
= now between-subject variability
– The number of subjects in the BE study

 Between-subject > WSV : wider the CI

 Higher variability: more subjects are needed to give

statistical power compared with when the WSV is low
Parallel study design:

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Parallel study design:

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Parallel study design:
• Only demographic characteristics were provided for all subjects
combined, not for the individual test or reference groups.
• Subjects are healthy South Asian young males, non-vegetarian,
non-alcoholics, normal height and weight and should fulfil
inclusion/exclusion criteria.
• Study is randomised; unlikely there is an imbalance in study
population between Test and Reference
• But an applicant should provide discussion on comparability of
study groups.

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Parallel study design:
• An additional statistical analysis was performed using two-sample t-Test
procedure to compare test and reference formulations at 5% level of
significance.

• No statistical analysis was performed on other known variables like sex, race
and smoking status as all the study population under investigation has the
same characteristics.

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Parallel study design:
• What happens in case the number of subjects are not equal in each
group, due to drop-outs?

• Will be ‘compensated’ in the standard error.

• In principle, an imbalance in number of subjects normally increases the
SE and thus it is more difficult to proof bioequivalence.

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 Example: 75 mg levonorgestrel implant
.comparator: Jadelle (Bayer)(

QUALITATIVE AND QUANTITATIVE COMPOSITION

The product consists of two implants. Each implant contains 75 mg levonorgestrel.

PHARMACEUTICAL FORM

Implant.

Therapeutic indications

Contraception.
Clinical efficacy and safety have been established in women aged 18 to 40 years.
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Posology and method of administration
For subcutaneous use.
Jadelle is a contraceptive method for long-term (up to five years) use

Pharmacokinetic properties
Levonorgestrel is released from the implants directly into tissue fluid. Maximum
serum levonorgestrel concentrations of approximately 772 pg/ml are reached 48
hours after insertion.

After the initial phase, levonorgestrel concentrations decline to 435 pg/ml within one
month, 355 pg/ml within six months, 341 pg/ml within one year, and 277 pg/ml within
five years.
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 5 years

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Highly variable drugs.

Problem:
Difficult to establish bioequivalence with
normal acceptance criteria (90% CI)

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Bioequivalence – highly variable drugs
Highly variable drugs.

?What are HVD

HVD drugs and products

How to establish BE HVD

 ?What are HVD

HVD are medicinal products which show

high inter occasional variability: CV > 30%

!Not the ANOVA CV

Occasion 1 Occasion 2
 HVD drugs and products

High Variable Drug 1200

Furosemide (ng/ml)
1000

High variability caused by intrinsic intra- 800

600

individual variability in the pharmacokinetic 400

200
0
response of the active compound 0 2 4 6
time (h)

High Variable Product

High variability caused by intra individual
variability in the pharmacokinetics caused by
formulation effects
 How to establish BE

CV=15%
Problem:
Difficult to establish bioequivalence with
normal acceptance criteria (90 % CI)
CV=30%

45%

N=88 subjects
 How to establish BE

 Increase number of subjects

 Replicate design to determine intra-individual variability for
Reference

- widen 90% CI 80-125%

 How to establish BE for HVD
• Replicate design to determine intra-individual variability

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Replicate study design:
• R-R-T and R-R-T-T study design both acceptable.
• R-R data only for estimation of the within-subject variability.
• Check R-R data on outliers; high variability should not be
caused due to outlier values not typical for the drug
substance or drug product
• For BE calculation, all R-T data will be taken into account
(included in the LSM):

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Two stage study design:
Problem:

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Two stage study design:
Options to obtain information on intra-subject variability:
•1. Carry out a pilot BE study and evaluate CV%.
To be noted:
• R-R: Information on variability in pharmacokinetics (intra-subject CV)
• R-T: Information on comparability between R and T and info on ANOVA CV
• R-R-T: Information on variability in pharmacokinetics (intra-subject CV) and
on comparability between R and T.
•Based upon the estimated CV and/or R-T data, full BE study can be
designed.

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Two stage study design:
2. Carry out a two-stage designed study:

•First stage: a number of subjects included based upon a likely intra-subject variance estimate

•Interim analysis

•BE proven: study can be stopped

•BE not proven: adding additional subjects based upon obtained variability in the first stage

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However…… Consumer Risk

 The risk of declaring two product BE when

they’re not is called the ‘consumer risk’
 In statistical terms, this is a Type I error
– The risk of rejecting the null hypothesis when it’s true

 The consumer risk is set at 5%

Choice:
- two one-side test procedure
- confidence interval ratio T/R 100 (1-2)
-  set at 5% (90% CI)
Two stage study design:
Of importance:

•Overall type I error should be maintained at 5%

•By looking into the data, the consumer risk increases

•5% should be divided over both stages (preserve the consumer risk)

•per stage confidence interval ratio T/R 100 (1-2α): α ≠ 5%

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Two stage study design:
93.93%

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Two stage study design:
How to spend alpha?

-Applicant planned to include 50 subjects on an estimated CV.

- After 25 subjects the applicant will look into the data
for analysis only for the CV. If the CV observed is in line
with the estimated CV, the study will be finished with the 50
subjects.
If observed CV is too high (too many subjects needed),
the study will be terminated.

α has not to be divided, final analysis on 90% CI.

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Two stage study design:
How to spend alpha?

-Applicant planned to include 25 subjects on an estimated CV.

After 25 subjects applicant evaluate the data for BE. They can
pass (BE proven) or it appeared that more subjects are needed
as BE not proven.

α should be divided over both stages; 93.93% CI first

stage. If BE not proven, additional 25 subjects should be
added and in second stage 93.93% CI should be applied
(on data of 50 subjects).
To be noted: if 3 subjects dropped out at first stage, than 22
60 subjects should be added in the second stage.
Two stage study design:
How to spend alpha?

-The same situation as mentioned before, however now the

BE did not pass (only marginally) on the first 25 subjects. Less
than the 25 subjects will be added.

α should be divided over both stages; 93.93% CI

first stage. If BE not proven, additional of less than 25
subjects will not inflate consumer risk and in second stage
93.93% CI can still be applied (on data of < 50 subjects).

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Two stage study design:
How to spend alpha?

-The applicant has no idea on CV. Applicant includes a

number of subjects in the study and looks into the data to
estimate CV. Based on this CV additional subjects will be
added.

applicant can decide how much α is spend in each

stage (not mandatory to use 93.93% + 93.93%) and
how to distribute sample size in the two stages (not
mandatory that both stages are equally sized)

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Two stage study design:
How to spend alpha?

-The applicant has no idea on CV. Applicant includes 25

subjects in the study and looks into the data to estimate
CV/BE. Based on this analysis BE can be proven without
adding additional subjects.

α should still be divided over both stages, depending

on number of subjects in first stage and second stage;
BE should be proven in first stage using the adapted CI.

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Two stage study design:
To be noted:
-All steps should be predefined and specified in the study
protocol, including spending the α over the two stages.

-The examples are just to give some direction in assessment.

-Discussions are ongoing in the PKWP on how to spend the

alpha in a correct way.

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End
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