Computer Methods and Programs in Biomedicine 70 (2003) 21 35

www.elsevier.com/locate/cmpb

Assessment of average, population and individual

bioequivalence in two- and four-period crossover studies
Herman P. Wijnand *
Sibeliuspark 754, 5343 BS Oss, The Netherlands
Received 3 July 2001; received in revised form 18 October 2001; accepted 25 January 2002

Abstract

The aim of bioequivalence studies is to assess the equivalence of two pharmaceutical formulations of the same
active drug substance. Currently three types of bioequivalence are distinguished: average, population and individual
bioequivalence. Average and population bioequivalence can be assessed in two-period (non-replicated) crossover
studies, whereas individual bioequivalence requires three- or four-period replicated studies, with a preference for
four-period studies. The PC-program BIOEQV80 is presented for the statistical analysis of average and population
bioequivalence from two-period crossover studies. The program BIOEQ2X2 is presented for the statistical analysis of
all three types of bioequivalence from four-period replicated crossover studies. The statistical aspects of population
and individual bioequivalence are based on a recent Guidance issued by the US Food and Drug Administration.
2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Average bioequivalence; Population bioequivalence; Individual bioequivalence; Two-period crossovers; Bootstrapping;
Four-period crossovers

1. Introduction parameter. Since the decision depends on a com-

In bioequivalence studies a new pharmaceutical
formulation (the Test, further abbreviated as T)
of an active drug substance is compared with a
standard formulation (the Reference, further re-
ferred to as R). If a 90%-confidence interval for
the median T/R ratio of a parameter of interest
lies fully within the predetermined bioequivalence
range (usually 80125%), the formulations are
concluded bioequivalent with respect to that
* Tel./fax: + 31-412-626693.
E-mail address: dr.wijnand@planet.nl (H.P. Wijnand).
parison of average values, the result is termed
average bioequivalence (ABE). In the early 1990s
the regulatory requirements for ABE have been
formalised starting in the USA [1] and the Eu-
ropean Union [2].
During the last decade two new concepts, popu-
lation bioequivalence (PBE) and individual bioe-
quivalence (IBE), have given rise to a vivid
discussion in the scientific community. This re-
sulted in numerous publications on study designs
and statistical models. In 1997 the US Food and
Drug Administration (FDA) issued the first Draft
Guidance on this matter [3], mainly based on a

0169-2607/02/$ - see front matter 2002 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 9 - 2 6 0 7 ( 0 2 ) 0 0 0 1 9 - 6
22 H.P. Wijnand / Computer Methods and Programs in Biomedicine 70 (2003) 2135
publication by Schall and Luus [4]. A second
Draft Guidance [5] followed this in 1999. The
final FDA General Considerations [6] for orally
administered drug products appeared in October
2000, including criteria for the selection of ABE,
PBE or IBE. It was followed by a separate Guid-
ance on statistical approaches of replicate studies
[7] in January 2001.
Both ABE and PBE can be assessed by two-
treatment two-period crossover studies, for which
the PC-program BIOEQV80 is presented. All three
types of bioequivalence can be assessed by four-
period replicate crossover studies, for which the
PC-program BIOEQ2X2 is presented.
2. Relevance for pharmaceutical companies
Replicate study designs such as four-period
studies in which both pharmaceutical formula-
tions are administered twice to the same subject,
are recommended [6] for modified-release dosage
forms and for highly variable drug products.
PBE is important for pharmaceutical compa-
nies when developing new drug substances. This is
because in popular terms as long as a new
drug substance has not been marketed, bioequiva-
lence studies on different pharmaceutical formula-
tions of the same active drug substance should
satisfy requirements of PBE. In such cases it is not
necessary to satisfy the requirements of ABE
based on 90%-confidence intervals. The much
weaker requirement [6,7] is that it is sufficient that
the point estimate of the geometric mean T/R
ratio falls within 80 125%. The additional re-
quirement of PBE should of course be satisfied. It
is interesting to note that in such cases two-period
studies may be sufficient.
IBE is important as soon as the active drug
substance has already been marketed. In this situ-
ation the same weaker requirements of ABE hold
[6,7] and the additional requirement of IBE
should be satisfied. This implies the use of study
designs with more than two periods, preferably
four.
The selection of the bioequivalence criterion
(ABE, PBE or IBE) should be documented in the
study protocol [6].
3. Theory for two-period bioequivalence studies
3.1. A6erage bioequi6alence from two-period
studies
All theoretical aspects of statistical models for
the assessment of ABE from two-period crossover
studies have been published earlier in this Journal
in a series of papers, the last of which [8] appeared
in 1994.
3.2. Population bioequi6alence from two-period
studies
In the two Draft Guidances [3,5] and in the
final Guidance [7] issued by the US FDA, the
statistical criteria for PBE have remained essen-
tially the same. In all equations to be given in this
paper, Greek letters denote population statistics
and roman letters denote sample statistics. For
log-transformed observations (e.g. the Area-Un-
der-the-plasma-concentration-Curve (AUC), or
the plasma peak concentration, Cmax), two criteria
are distinguished.
Reference-scaled:
(vT vR )2 + (| 2TT | 2TR)
q1 = 5 qp (1)
| 2TR
Constant-scaled:
(vT vR )2 + (| 2TT | 2TR)
q1 = 5 qp (2)
| 2T0
where vT is population average response of the
log-transformed measure for T; vR, population
average response of the log-transformed mea-
sure for R; | 2TT, total variance (sum of within-
and between-subject variances) of T; | 2TR, total
variance (sum of within- and between-subject
variances) of R; | 2T0, specified constant total
variance and qP is the PBE limit.
In Eq. (1), reference-scaling means that the
criterion used is scaled to the variability of the
reference product, which effectively widens the
bioequivalence limit for more variable reference
products. This could unnecessarily narrow the
bioequivalence limit for drugs or drug products
with low variability but wide therapeutic range.
 H.P. Wijnand / Computer Methods and Programs in Biomedicine 70 (2003) 2135
Therefore, a form of constant scaling has been
introduced in Eq. (2), allowing larger values for
the reference variance.
The determination of both qP and | 2T0 is based
on the consideration of the ABE criterion and the
addition of variance terms to the PBE criterion:
ABE limit +variance factor (ln 1.25)2 +mp
qp = = Guidance [5] and in the final Guidance [7] calcula-
variance | 2T0
(3)
The US FDA recommends [7] contacting them
for further information on mP and | 2T0. In the
appendices to the Draft Guidances [3,5] and the
final Guidance [7], values of 0.02 for mP and 0.04
for | 2T0 have been used in a number of numerical
examples.
In actual practice the population parameters
vT, vR, |TT and |TR are unknown. They are
estimated by the sample statistics MT, MR, sT and
sR, respectively. Let n1 denote the number of
subjects with sequence RT and n2 the number of
subjects with sequence TR, and let n =n1 +n2.
Then MT and MR, adjusted for unequal group
sizes n1 and n2, are unbiased estimates of vT and
vR, respectively. The sample statistics s 2T and s 2R,
pooled across sequences and each, therefore,
known with n1 +n2 2 degrees of freedom, are
unbiased estimates of | 2TT and | 2TR, respectively.
As stated explicitly by Schall and Luus [4], an
unbiased estimate of the two identical numerators
in Eqs. (1) and (2) is (MT MR )2 s 2d/n + s 2T
s 2R, so that:
! "
s 2d
E (MT MR )2 + s 2T s 2R
n subject may occur more than once, or may not
= (vT vR )2 + (| 2TT | 2TR) (4)
2
where s is the sample variance of the intra-sub-
d 2. Calculate the estimate of q1 in Eqs. (5) and (6),
ject differences. The criteria set by Eqs. (1) and (2)
now transfer into:
Reference-scaled:
(MT MR )2 s 2d/n + s 2T s 2R
q1 = 5qp
s 2R
Constant-scaled:
(MT MR )2 s 2d/n + s 2T s 2R
q1 = 5qp
| 2T0
23
3.3. Computational methods for PBE from
two-period studies
In FDAs first Draft Guidance [3], as well as in
Schall and Luus publication [4], the method of
bootstrapping has been recommended for evaluat-
ing the criteria for PBE. In FDAs second Draft
tion schemes based on the method of moments
were given for a four-period replicated crossover
design, but not for a two-period non-replicated
crossover design. This leaves bootstrapping as the
validated method for evaluating the criteria for
PBE. A lucid introduction to bootstrapping was
given by Efron and Tibshirani [9].
Eqs. (5) and (6) are scaled with respect to the
variance of the reference formulation, whereas
Schall and Luus original equation for PBE [4] is
an unscaled criterion. These authors, however,
clearly discussed the possibility of extending their
criterion to scaling with respect to the variance of
the reference. Schall also explored this in a later
publication on scaling [10]. The procedures of
bootstrapping specified by the FDA [3] and those
given by Schall and Luus [4] are essentially equal.
1. Generate a bootstrap sample (often also called
bootstrap replicate) by sampling with replace-
ment n1 pairs of observations (e.g. AUC or
Cmax) from sequence RT and n2 pairs of obser-
vations from sequence TR. This means that
for each subject the intra-subject pair of obser-
vations remains unchanged. Furthermore, a
occur at all, in a bootstrap replicate, due to
sampling with replacement.
where the denominator s 2R or | 2T0 should keep
its value from the original non-bootstrapped
observations or the specified constant vari-
ance, respectively.
3. Repeat steps 12 a large number of times
(5) (some thousands of replicates are usually ade-
quate). Let B denote the total number of
bootstrap replicates.
4. Sort the B estimates of q1 in ascending order.
(6)
The 100(1 h) percentile of the B ordered
24 H.P. Wijnand / Computer Methods and Programs in Biomedicine 70 (2003) 2135

values of q1 is the upper bound of a one-sided tained from two pairs of cells. Within each pair of
100(1 h) per cent confidence interval for q1. cells, one cell mean changes by the addition of the
Usually h is chosen as 0.05, which results in a same constant, whereas the other cell mean re-
one-sided 95%-confidence interval. mains unchanged. Therefore, the difference of the
sample means, MT MR, remains unchanged.
3.4. The influence of period effects It follows that, by pooling across sequences, the
denominator and all terms in the nominator of
The statistical distribution function of log- the Eqs. (5) and (6) remain unaffected by fixed
transformed characteristics is multiplicative in the period effects, so that the criteria for PBE are not
untransformed domain. A period effect in the sensitive to such effects.
untransformed domain originates when all values This can easily be verified numerically. Table 1
of one period are multiplied by the same factor. shows the AUC-values of theophylline used as an
Their log-transforms then become the logarithms example in papers referenced earlier [8]. The anal-
of the original values plus or minus the logarithm ysis of variance (ANOVA) on log-transforms
of a constant. If a cell is defined as a set of values given in Table 2 shows the absence of a significant
in a unique combination of sequence and period, period effect. In ten data sets significant period
the variance of the cell values will not change by effects were introduced by multiplying all AUC-
adding a constant to each value. Since the sample values of the second period by 0.5, 0.6, 0.7, 0.8,
variances s 2T and s 2R are pooled across sequences, 0.9, 1.1, 1.2, 1.3, 1.4 and 1.5. In this way the
they will not change either. Similarly, s 2d/n will not Period 2/Period 1 ratio, which originally was 1.07,
change. ranged from 0.54 to 1.61 in the ten simulated data
The sample means MT and MR do change in sets, with probabilities going down to as low as
the logarithmic domain. They are, however, ob- B 0.00001. In the logarithmic domain the sample
mean MT ranged from 5.08399 to 5.63329 and the
sample mean MR ranged from 5.08209 to 5.63139,
Table 1
Theophylline AUC(0-inf) data from a two-period crossover
but their difference was 0.00190 for all data sets.
study [8] Similarly, the CV remained 13.75%, and the vari-
ances, s 2T and s 2R remained 0.036253 and 0.051972,
Subject Reference Test Ratio respectively.
Each data set was bootstrapped in ten runs
(Period 1) (Period 2)
2 339.03 329.76 0.97266 each with 8000 bootstrap replicates. The resulting
4 242.64 258.19 1.06409 mean q1(0.95) from 80 000 bootstrap replicates
5 249.94 201.56 0.80643 was approximately 0.37 for all ten data sets (plus
8 184.32 249.64 1.35438 the original data set). No statistically significant
12 209.30 231.98 1.10836
differences between data sets were found, given
13 207.40 234.19 1.12917
15 239.84 241.25 1.00588 the variability between runs within data sets.
16 211.24 255.60 1.21000
18 230.36 256.55 1.11369 3.5. The influence of group ties
(Period 2) (Period 1)
In small sequence groups an interesting type of
1 288.79 228.04 0.78964
3 343.37 288.21 0.83936 tie can be found. For example, the probability
6 225.77 217.97 0.96545 that sampling with replacement from a sequence
7 235.89 133.13 0.56437 group of four subjects generates a bootstrap repli-
9 215.14 213.78 0.99368 cate of four identical subjects is (1/4)4 1 = 0.0156.
10 245.48 248.98 1.01426
In general, for sequence groups with ni subjects
11 134.89 163.93 1.21529
14 223.39 245.92 1.10086 the probability that a group replicate will consist
17 169.70 188.05 1.10813 of ni identical subjects, is (1/ni )ni 1. Since boot-
strapping with the aim of obtaining confidence
 H.P. Wijnand / Computer Methods and Programs in Biomedicine 70 (2003) 2135 25

Table 2
ANOVA on ln-transforms of the theophylline AUC(0-inf) data given in Table 1, with 90%- and 95%-confidence intervals for the
median treatment and period ratios, as obtained by the program BIOEQV80

Arithmetic means of ln-transforms:

Treatment number 1 +5.42866E+00
(Reference)
Treatment number 2 +5.43056E+00
(Test)
Geometric means of untransformed 6alues:
Treatment number 1 +2.27844E+02
(Reference)
Treatment number 2 +2.28277E+02 100.19% of reference mean
(Test)
Period 1 +2.20167E+02
Period 2 +2.36237E+02 107.30% of period 1 mean
Variable source DF Sum of squares Mean square F-value Prob(F)

Subjects 17 +1.208092E+00 +7.106423E02 3.7915 0.00530

Subject/Gr. 16 +1.111719E+00 +6.948243E02 3.7071 0.00626
Groups 1 +9.637288E02 +9.637288E02 1.3870 0.25613
Periods 1 +4.466680E02 +4.466680E02 2.3831 0.14220
Treatments 1 +3.247940E05 +3.247940E05 0.0017 \0.30
Residue 16 +2.998917E01 +1.874323E02
Total 35 +1.552683E+00
Residual variation 13.75% of untransformed
coefficient reference mean
Test-to-Reference ratio Period 2-to-Period 1 ratio

Point estimate 1.0019 1.0730

90%-Confidence Interval 0.9252; 1.0850 0.9908; 1.1620
95%-Confidence Interval 0.9095; 1.1037 0.9740; 1.1820

intervals commonly employs some thousands of
bootstrap replicates [9], the occurrence of boot-
strapped sequence groups containing only one
re-sampled subject is almost inevitable when
handling small groups. In such cases all inter-
subject differences and variances vanish and the
cell mean reduces to the re-sampled subjects
Test or Reference value. Such replicates of a
sequence group are considered as biased and
non-informative. In the program to be discussed
in Section 5.1 they are rejected and sampling
with replacement is restarted.
Fortunately, such unwanted group ties can
only occur when handling small sequence
groups. Current standards of bioequivalence
testing with two-period crossover studies require
a minimum number of 12 evaluable subjects [7].
With two groups each of six subjects the proba-
bility of a group tie to occur is 2(1/6)5 =
0.00026, so that only one group tie in 3900
bootstrap replicates is expected. With two
groups each of seven subjects, one group tie in
no less than 59 000 bootstrap replicates is ex-
pected.
Obviously such small numbers of rejected and
re-sampled sequence groups cannot seriously
bias the final results of bootstrapping. They
might, however, be of interest when re-analysing
older studies with less than six subjects per
group. It may add to the completeness of infor-
mation that the number of group ties be stated
in the results of each bootstrap run.
26 H.P. Wijnand / Computer Methods and Programs in Biomedicine 70 (2003) 2135
4. Theory for four-period studies
4.1. General
In four-period two-treatment crossover studies
with replication of the formulations T and R, the
six possible sequence groups are RTTR, TRRT,
TRTR, RTRT, RRTT and TTRR. For the sake
of completeness, the program to be discussed in
Section 5.2 allows the inclusion of all possible
sequences, consecutively numbered 1 to 6, with
group sizes n1 to n6 and total study size N =ni,
with N5 300. Within each participating subject
no missing values are allowed. The number of
non-empty groups is denoted as s. It is obvious
that the analysis requires that 25s 5 6.
The US FDA [7] recommends studies with only
two sequence groups, TRTR and RTRT. In this
case, N = n3 + n4 and s =2.
The analyses of the three types of bioequiva-
lence (on ln-transforms) have in common that the
formulation means, MT and MR, are adjusted for
unequal sequence group sizes by dividing the sum
of the sequence group means of either formula-
tion by the number of non-empty groups, s. This
results in unbiased estimates of vT and vR. It
should be noted that calculation schemes for
statistics other than MT and MR in the three types
of bioequivalence analysis are fully independent
of one another.
4.2. A6erage bioequi6alence from four-period
studies
The assessment of ABE consists of performing
an ANOVA on the ln-transformed characteristics
(e.g. AUC, Cmax), followed by computing the
point estimate of the geometric mean T/R ratio
and the 90%-confidence interval of this ratio.
In the ANOVA the total variance (with DF=
degrees of freedom= 4N 1) is split up into the
variance between subjects (DF=N 1) and the
variance within subjects (DF=3N).
The variance between subjects distinguishes the
variance between groups (DF=s 1), tested in
the F-test against the variance within groups
(DF =N s).
The variance within subjects distinguishes the
variance between formulations (DF=1), the vari-
ance between periods (DF=3), and the Subject-
by-Formulation interaction variance across
sequences (DF= N s), all tested in the F-test
against the residual variance (DF= 2N+ s 4).
The point estimate of the difference of the mean
Test and the mean Reference, as well as the
90%-confidence interval of this difference, are cal-
culated in the ln-logarithmic domain. Back trans-
formation by exponentiation results in the
geometric mean T/R ratio and its 90%-confidence
interval.
4.3. Population bioequi6alence from four-period
studies
In the two Draft Guidances [3,5] and in the
final Guidance [7] issued by the US FDA, the
statistical criteria for PBE from four-period stud-
ies have remained essentially equal. For popula-
tion statistics from four-period studies the
equations are the same as Eqs. (1) and (2) given in
Section 3.2 for two-period studies. The same
holds true for the numerical values of the specified
constant total variance | 2T0 and the PBE limit qP.
For four-period studies the Eqs. (1) and (2) are
rearranged to result into the following linearised
criteria:
Reference-scaled:
p1 = (vT vR )2 + (| 2TT | 2TR) qP| 2TR 5 0
(7)
Constant-scaled:
p2 = (vT vR )2 + (| 2TT | 2TR) qP| 2T0 5 0 (8)
In actual practice the population parameters
vT, vR, | 2TT and | 2TR are unknown. They are
estimated by the sample statistics MT, MR, s 2TT
and s 2TR, respectively. In addition to the sample
statistics s 2TT and s 2TR, the following sample vari-
ances are required for four-period studies [7]: s 2WT
and s 2WR are the within-subject variances of T and
R respectively, and s 2BT and s 2BR are the between-
subject variances of T and R, respectively. Each of
these variances is obtained by pooling across se-
quence groups and is, therefore, known with N
s degrees of freedom.
 H.P. Wijnand / Computer Methods and Programs in Biomedicine 70 (2003) 2135 27

Since s 2TT = s 2BT +0.5s 2WT [7] and similarly
s =s 2BR +0.5s 2WR, the linearised criteria of Eqs.
2
TR
(7) and (8) are estimated by:
Reference-scaled:
eta1 = (MT MR )2 +s 2BT +0.5s 2WT
(1+ qP)s 2BR 0.5(1 +qP)s 2WR 50
Constant-scaled:
eta2 = (MT MR )2 +s 2BT +0.5s 2WT s 2BR
0.5s 2WR qP| 2T0 50 (10)
for the term (MT MR )2 and the 2-distribution
is used for the variance terms. Each term itself is
taken as point estimate Ei. Next, for each term the
value of Ui = (Hi Ei )2 is calculated. Ultimately,
the 95% upper confidence bounds, designated
H(eta1) and H(eta2) for PBE, are obtained as
follows:
(9)
Reference-scaled:
H(eta1)= %Ei + (%Ui )1/2 (11)
Constant-scaled:

As specified in Appendix F of FDAs Guideline H(eta2)= %Ei + (%Ui )1/2 qP| 2T0 (12)
[7], the calculation of the 95% upper confidence
bound for eta1 or eta2 starts with calculating the
95% upper confidence bound Hi for each of the Using the mixed-scaling approach [7], the selec-
five terms in the expression of eta1 in Eq. (9) or tion of either the reference-scaled or the constant-
for each of the first five terms in the expression of scaled approach depends on the study estimate of
eta2 in Eq. (10). The central t-distribution is used the total variance of the reference, estimated by
s 2BR + 0.5s 2WR in the four-period design. If this
variance is 5| 2T0 (in many examples of the FDA
Table 3
Results of one bootstrapping run for the assessment of PBE of
a value of 0.04 has been used), the constant-scaled
the theophylline AUC(0-inf) data given in Table 1, as obtained criterion and its confidence bound H(eta2) should
by the program BIOEQV80 be computed. Otherwise the reference-scaled crite-
rion and its confidence bound H(eta1) should be
Calculations are Reference-scaled Run number 1 computed. If the upper bound for the appropriate
(ln-metric)
criterion is 5 0, PBE is concluded. Otherwise
Variance of original Ref. formulation 5.19723E02 PBE is not concluded.
(ln-metric) (DF= 16)
Variance of original Test formulation 3.62534E02 4.4. Indi6idual bioequi6alence from four-period
(ln-metric) (DF = 16) studies
Test-to-Reference variance ratio 0.6976
(ln-metric)
Variance factor =Epsilon (for critical 0.020 In Appendix G of FDAs Guidance on replicate
Theta) models and analysis [7] the statistical criteria for
Constant total variance (for critical 0.040 IBE have been specified, based on a publication
Theta) by Hyslop et al. [11]. For ln-transformed observa-
Theta1(Min.) 1.3623
Theta1(0.025) 0.9213
tions (e.g. AUC, Cmax) two criteria are distin-
Theta1(0.050) 0.8330 guished, based on a reparametrisation. This
Theta1(0.500) 0.2897 reparametrisation makes the analysis for IBE fully
Theta1(0.950) 0.3705 (critical independent of the analyses for ABE and PBE
value= 1.7448) described in the previous sections.
Theta1(0.975) 0.5199
Theta1(Max.) 1.4254
Reference-scaled:
Total number of 8000 bootstrap samples successfully pro-
(vT vR )2 + | 2S F + (| 2WT | 2WR)
cessed (plus 0 rejected sequence groups containing only one 5 qI (13)
repeated subject). | 2WR
28 H.P. Wijnand / Computer Methods and Programs in Biomedicine 70 (2003) 2135

Table 4 The US FDA [7] recommends mI = 0.05 and

Erythromycin AUC(0-inf) data from a two-period crossover
| 2W0 = 0.04. These numerical values result in
study [19]
qI = 2.495.
Subject Reference Test Ratio The Eqs. (13) and (14) are rearranged to re-
sult in the following linearised criteria:
(Period 1) (Period 2)
10 4.98 3.19 0.64056
Reference-scaled:
11 7.14 9.83 1.37675
12 1.81 2.91 1.60773 p1 = (vT vR )2 + | 2S F + (| 2WT | 2WR)
13 7.34 4.58 0.62398
14 4.25 7.05 1.65882 qI| 2WR 5 0 (16)
15 6.66 3.41 0.51201
16 4.76 2.49 0.52311 Constant-scaled:
17 7.16 6.18 0.86313
18 5.52 2.85 0.51630 p2 = (vT vR )2 + | 2S F + (| 2WT | 2WR)

(Period 2) (Period 1)
qI| 2W0 5 0 (17)
01 5.47 2.52 0.46069
02 4.84 8.87 1.83264 The criteria p1 and p2 are estimated by eta1
03 2.25 0.79 0.35111
04 1.82 1.68 0.92308
and eta2 as follows, where all variances are
05 7.87 6.95 0.88310 pooled across sequence groups (the notation is
06 3.25 1.05 0.32308 similar to that for PBE).
07 12.39 0.99 0.07990
08 4.77 5.60 1.17400 Reference-scaled:
09 1.88 3.16 1.68085
eta1 = (MT MR )2 + s 2S F + 0.5s 2WT
(1.5+ qI)s 2WR 5 0 (18)

Constant-scaled:
(vT vR )2 + | 2S F +(| 2WT | 2WR)
5qI
| 2W0
where vT is population average response of the
ln-transformed measure for T; vR, population
average response of the ln-transformed measure
for R; | 2S F, subject-by-formulation interaction
variance component; | 2WT, within-subject vari-
ance of T; | 2WR, within-subject variance of R;
| 2W0, specified constant within-subject variance
and qI is IBE limit.
The determination of both qI and | 2W0 is
based on the consideration of the ABE criterion
and the addition of variance terms to the IBE
criterion:
Constant-scaled:
eta2 = (MT MR )2 + s 2S F + 0.5s 2WT 1.5s 2WR
qI| 2W0 5 0 (19)
(14)
Similar to the calculations for PBE, the calcu-
lation of the 95% upper confidence bound for
eta1 and eta2 of IBE starts with calculating the
95% upper confidence bound Hi for each of the
four terms in the expression of eta1 (Eq. (18)) or
for each of the first four terms in the expression
of eta2 (Eq. (19)). The central t-distribution is
used for the term (MT MR )2 and the 2-distri-
bution is used for the variance terms. Each term
is taken as point estimate Ei. Next, for each
term the value of Ui = (Hi Ei )2 is calculated
and the 95% upper confidence bound H(eta1)
and H(eta2) for IBE are obtained as follows:

ABE limit +variance factor (ln 1.25)2 +mI Reference-scaled:

qI = =
variance | 2W0
(15) H(eta1)= %Ei (%Ui )1/2 (20)
 H.P. Wijnand / Computer Methods and Programs in Biomedicine 70 (2003) 2135 29

Constant-scaled: 5. Description of programs

H(eta2)= %Ei (%Ui )1/2 qI| 2W0 (21)

Using the mixed-scaling approach [7], the selec-
tion of either the reference-scaled or the constant-
scaled approach depends on the study estimate of
the within-variance of the reference product, esti-
mated by s 2WR in the four-period design. If the
study estimate of s 2WR is smaller than | 2W0 (usually
taken as 0.04), the constant-scaled criterion and
its associated confidence interval should be com-
puted. Otherwise, the reference-scaled criterion
and its confidence interval should be computed. If
the upper confidence bound for the appropriate
criterion is 5 0, IBE is concluded. If the upper
bound is \0, IBE is not concluded.
5.1. Program BIOEQV80 for two-period studies
The procedures outlined in Section 3 have been
implemented in a new version of the PC-program
BIOEQV36 published earlier [8] for the statistical
analysis of two-treatment two-period crossover
studies for the assessment of ABE. The latter
program has been updated several times, ulti-
mately resulting in Version 7.0 (program
BIOEQV70). This version included a statistical anal-
ysis published by Gould [12]. This author claimed
that, by using individual residues per sequence-by-
period cell, all three types of bioequivalence could
be assessed from two-period crossover studies. It

Table 5
ANOVA on ln-transforms of the erythromycin AUC(0-inf) data given in Table 4, with 90%- and 95%-confidence intervals for the
median treatment and period ratios, as obtained by the program BIOEQV80

Arithmetic means of ln-transforms:

Treatment number 1 +1.52090E+00
(Reference)
Treatment number 2 +1.17982E+00
(Test)
Geometric means of
untransformed values:
Treatment number 1 +4.57633E+00
(Reference)
Treatment number 2 +3.25380E+00 71.10% of reference mean
(Test)
Period 1 +3.58443E+00
Period 2 +4.15420E+00 115.90% of period 1 mean
Variable source DF Sum of squares Mean square F-value Prob(F)

Subjects 17 +9.648715E+00 +5.675715E01 1.8368 0.11543

Subject/Gr. 16 +8.343429E+00 +5.214643E01 1.6876 0.15275
Groups 1 +1.305286E+00 +1.305286E+00 2.5031 0.13319
Periods 1 +1.958572E01 +1.958572E01 1.6338 \0.30
Treatments 1 +1.046995E+00 +1.046995E+00 3.3883 0.08428
Residue 16 +4.943980E+00 +3.089988E01
Total 35 +1.583555E+01
Residual variable 13.75% of untransformed
coefficient reference mean
Test-to-Reference ratio Period 2-to-Period 1 ratio

Point estimate 0.7110 1.1590

90%-Confidence Interval 0.5145; 0.9826 0.8386; 1.6016
95%-Confidence Interval 0.4800; 1.0531 0.7825; 1.7166
30 H.P. Wijnand / Computer Methods and Programs in Biomedicine 70 (2003) 2135

Table 6
Results of ten bootstrap runs each with 8000 replicates for the assessment of PBE, as obtained by the program BIOEQV80, of the
erythromycin AUC(0-inf) data given in Table 4

Calculations are reference-scaled (ln-metric)

Total variance of reference formulation 3.13274E01

(ln-metric)
Variance factor Epsilon (for critical 0.020
Theta)
Constant total variance (for critical 0.040
Theta)
Number of bootstrap samples 8000 in each of
ten runs
Mean Standard Variation coefficient Variation coefficient of
deviation single single run (%) mean (%)
run

Theta1 (Min.) 0.8754 0.1187 13.56 4.29

Theta1 (0.025) 0.1138 0.0094 8.30 2.62
Theta1 (0.050) 0.0349 0.0115 32.80 10.37
Theta1 (0.500) 0.9291 0.0054 0.59 0.19
Theta1 (0.950) 1.9175 0.0064 0.33 0.11
Theta1 (0.975) 2.1213 0.0183 0.86 0.27
Theta1 (Max.) 3.8703 0.2896 7.48 2.37

may, however, be assumed from FDAs final
Guidance [7], that Goulds approach has not
found regulatory approval. The program version
BIOEQV70 is still available for those interested in
exploring Goulds approach.
The new program BIOEQV80 is suitable for the
analysis of both average and PBE from two-pe-
riod studies, following the procedures outlined in
Section 3. It does not include Goulds analysis.
The maximum total study size (n1 +n2) is 60. All
results are displayed screen by screen and can
optionally be saved to a user-defined results file.
ANOVA on ln-transforms is performed on
parameters such as AUC and Cmax, followed by
back-transformation by exponentiation. Estab-
lishing 90%-confidence intervals for the median
Test-to-Reference ratio assesses ABE. Similar
confidence intervals can be obtained for charac-
teristics supposed to be normally distributed and,
therefore, without ln-transformation. All analyses
for ABE can also be performed by non-paramet-
ric methods.
A number of statistical analyses for ABE from
two-period studies are not included any longer,
since they may be considered as obsolete or sim-
ply out of use: Westlakes symmetrical intervals
[13], Mandallaz and Maus analysis [14], Lockes
confidence intervals for untransformed character-
istics [15], Rodda and Davies probabilities [16],
Hauck and Andersons probabilities [17], and the
two one-sided tests [18], the latter because confi-
dence intervals are of equal value in decision
making. If any of these currently excluded tech-
niques is desired, the PC-program BIOEQV62,
which is the latest update containing these proce-
dures, is still available from the author.
In the program BIOEQV80 the minimum num-
ber of bootstrap replicates per run is 200, but the
precision of the ultimate result is then very poor.
The maximum number of bootstrap replicates
per run is 8000; in many cases this will be suffi-
cient.
The final result of the bootstrapping procedure
not only consists of the upper limit of the one-
sided 95%-confidence interval, which can be sym-
bolised as q1(0.95). Each bootstrapping run of
log-transforms, resulting in a total number of B
ordered bootstrap replicates ranging from
q1(min.) to q1(max.), provides the following
values:
 H.P. Wijnand / Computer Methods and Programs in Biomedicine 70 (2003) 2135 31

q1(min.), q1(0.025), q1(0.05), q1(0.50), q1(max.). However, no valid conclusions can be

q1(0.95), q1(0.975), q1(max.),
and the critical value of qP(0.95).
If q1(0.95)5qP(0.95), the two formulations are
concluded population bioequivalent.
In order to obtain an estimate of the precision
of the bootstrapping process, repeat runs using
the same original data and the same auxiliary
settings are possible, from which overall means
and variation coefficients of q1(min.) to q1(max.)
are obtained.
Bootstrapping can also be performed by
BIOEQV80 with untransformed characteristics. This
similarly results in values ranging from q1(min.) to
drawn, since critical values of qP(0.95) have not
been established yet for characteristics without
logarithmic transformation.
The table of values ranging from q1(min.) to
q1(max.) may already give some idea about the
distribution of q1. Complete frequency distribu-
tions of each bootstrapping run can be obtained
optionally in two different ways: by automatic
and by user-defined lower and upper boundaries
and class width. In the automatic procedure noth-
ing has to be specified, but the resulting mid-class
values are almost never smooth, so that fre-
quency distributions of repeat runs cannot easily

Table 7
Ln-transformed Cmax-values from Shumaker & Metzlers phenytoin study [20]

Sequence (group) Subject code Test 1 Test 2 Reference 1 Reference 2

(Period 2) (Period 3) (Period 1) (Period 4)

RTTR 1 0.4383 0.7885 0.4886 0.7372
2 0.9163 0.6831 0.8154 0.8796
5 0.8329 0.8242 0.6780 0.6313
8 1.0543 0.7793 0.6152 0.8372
9 0.5933 0.6471 0.7747 0.7975
12 0.8544 0.8372 0.6881 0.5481
14 0.8020 0.6831 0.5933 0.7655
15 0.4447 0.4383 0.4511 0.5878
17 0.4187 0.4574 0.3293 0.2852
19 0.9969 0.9042 0.9243 0.6419
20 0.7655 0.8502 0.6627 0.5539
24 0.7372 0.6259 0.4383 0.5596
25 0.4383 0.7561 0.5481 0.4187

(Period 1) (Period 4) (Period 2) (Period 3)

TRRT 3 0.4055 0.4187 0.5423 0.3148
4 0.9708 0.8459 0.8838 0.7885
6 0.4824 0.7655 0.6471 0.5247
7 1.0403 1.2179 1.1725 0.9555
10 0.5539 0.7747 0.5068 0.8020
11 0.8329 0.9282 0.8109 0.9555
13 0.6523 0.7227 0.6981 0.6098
16 0.7419 0.8629 0.9478 0.8416
18 0.7975 1.0332 0.7467 0.5822
21 0.8198 0.7080 0.6729 0.6313
22 0.8065 0.9282 0.8154 0.5822
23 0.5128 0.9083 0.4824 0.7178
26 0.5653 0.4700 0.5481 0.3716
Grand mean test +7.3718E01
Grand mean ref. +6.6159E01
Delta (Test-Reference) +7.5588E02
32 H.P. Wijnand / Computer Methods and Programs in Biomedicine 70 (2003) 2135

Table 8
ANOVA on the ln-transformed data of Table 7, as obtained by the program BIOEQ2X2

Source of variation Sum of squares DF Mean square F-ratio Tail probability

Between subjects +2.6190E+00 25 +1.0476E01

Within groups +2.5266E+00 24 +1.0527E01
Between groups +9.2438E02 1 +9.2438E02 0.8781 0.358
Within subjects +1.2222E+00 78 +1.5669E02
Residual +7.2786E01 50 +1.4557E02
Between formulations +1.4855E01 1 +1.4855E01 10.2047 0.002
Between periods +6.9183E02 3 +2.3061E02 1.5842 0.205
Subjectformulations (Seq) +2.7657E01 24 +1.1524E02 0.7916 0.714
Grand total +3.8412E+00 103
Testing for ABE
(Median Test-to-Reference formulation ratio, after
back-transformation by exponentiation):
Point estimate =107.85; 90%-confidence
interval = 103.66, 112.22%

Table 9
Analysis for PBE of the ln-transformed data of Table 7, according to Appendix F of the January 2001 Guidance of the US FDA,
as obtained by the program BIOEQ2X2

Delta=Adjusted mean formulation difference +7.5588E02

DF for variances pooled across Sequences 24
Formulasubject (Seq.) interaction variance 1.1524E02
Intersubject variance of Test (across Seq.) 3.1415E02
Intersubject variance of Ref. (across Seq.) 2.6983E02
Intrasubject variance of Test (across Seq.) 1.4639E02
Intrasubject variance of Ref. (across Seq.) 1.4113E02
Total variance of reference (across Seq.) 3.4040E02
Analysis is constant-scaled
This results in criterion ThetaP 1.7448E+00
Specified constant variance 4.0000E02
Variance factor ( =Epsilon) 2.0000E02
Parameter function (S2 =variance) 95% Upper confidence bound (H) Point estimate (E) U = (HE)2

Delta +1.2456E02 +5.7135E03 +4.5463E05

Intersubject S2 Test +5.4444E02 +3.1415E02 +5.3033E04
Intrasubject S2 Test +1.2685E02 +7.3193E03 +2.8788E05
Intersubject S2 Ref. 1.7784E02 2.6983E02 +8.4630E05
Intrasubject S2 Ref. 4.6508E03 7.0566E03 +5.7879E06
(+) (+)
+1.0408E02 +6.9500E04
Eta2

Point estimate 5.9385E02

95% Upper confidence bound 3.3022E02
Critical value 0.0
 H.P. Wijnand / Computer Methods and Programs in Biomedicine 70 (2003) 2135 33

be compared. The user-defined procedure facili-
tates comparison of repeat runs in those cases
where repeat runs have been given the same
boundaries and class width.
The PC-program BIOEQV80, written and com-
piled with TURBOBASIC (Borland), occupies 137
kbytes when stored on disk and 388 kbytes of
conventional internal memory when running. In
accordance with FDAs final Guidance [7], the
parameters mP and | 2T0 can be given different
numerical values, depending on the information
given by appropriate FDA staff to sponsors or
applicants. A Pentium PC of ] 200 MHz is rec-
ommended to prevent long run times for
bootstrapping.
5.2. Program BIOEQ2X2 for four-period studies
The procedures outlined in Section 4 have
been implemented in the PC-program BIOEQ2X2
for the statistical analysis of two-treatment four-
period crossover studies with replication for the
assessment of average, population and IBE. The
maximum number of subjects is 300. The mini-
mum and maximum number of sequence groups
are two and six, respectively. No missing values
are allowed, since the analyses are based on the
assumption that each subject has been treated
two times with either pharmaceutical formula-
tion. All entered values and calculated results
are saved to a user-defined results file on disk.
The three types of bioequivalence analysis are
only suitable for ln-transforms of characteristics
such as AUC and Cmax. If necessary, non-trans-
formed data can be ln-transformed after data
entry.
As stated already in Section 4.2, the formula-
tion means of ln-transforms (MT and MR ), ad-
justed for unequal group sizes, are the only
statistics being the same for all three types of
bioequivalence analysis. These statistics are cal-
culated following the schemes of the Appendices
F and G of FDAs recent Guidance [7].
The main results of each analysis are dis-

Table 10
Analysis for IBE of the ln-transformed data of Table 7, according to Appendix G of the January 2001 Guidance of the US FDA,
as obtained by the program BIOEQ2X2

Delta=Adjusted mean formulation difference +7.5588E02

DF for variances pooled across Sequences 24
FormulaSubject (Seq.) interaction variance 1.1524E02
Intrasubject variance of Test (across Seq.) 1.4639E02
Intrasubject variance of Reference (across Seq.) 1.4113E02
Analysis is constant-scaled
This results in criterion ThetaI 2.4948E+00
Specified constant intrasubject variance 4.0000E02
Variance factor ( = Epsilon) 5.0000E02
Parameter function (S2 = variance) 95% Upper confidence bound Point estimate (E) U = (HE)2
(H)

Delta +1.2456E02 +5.7135E03 +4.5463E05

Interaction S2 +1.9971E02 +1.1524E02 +7.1361E05
Intrasubject S2 Test +1.2685E02 +7.3193E03 +2.8788E05
Intrasubject S2 Reference 1.3952E02 2.1170E02 +5.2091E05
(+) (+)
+3.3868E03 +1.9770E04
Eta2

Point estimate 9.6406E02

95% Upper confidence bound 8.2345E02
Critical value 0.0
34 H.P. Wijnand / Computer Methods and Programs in Biomedicine 70 (2003) 2135
played on screen; all necessary details can be
found in the user-defined results file on disk.
After data entry an ANOVA as scheduled in
Section 4.2 is always performed, followed by
back-transformation by exponentiation of the
parameters of interest. Establishing the point esti-
mate and the 90%-confidence interval for the me-
dian T/R formulation ratio assesses ABE.
The analyses for PBE and IBE are optional.
These two analyses are not only fully independent
of each other, but also independent of the preced-
ing ANOVA. It should be noted that all variances
used in the assessment of population or IBE are
pooled across sequences. This is in contrast to the
ANOVA, which except for the Subject-by-For-
mulation interaction componentis based on
overall variances within and between subjects,
without distinguishing within-subject variances
per formulation.
In accordance with FDAs final Guidance [7],
the parameters necessary to obtain the criterion
[P or [I can be given different numerical values,
depending on the information given by appropri-
ate FDA-staff to sponsors or applicants.
For PBE as well as for IBE the analyses ulti-
mately result in the 95% upper confidence bound
Heta1 if the analysis is reference-scaled, or Heta2
if the analysis is constant-scaled. If the confidence
bound is 5 0, PBE or IBE, respectively, is
concluded.
The PC-program BIOEQ2X2, written and com-
piled with TURBOBASIC (Borland), occupies 111
kbytes when stored on disk and 341 kbytes of
conventional internal memory when running.
6. Sample runs
6.1. Sample runs from two-period studies
Table 1 shows the theophylline data used as an
example in a number of papers referenced earlier
[8]. The ANOVA as presented by BIOEQV80 is
given in Table 2, which shows that the formula-
tions satisfy ABE requirements. These two tables
have also been used in Section 3.4 to demonstrate
that the results of bootstrapping are independent
of (deliberately introduced) period effects.
The results of a bootstrapping run on the origi-
nal data of Table 1 are given in Table 3. As can
be seen from this table, the experimental value of
q1(0.95) is far below the critical value of qP(0.95),
so that the formulations satisfy the requirements
of PBE, in addition to those of ABE.
Table 4 gives the AUC-values of erythromycin
used by Chow and Liu [19] as an example. The
ANOVA on log-transforms given in Table 5
shows a lack of ABE. The results of a multiple
bootstrap run are given in Table 6. As appears
from the large value of q1(0.95), which exceeds the
critical value of 1.7448, the formulations are not
population bioequivalent either.
Table 6 also shows how the precision of boot-
strapping can be estimated from repeat runs. This
may be interesting since the technique of boot-
strapping has often been criticised, because of a
lack of information on the precision of its results.
6.2. Sample runs from four-period studies
Shumaker and Metzler [20] published a two-
formulation four-period replicate bioequivalence
study with phenytoin. They compared a single
dose of 125 mg of a test phenytoin oral suspen-
sion formulation with a commercially available
reference drug formulation (Dilantin-125) in two
sequence groups (RTTR and TRRT) each with 13
fasted healthy adult male volunteers. This resulted
in values of AUC and Cmax without missing data.
The authors concluded ABE from the 90%-confi-
dence intervals of the median T/R formulation
ratios of either parameter, based on analyses of
variance using the SAS package. In addition, they
concluded IBE based on the close similarity of the
formulation means and of the within-subject vari-
ances per formulation for both parameters. Shu-
maker and Metzler reasoned that the proposed
IBE criteria were not computed since there was
little difference between the two products, and the
estimate of the interaction term was zero. This
means that although they did state the original
FDA criteria for IBE, they did not estimate them.
It should, however, be noted that at the time of
their publication (1998) no decision had been
made by any regulatory body with respect to the
best procedure of evaluating these criteria.
 H.P. Wijnand / Computer Methods and Programs in Biomedicine 70 (2003) 2135
Table 7 shows the ln-transforms of Shumaker
and Metzlers Cmax-values. The ANOVA using the
program BIOEQ2X2 is given in Table 8. The result-
ing point estimate (107.85%) and the 90%-confi-
dence interval of the median Test-to-Reference
formulation ratio (103.66%, 112.22%) exactly
match Shumaker and Metzlers results obtained
with the SAS analysis.
The results of the analyses for PBE and IBE
from the program BIOEQ2X2 are given in Tables 9
and 10, respectively. Since in both cases the intra-
subject variance of the test formulation (across
sequences) is smaller than 0.04, the analyses are
constant-scaled. In both cases the 95% upper
confidence bound for eta2 is below zero, which
leads to the conclusion of PBE as well as of IBE
for the parameter Cmax. The results of the statisti-
cal analyses for the parameter AUC (not shown
here) are similar.
Since phenytoin is a drug that has already been
on the market for a considerable period of time,
the assessment of PBE would not have been rele-
vant in actual practice. Its results are included in
order to show the details of the statistical analysis.
7. Availability of programs
Free copies of the programs BIOEQV80 and
BIOEQ2X2 can be obtained from the author by
sending a request either by e-mail or alternatively
by a letter with a formatted blank 90 mm (3.5 in.)
1.4 Mbytes diskette.
References
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Evaluation and Research (CDER): Statistical procedures
for bioequivalence studies using a standard two-treatment
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1992.
[2] Commission on the European Communities, CPMP Work-
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Guidance, Investigation of Bioavailability and Bioequiva-
lence, December 1991, III/54/89-EN.
[3] US Food and Drug Administration, Center for Drug
Evaluation and Research (CDER): Draft Guidance. In
Vivo Bioequivalence Studies Based on Population and
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[6] US Food and Drug Administration, Center for Drug
Evaluation and Research (CDER): Guidance: Bioavailabil-
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Drug Products General Considerations. October 2000.
[7] US Food and Drug Administration, Center for Drug
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[10] R. Schall, A unified view of individual, population and
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