Prof. Tasneem Ahmad B.Pharm, M.Pharm, Ph.D.

Project Director
Center for Bioequivalence Studies and Bioassay Research
International Center for Chemical and Biological Sciences
University of Karachi - Pakistan

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 1

 Selection of BE Study Design
Estimation of Sample Size (number of
1000
volunteers)
800
Calculation of Bioavailability (BA) and
mcg/mL

600

400
Pharmacokinetic (PK) Parameters
 AUC0-t Area under the curve 0-t
200
 Elimination Rate Constant and Half Life
0
0 5 10 15
 AUC0- ∞ Area under the curve 0-∞
hr  tmax Time of Cin the plasma profile
max

 Cmax Maximum concentration

Logarithmic Transformation
Measuring Confidence Interval
Testing the initial Hypothesis and Power
assumptions
02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 2
 Selection of BE Study Design

Before a clinical trial is conducted , design of

study has to be selected .
The choice of the design depends primarily on the
variability in the observations i.e., the individuals
subjects may differ very widely in their responses
to the drug products.
Thus , one major source o f variability arises from
differences between subjects. As a result criterion
for choosing an appropriate design is whether or
not the selected design can identify, estimate and
isolate the intersubject variability in the data
analysis.
Any design that can remove this variation from
the comparison in average bioavailability between
formulations would be the appropriate one.
Crossover design is also a design of that type and
commonly used in bioequivalence studies.
02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 3
 Minimize variability not
attributable to formulations

Minimize bias

REMEMBER: goal is to compare

performance of the two products

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 4

 The Standard 2x2 Crossover Design

Period I Washout Period II

 Seq1 Test Reference
Subjects → ®
 Seq2 Reference Test

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 5

 Sample size Calculation

Sample size calculation plays an important

role in biobpharmaceutical research and
development .

In bioequivalence studies how large a

sample size to have a desired power to
established average bioequivalence within
meaningful limits is a question of
particular to the investigator.

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 6

We first consider the case where   T   R  0 and
n1  n2  n

 t (  / 2, 2n  2)  t ( , 2n  2)
2 2
 CV 
n(0)   
 20 

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 7

When   0 we will consider a sample size
determination where   0 ,     0
0
     0
T R

 t (  , 2n  2)  t ( , 2n  2)
2 2
 CV 
n( 0 )  
 20   0 

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 8

 Bioavailability (BA) and
Pharmacokinetic (PK) Parameters
The plasma or blood concentration –time
curve can be characterized by taking
blood samples immediately before and at
various time points after the adminis-
tration of drug. The profile of the plasma
or blood concentration –time curve is
then studies by means of several
pharmacokinetic parameters ,such as
area under the curve (AUC) , maximum
concentration (Cmax) and time to
achieve the maximum concentration
(tmax) ,(t-half) and elimination rate
constant .
02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 9
 AUC0-t

AUC

C0 , C1 , C2 ,... Ck

t0 , t1 , t 2 ,...t k
02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 10
Now k
 Ci 1  Ci 
AUCt     ti  ti1 
i 1  2 
.
The area under the curve may also be extrapolated and calculated
from zero time up to related to the complete elimination of the
drug . This is referred to as area under the curve from time zero to
infinite and can be calculated as

 C C 
k
C
AUC     t  t
i 1 i k

t
 2
i 1 
i i 1
e k

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 11

 BA & PK Parameters Required in
a BE Study
ke
GENTAMICIN IV BOLUS
A
y = 7.6135e-0.1394x
12

10
8
CONC(MG/L)

4
2
0
0.00 5.00 10.00 15.00 20.00 25.00 30.00
TIME(HRS)

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 12

 BA & PK Parameters Required in
a BE Study

Conc Vs Time on regular scale(cartesian)

A
50
45
y = 57.331e-0.0634x
40
35
Conc(Mcg/ml)

K
30
25
20
15
10
5
0
0 10 20 30 40 50
Time(min)

log 2
t1 
2 ke

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 13

 Logarithmic
Transformation

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 14

 The classic confidence
interval for T   R can be
obtained as .
 1 1
l.l  (YT  YR )  t ( , n1  n2  2)ˆ d   
 n1 n2 

80%T/R 125%  1 1
u.l  (YT  YR )  t ( , n1  n2  2)ˆ d   
(%)
 n1 n2 

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 15

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 16
Before understanding power analysis we have to consider some
statistical terms

Hypothesis is a statement that something is true

Hypothesis

Null Hypothesis Alternative Hypotheses

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 17

 Null Hypothesis
A hypothesis to be tested. We use the symbol
H0 to represent the null hypothesis. In BE study
it states the two products are inbioequivalent

Alternative Hypothesis
Hypothesis to be considered as an alternative to
the null hypothesis. We use the symbol Ha to
represent the alternative hypothesis.
The alternative hypothesis is the one believe to
be true, or what you are trying to prove is true.
In a BE study it reveals that the two products are
bioequivalent

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 18

 Acceptance
region,
Bioequivalent
Critical
region; Critical
Inbioequivalent region;
Inbioequivalent

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 19

 
Declare an inbioequivalent product as bioequivalent


Declare a bioequivalent product as inbioequivalent

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 20

 
Declare an inbioequivalent (1   )
product as bioequivalent

(1   ) 
Declare a bioequivalent product
as inbioequivalent

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 21

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 22
02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 23
 The ability of a statistical test to detect an effect, given that the
effect actually exists

Power is “the probability of rejecting a null hypothesis when it is

false — and therefore should be rejected.”

The probability that a statistical significance test will reject the null
hypothesis for a specified value of an alternative hypothesis.”

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 24

 The sample size you need to have adequate
power to correctly detect a sig. difference

If you have a limited number of participants,

you can determine if sufficient power exists
for the study to be worth conducting

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 25

 Alpha and n are negatively correlated
very small alpha requires large n

Power and n are positively correlated in a

nonlinear manner (typically it is a logistic or
ogive relationship – hence the term power
curves)

Sample differences from a power .75 to .80

are much smaller than from power of .92 to
.97 very large power requires large n

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 26

 Effect size ( How large is the effect in the

population )
Sample size (You are using a sample to
make inference about the population, how
is sample )
Decision criteria (Alpha)

Higher power can be obtained with low

coefficient of variation (CV)

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 27

 the significance
criterion (α
( )

the sample size (N)

the effect size

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 28

 Higher Value

Sampling
P(T) distribution if
H0 were true alpha 0.1

POWER = 1 - β ↑

β α T
02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 29
 Smaller Value

Sampling
P(T) distribution if
H0 were true alpha 0.01

POWER = 1 - β↓

β α T
02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 30
 Sampling
P(T) distribution if
H0 were true alpha 0.05

POWER = 1 - β

β α T

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 31

 There is some relationship between 
and
 that is if we increase

than  decreases and if we
decrease  than  is increase.
There is only way to decreasing
both and is to increase the sample
size. In practice, because type I
error is usually considered to be a
more important or serious error,
which one would to avoid, a
typical approach in hypothesis
testing is to control at an
acceptable level and try to
minimize by choosing an
appropriate sample size

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 32

 There is no closed algebra formula
to calculate the statistical power for
the two one sided test procedures.
In crossover design Schuirmann
used a numerical integration
approach.

Which is complicated to implement

in practice, while Chow and Liu
suggested using the Monte Carlo
simulation to obtain the empirical
power .

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 33

 The Monte Carlo simulation
involves sampling techniques to
generate a series of random
samples from a distribution that
represents the study population
of interest (e.g., the population
under ). For each generated
random sample, tow one sided
test procedure is applied and the
conclusion of rejecting or
accepting is made. The empirical
power is then calculated as the
proportion of the replications in
which is rejected

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 34

 Generate a random sample of size n1  n2
according to the normal distribution with pre-
specified  d and  2 where  d  T   R

X  YT  YR X : N ( d , ) 2

Calculate the 100  (1  2 ) % confidence interval for .

assuming logarithmically transformed data is used,

confidence interval for d can be defined as

 ˆ d  t ( , n  1) seˆ , ˆ d  t ( , n  1) seˆ 
02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 35
where
ˆ d  ˆT  ˆ R

ˆ where n  n1  n2
and seˆ 
n

Repeat first two steps N times e.g., N=1000

The empirical power is calculated as the

proportion of N random samples in which
the confidence interval falls entirely within
pre-specified limits i.e., (ln( L ),ln(U ))
 L  0.80 U 1.25
02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 36
 Sample size and power table
d  0

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 37

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02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 41
 Example
We performed a single –dose study
with two- period, two sequence
crossover study in N=18 healthy
volunteers, two controlled release
theophylline formulations were
compared with regard to rate of
extent of absorption.
Here we consider the PK
parameter AUC (o-t)
Now what is the power ?
02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 42
Sample size CV ALPHA
18 8% 0.05

Power
1

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 43

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 44
 Table I
Table II

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 45

Figure II

%CV for power values at different sample size, %CV and iterations.

It reveals interesting characteristics of power estimation on the basis of

randomization techniques. It clearly indicates that at any number of
iteration, the computed estimates of power will be invariable associated
with a degree of variability. It also shows that no gain, regarding the
accuracy of such estimates, could be attained beyond 4000 iterations.
In addition, a significant loss in accuracy of the power estimates
becomes very likely if the number of iteration set at 1000 or less.

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 46

 The mean power values and their corresponding %CV at 10000
iterations obtained by using Monte Carlo simulation are presented in
the table III below

Table III
Power
CV\SZ (Means) Bias(%CV)

45/24 0.08817 3.4851061

45/36 0.32598 1.3176379

35/24 0.386047368 1.1707137

45/48 0.5482 0.699941

35/36 0.6862 0.4900383

25/24 0.823975 0.3451102

35/48 0.849015 0.3395091

25/36 0.961585 0.2205879

25/48 0.99242 0.0967929

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 47

 And the respective diagrammatic representation is shown in the
figure III below.

Figure III

This figure indicates that the magnitude of variability could be any where
0.3 to 3.5 %. It further indicates that the variability or bias in the power
estimates is inversely proportional, such clear correlation could not be
established with any one factor or influencing the power estimation.

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 48

  nQurrey
 PASS
 SASS
 SAMPL SIZE 2
 PPstats http://www.ppstats.com/

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 49

02/18/09 Prof. Dr. Tasneem Ahmad, CBSBR, University of Karachi 50
 http://www.ppstats.com/

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