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Bioequivalence: An overview of statistical concepts
Indian J Pharmacol

August 2004

Vol 36

Issue 4

209216
the data. Replication is used concomitantly with error controlto reduce the experimental error or error variability.
Analysis of variance (ANOVA)
The various pharmacokinetic parameters (AUC, C
max
) derived from the plasma concentrationtime curve are subjectedto ANOVA in which the variance is partitioned into components due to subjects, periods and treatments. The classicalnull hypothesis test is the hypothesis of equal means,
H
0
:
µ
T
=
µ
R
(i.e. products are bioequivalent), where
µ
T
and
µ
R
represent the expected mean bioavailabilities of the test andreference formulations, respectively. The alternate hypothesistherefore is
H
1
:
µ
T
≠
µ
R
(i.e. products are bioinequivalent). Fora crossover trial with n subjects and t treatments, the ANOVAtakes the form as shown in Table 2.Table 3 illustrates the dependence of error variability in ANOVA on the study design. Suppose two treatments T
1
and T
2
are to be compared using a group of subjects. There are two ways of designing the experiment: (i) Design 1 (parallel groupdesign): divide the subjects into two groups and assign onetreatment to each group and; (ii) Design 2 (crossover design):consider each subject as a block and then apply both the treatments to each block (subject) on two different occasions. In aparallel group design, only the variability due to the treatmentis separated out, whereas in the crossover design, variability due to treatment, block (subject) and period are separatedout from error variability. Consequently, the error sum of squares (SSE) is greater in the parallel design for a specificsample size (Error sum of squares in Design 1 (SSE
1
) =
Errorsum of squares in Design 2 (SSE
2
) + (subjects sum of squares)SSS
2
+ (periods sum of squares) PSS
2
. As degrees of freedomfor SSE are the same in both the designs (for twotreatmentcase), the error mean sum of square for Design 1 (MSE
1
) willbe greater than the error mean sum of square for Design 2(MSE
2
),
18
i.e. error variability is greater in the parallel groupdesign compared to the crossover design.In ANOVA, the mean sum of squares due to a factor iscompared with the mean sum of squares due to error (e.g. F=MST / MSE), and if these are comparable, no difference between the levels of a factor is concluded, otherwise a difference is concluded. Suppose there is a difference in treatmentsi.e. the treatment mean sum of squares is larger than the error mean sum of squares. Then the chances of the treatmentmean sum of squares being larger than the error mean sum of squares are more in Design 2 compared to Design 1, sinceMSE
2
< MSE
1
.
Therefore, chances of showing a statistically significant difference (when actually there is a difference) arehigher in Design 2 compared to Design 1. This is equivalent tosaying that Design 2 is more powerful than Design 1. This re veals how the power of a test is influenced by the design of theexperiment.In ANOVA, the ratio of the formulations’ mean sum of squares to the error mean sum of squares gives an Fstatisticto test the null hypothesis
H
0
:
µ
T
=
µ
R
. This provides a test of whether the mean amount of drug absorbed from the test formulation is identical to the mean amount of drug absorbedfrom the reference. The test of this simple null hypothesis of identity is of little interest in bioequivalence studies, since the
Table 3
A comparison of ANOVA for parallel group design and 2treatment, 2period crossover design with n subjects
Design 1Design 2 SourcesSumDegree ofMean sumFSourcesSumDegree of Mean sumF ofof squaresfreedomof squaresStatisticofof squaresfreedomof squaresStatistic variation(SS)(DF)(MS)variation(SS)(DF)(MS)
Between
SST
1
1MST
1
MST
1
/MSE
1
Between
SST
2
1MST
2
MST
2
/MSE
2
treatmentstreatmentsBetween
SSS
2
n
a
1MSS
2
blocks(subjects)Between
SSP
2
1MSP
2
periodsError
SSE
1
n
a
 2MSE
1
Error
SSE
2
n2MSE
2
Total
n  1
Total
2n1
a
n is number of subjects
Table 2
Analysis of variance (ANOVA) table for tperiod, ttreatmentcrossover design
Sources ofDegree ofSum ofMean sumF variationfreedomsquaresof squaresStatistic (DF)(SS)(MS)
Treatment
t
a
1SSTMSTMST/MSE
Subject
n
b
1SSSMSSMSS/MSE
Period
t1SSPMSPMSP/MSE
Error
(t1)(n2)SSEMSE
Total
tn1
a
t is number of treatments
b
n is number of subjectsSSTSum of squares due to treatments; SSSSum of squares due to subjects;SSPSum of squares due to period; SSESum of squares due to error; MSTMean sum of squares due to treatments; MSSMean sum of squares due tosubjects; MSPMean sum of squares due to period; MSEMean sum of squaresdue to error