Lesson 15 - Crossover Designs

19/07/2017 Lesson 15: Crossover Designs
Lesson 15: Crossover Designs

Introduction
A crossover 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 the trial. This is in contrast to a parallel design in which patients are randomized
to a treatment and remain on that treatment throughout the duration of the trial.
The reason to consider a crossover design when planning a clinical trial is that it could yield a more efficient
comparison of treatments than a parallel design, i.e., fewer patients might be required in the crossover design
in order to attain the same level of statistical power or precision as a parallel design.(This will become more
evident later in this lesson...) Intuitively, this seems reasonable because each patient serves as his/her own
matched control. Every patient receives both treatment A and B. Crossover designs are popular in medicine,
agriculture, manufacturing, education, and many other disciplines. A comparison is made of the subject's
response on A vs. B.
Although the concept of patients serving as their own controls is very appealing to biomedical investigators,
crossover designs are not preferred routinely because of the problems that are inherent with this design. In
medical clinical trials the disease should be chronic and stable, and the treatments should not result in total
cures but only alleviate the disease condition. If treatment A cures the patient during the first period, then
treatment B will not have the opportunity to demonstrate its effectiveness when the patient crosses over to
treatment B in the second period. Therefore this type of design works only for those conditions that are
chronic, such as asthma where there is no cure and the treatments attempt to improve quality of life.
Crossover designs are the designs of choice for bioequivalence trials. The objective of a bioequivalence trial
is to determine whether test and reference pharmaceutical formulations yield equivalent blood concentration
levels. In these types of trials, we are not interested in whether there is a cure, this is a demonstration is that a
new formulation, (for instance, a new generic drug), results in the same concentration in the blood system.
Thus, it is highly desirable to administer both formulations to each subject, which translates into a crossover
design.
Learning objectives & outcomes
Upon completion of this lesson, you should be able to do the following:
Distinguish between situations where a crossover design would or would not be advantageous.
Use the following terms appropriately: first-order carryover, sequence, period, washout, aliased effect.
State why an adequate washout period is essential between periods of a crossover study in terms of
aliased effects.
Evaluate a crossover design as to its uniformity and balance and state the implications of these
characteristics.
Understand and modify SAS programs for analysis of data from 2 2 crossover trials with continuous
or binary data.
Provide an approach to analysis of event time data from a crossover study.
Distinguish between population bioequivalence, average bioequivalence and individual
bioequivalence.
Relate the different types of bioequivalence to prescribability and switchability.
Reference
Piantadosi Steven. (2005) Crossover Designs. In: Piantadosi Steven. Clinical Trials: A Methodologic
Perspective. 2nd ed. Hobaken, NJ: John Wiley and Sons, Inc.
https://onlinecourses.science.psu.edu/stat509/book/export/html/123 1/20
15.1 - Overview of the Crossover Designs

The order of treatment administration in a crossover experiment is called a sequence and the time of a
treatment administration is called a period. Typically, the treatments are designated with capital letters, such
as A, B, etc.
The sequences should be determined a priori and the experimental units are randomized to sequences. The
most popular crossover design is the 2-sequence, 2-period, 2-treatment crossover design, with sequences AB
and BA, sometimes called the 2 2 crossover design.
In this particular design, experimental units that are randomized to the AB sequence receive treatment A in
the first period and treatment B in the second period, whereas experimental units that are randomized to the
BA sequence receive treatment B in the first period and treatment A in the second period.
We express this particular design as AB|BA or diagram it as:
[Design 1] Period 1 Period 2

Sequence AB A B
Sequence BA B A
Examples of 3-period, 2-treatment crossover designs are:
[Design 2] Period 1 Period 2 Period 3

Sequence ABB A B B
Sequence BAA B A A
and

Sequence AAB A A B
Sequence ABA A B A
Sequence BAA B A A
Examples of 3-period, 3-treatment crossover designs are

Sequence ABC A B C
Sequence BCA B C A
Sequence CAB C A B
and

Sequence ABC A B C
Sequence BCA B C A
Sequence CAB C A B
Sequence ACB A C B
Sequence BAC B A C
Sequence CBA C B A
Some designs even incorporate non-crossover sequences such as Balaam's design:

Sequence AB A B
Sequence BA B A
Sequence AA A A
Sequence BB B B
Balaams design is unusual, with elements of both parallel and crossover design. There are advantages and
disadvantages to all of these designs; we will discuss some and the implications for statistical analysis as we
continue through this lesson.
15.2 - Disadvantages
The main disadvantage of a crossover design is that carryover effects may be aliased (confounded) with
direct treatment effects, in the sense that these effects cannot be estimated separately. You think you are
estimating the effect of treatment A but there is also a bias from the previous treatment to account for.
Significant carryover effects can bias the interpretation of data analysis, so an investigator should proceed
cautiously whenever he/she is considering the implementation of a crossover design.
A carryover effect is defined as the effect of the treatment from the previous time period on the response at
the current time period. In other words, if a patient receives treatment A during the first period and treatment
B during the second period, then measurements taken during the second period could be a result of the direct
effect of treatment B administered during the second period, and/or the carryover or residual effect of
treatment A administered during the first period. These carryover effects yield statistical bias.
What can we do about this carryover effect?
The incorporation of lengthy washout periods in the experimental design can diminish the impact of
carryover effects. A washout period is defined as the time between treatment periods. Instead of immediately
stopping and then starting the new treatment, there will be a period of time where the treatment from the first
period where the drug is washed out of the patient's system.
The rationale for this is that the previously administered treatment is washed out of the patient and,
therefore, it can not affect the measurements taken during the current period. This may be true, but it is
possible that the previously administered treatment may have altered the patient in some manner, so that the
patient will react differently to any treatment administered from that time onward. An example is when a
pharmaceutical treatment causes permanent liver damage so that the patients metabolize future drugs
differently. Another example occurs if the treatments are different types of educational tests. Then subjects
may be affected permanently by what they learned during the first period.
How long of a wash out period should there be?
In a trial involving pharmaceutical products, the length of the washout period usually is determined as some
multiple of the half-life of the pharmaceutical product within the population of interest. For example, an
investigator might implement a washout period equivalent to 5 (or more) times the length of the half-life of
the drug concentration in the blood. The figure below depicts the half-life of a hypothetical drug.
Actually, it is not the presence of carryover effects per se that leads to aliasing with direct treatment effects in
the AB|BA crossover, but rather the presence of differential carryover effects, i.e., the carryover effect due to
treatment A differs from the carryover effect due to treatment B. If the carryover effects for A and B are
equivalent in the AB|BA crossover design, then this common carryover effect is not aliased with the
treatment difference. So, for crossover designs, when the carryover effects are different from one another,
this presents us with a significant problem.
In the example of the educational tests, differential carryover effects could occur if test A leads to more
learning than test B. Another situation where differential carryover effects may occur is in clinical trials
where an active drug (A) is compared to placebo (B) and the washout period is of inadequate length. The
patients in the AB sequence might experience a strong A carryover during the second period, whereas the
patients in the BA sequence might experience a weak B carryover during the second period.
The recommendation for crossover designs is to avoid the problems caused by differential carryover effects
at all costs by employing lengthy washout periods and/or designs where treatment and carryover are not
aliased or confounded with each other. It is always much more prudent to address a problem a priori by
using a proper design rather than a posteriori by applying a statistical analysis that may require unreasonable
assumptions and/or perform unsatisfactorily. You will see this later on in this lesson...
For example, one approach for the statistical analysis of the 2 2 crossover is to conduct a preliminary test
for differential carryover effects. If this is significant, then only the data from the first period are analyzed
because the first period is free of carryover effects. Essentially you be throwing out half of your data!
If the preliminary test for differential carryover is not significant, then the data from both periods are
analyzed in the usual manner. Recent work, however, has revealed that this 2-stage analysis performs poorly
because the unconditional Type I error rate operates at a much higher level than desired. We won't go into the
specific details here, but part of the reason for this is that the test for differential carryover and the test for
treatment differences in the first period are highly correlated and do not act independently.
Even worse, this two-stage approach could lead to losing one-half of the data. If differential carryover effects
are of concern, then a better approach would be to use a study design that can account for them.
Prior to the development of a general statistical model and investigations into its implications for, we require
more definitions.
15.3 - Definitions with a Crossover Design

First-order and Higher-order Carryover Effects
Within time period j, j = 2, ... , p, it is possible that there are carryover effects from treatments administered
during periods 1, ... , j - 1. Usually in period j we only consider first-order carryover effects (from period j -
1) because:
1. if first-order carryover effects are negligible, then higher-order carryover effects usually are negligible;
2. the designs needed for eliminating the aliasing between higher-order carryover effects and treatment
effects are very cumbersome and not practical. Therefore, we usually assume that these higher-order
carryover effects are negligible.
In actuality, the length of the washout periods between treatment administrations may be the determining
factor as to whether higher-order carryover effects should be considered. We focus on designs for dealing
with first-order carryover effects, but the development can be generalized if higher-order carryover effects
need to be considered. We will focus on:
Uniformity
A crossover design is labeled as:
1. uniform within sequences if each treatment appears the same number of times within each sequence,
and
2. uniform within periods if each treatment appears the same number of times within each period.
For example, AB/BA is uniform within sequences and period (each sequence and each period has 1 A and 1
B) while ABA/BAB is uniform within period but is not uniform within sequence because the sequences
differ in the numbers of A and B.
If a design is uniform within sequences and uniform within periods, then it is said to be uniform. If the
design is uniform across periods you will be able to remove the period effects. If the design is uniform across
sequences then you will be also be able to remove the sequence effects. An example of a uniform crossover is
ABC/BCA/CAB.
Latin Squares
Latin squares historically have provided the foundation for r-period, r-treatment crossover designs because
they yield uniform crossover designs in that each treatment occurs only once within each sequence and once
within each period. As will be demonstrated later, Latin squares also serve as building blocks for other types
of crossover designs. Latin squares for 4-period, 4-treatment crossover designs are:
[Design 7] Period 1 Period 2 Period 3 Period 4

Sequence ABCD A B C D
Sequence BCDA B C D A
Sequence CDAB C D A B
Sequence DABC D A B C
and

Sequence ABCD A B C D
Sequence BDAC B D A C
Sequence CADB C A D B
Sequence DCBA D C B A
Latin squares are uniform crossover designs, uniform both within periods and within sequences. Although
with 4 periods and 4 treatments there are 4! = (4)(3)(2)(1) = 24 possible sequences from which to choose, the
Latin square only requires 4 sequences.
Balanced Designs
The Latin square in [Design 8] has an additional property that the Latin square in [Design 7] does not have.
Each treatment precedes every other treatment the same number of times (once). For example, how many
times is treatment A followed by treatment B? Only once. How many times do you have one treatment B
followed by a second treatment? Only once. This is an advantageous property for Design 8. This same
property does not occur in [Design 7]. When this occurs, as in [Design 8], the crossover design is said to be
balanced with respect to first-order carryover effects.
Think About It!
Come up with an answer to this question by yourself and then click on the icon to the left to reveal
the solution.
Look back through each of the designs that we have looked at thus far and determine whether or not it is
balanced with respect to first-order carryover effects.
When r is an even number, only 1 Latin square is needed to achieve balance in the r-period, r-treatment
crossover. When r is an odd number, 2 Latin squares are required. For example, the design in [Design 5] is a
6-sequence, 3-period, 3-treatment crossover design that is balanced with respect to first-order carryover
effects because each treatment precedes every other treatment twice.
Strongly Balanced Designs
A crossover design is said to be strongly balanced with respect to first-order carryover effects if each
treatment precedes every other treatment, including itself, the same number of times. A strongly balanced
design can be constructed by repeating the last period in a balanced design.
Here is an example:
[Design 9] Period 1 Period 2 Period 3 Period 4 Period 5

Sequence ABCDD A B C D D
Sequence BDACC B D A C C
Sequence CADBB C A D B B
Sequence DCBAA D C B A A
This is a 4-sequence, 5-period, 4-treatment crossover design that is strongly balanced with respect to first-
order carryover effects because each treatment precedes every other treatment, including itself, once.
Obviously, the uniformity of the Latin square design disappears because the design in [Design 9] is no longer
is uniform within sequences.
Uniform and Strongly Balanced Design
Latin squares yield uniform crossover designs, but strongly balanced designs constructed by replicating the
last period of a balanced design are not uniform crossover designs. The following 4-sequence, 4-period, 2-
treatment crossover design is an example of a strongly balanced and uniform design.

Sequence ABBA A B B A
Sequence BAAB B A A B
Sequence AABB A A B B
Sequence BBAA B B A A
15.4 - Statistical Bias

Why are these properties important in statistical analysis?
We now investigate statistical bias issues. In other words, does a particular crossover design have any
nuisance effects, such as sequence, period, or first-order carryover effects, aliased with direct treatment
effects? We consider first-order carryover effects only. If the design incorporates washout periods of
inadequate length, then treatment effects could be aliased with higher-order carryover effects as well, but let
us assume the washout period was adequate for eliminating carryover beyond 1 treatment period.
The approach is very simple in that the expected value of each cell in the crossover design is expressed in
terms of a direct treatment effect and the assumed nuisance effects. Then these expected values are averaged
and/or differenced to construct the desired effects.
For example, in the 2 2 crossover design in [Design 1], if we include nuisance effects for sequence, period,
and first-order carryover, then model for this would look like:

Sequence AB A + + B + - + A
Sequence BA B - + A - - + B
where A and B represent population means for the direct effects of treatments A and B, respectively,
represents a sequence effect, represents a period effect, and A and B represent carryover effects of
treatments A and B, respectively.
A natural choice of an estimate of A (or B) is simply the average over all cells where treatment A (or B) is
assigned: [12]
\[\hat{\mu}_A=\frac{1}{2}\left( \bar{Y}_{AB, 1}+ \bar{Y}_{BA, 2}\right) \text{ and }

\hat{\mu}_B=\frac{1}{2}\left( \bar{Y}_{AB, 2}+ \bar{Y}_{BA, 1}\right)\]
Will this give us a good estimate of the means across the treatment? Not quite...
The mathematical expectations of these estimates are as follows: [13]
\(E(\hat{\mu}_A)=\frac{1}{2}\left( \mu_A+\nu+\rho+\mu_A-\nu-\rho+ \lambda_B

\right)=\mu_A +\frac{1}{2}\lambda_B\)
\(E(\hat{\mu}_B)=\frac{1}{2}\left( \mu_B+\nu-\rho+\mu_B-\nu+\rho+ \lambda_A
\right)=\mu_B +\frac{1}{2}\lambda_A\)
\(E(\hat{\mu}_A-\hat{\mu}_B) = ( \mu_A-\mu_B) - \frac{1}{2}( \lambda_A-
\lambda_B) \)
From [Design 13] it is observed that the direct treatment effects and the treatment difference are not aliased
with sequence or period effects, but are aliased with the carryover effects.
The treatment difference, however, is not aliased with carryover effects when the carryover effects are equal,
i.e., A = B. The results in [13] are due to the fact that the AB|BA crossover design is uniform and balanced
with respect to first-order carryover effects. Any crossover design which is uniform and balanced with
respect to first-order carryover effects, such as the designs in [Design 5] and [Design 8], also exhibits these
results.
Example
Consider the ABB|BAA design, which is uniform within periods, not uniform with sequences, and is
strongly balanced.
[14] Period 1 Period 2 Period 3

Sequence ABB A + + 1 B + +2 + A B + - 1 - 2 + B
Sequence BAA B - + 1 A - +2 + B A - - 1 - 2 + A
A natural choice of an estimate of A (or B) is simply the average over all cells where treatment A (or B) is
assigned: [15]
\[\hat{\mu}_A=\frac{1}{3}\left( \bar{Y}_{ABB, 1}+ \bar{Y}_{BAA, 2}+ \bar{Y}_{BAA, 3}\right) \text{

and } \hat{\mu}_B=\frac{1}{3}\left( \bar{Y}_{ABB, 2}+ \bar{Y}_{ABB, 3}+ \bar{Y}_{BAA, 1}\right)\]
The mathematical expectations of these estimates are solved to be: [16]
\( E(\hat{\mu}_A)=\mu_A+\frac{1}{3}(\lambda_A+ \lambda_B-\nu)\)
\( E(\hat{\mu}_B)=\mu_B+\frac{1}{3}(\lambda_A+ \lambda_B+\nu)\)
\( E(\hat{\mu}_A-\hat{\mu}_B)=(\mu_A-\mu_B)-\frac{2}{3}\nu\)
From [16], the direct treatment effects are aliased with the sequence effect and the carryover effects, whereas
the treatment difference only is aliased with the sequence effect. The results in [16] are due to the ABB|BAA
crossover design being uniform within periods and strongly balanced with respect to first-order carryover
effects.
15.5 - Higher-order Carryover Effects

The lack of aliasing between the treatment difference and the first-order carryover effects does not guarantee
that the treatment difference and higher-order carryover effects also will not be aliased or confounded. For
example, let 2A and 2B denote the second-order carryover effects of treatments A and B, respectively, for
the design in [Design 2] (Second-order carryover effects looks at the carryover effects of the treatment that
took place previous to the prior treatment.):

Sequence ABB A + + 1 B + + 2 + A B + - 1 - 2 + B + 2A
Sequence BAA B - + 1 A - + 2 + B A - - 1 - 2 + A + 2B
[18] \( E(\hat{\mu}_A-\hat{\mu}_B)=(\mu_A-\mu_B)-\frac{2}{3}\nu-\frac{1}{3}(\lambda_{2A}-
\lambda_{2B}) \)
The expectation of the treatment mean difference indicates that it is aliased with second-order carryover
effects.
Summary of Impacts of Design Types
The ensuing remarks summarize the impact of various design features on the aliasing of direct treatment and
nuisance effects.
1. If the crossover design is uniform within sequences, then sequence effects are not aliased with
treatment differences.
2. If the crossover design is uniform within periods, then period effects are not aliased with treatment
differences.
3. If the crossover design is balanced with respect to first-order carryover effects, then carryover effects
are aliased with treatment differences. If the carryover effects are equal, then carryover effects are not
aliased with treatment differences.
4. If the crossover design is strongly balanced with respect to first- order carryover effects, then carryover
effects are not aliased with treatment differences.
Complex Carryover
The type of carryover effects we modeled here is called simple carryover because it is assumed that the
treatment in the current period does not interact with the carryover from the previous period. Complex
carryover refers to the situation in which such an interaction is modeled. For example, suppose we have a
crossover design and want to model carryover effects. With simple carryover in a two-treatment design, there
are two carryover parameters, namely, A and B.
With complex carryover, however, there are four carryover parameters, namely, AB, BA, AA and BB,
where AB represents the carryover effect of treatment A into a period in which treatment B is administered,
BA represents the carryover effect of treatment B into a period in which treatment A is administered, etc. As
you might imagine, this will certainly complicate things!
15.6 - Implementation Overview

Obviously, it appears that an ideal crossover design is uniform and strongly balanced.
There are situations, however, where it may be reasonable to assume that some of the nuisance parameters
are null, so that resorting to a uniform and strongly balanced design is not necessary (although it provides a
safety net if the assumptions do not hold).
For example, some researchers argue that sequence effects should be null or negligible because they
represent randomization effects. Another example occurs in bioequivalence trials where some researchers
argue that carryover effects should be null. This is because blood concentration levels of the drug or active
ingredient are monitored and any residual drug administered from an earlier period would be detected.
The message to be emphasized is that every proposed crossover trial should be examined to determine
which, if any, nuisance effects may play a role. Once this determination is made, then an appropriate
crossover design should be employed that avoids aliasing of those nuisance effects with treatment effects.
This is a decision that the researchers should be prepared to address.
For example, an investigator wants to conduct a two-period crossover design, but is concerned that he will
have unequal carryover effects so he is reluctant to invoke the 2 2 crossover design. If the investigator is
not as concerned about sequence effects, then Balaams design in [Design 8] may be appropriate. Balaams
design is uniform within periods but not within sequences, and it is strongly balanced. Therefore, Balaams
design will not be adversely affected in the presence of unequal carryover effects.
Some researchers consider randomization in a crossover design to be a minor issue because a patient
eventually undergoes all of the treatments (this is true in most crossover designs). Obviously, randomization
is very important if the crossover design is not uniform within sequences because the underlying assumption
is that the sequence effect is negligible. Randomization is important in crossover trials even if the design is
uniform within sequences because biases could result from investigators assigning patients to treatment
sequences.
At a minimum, it always is recommended to invoke a design that is uniform within periods because period
effects are common. Period effects can be due to:
1. increased patient comfort in later periods with trial processes;

2. increased patient knowledge in later periods;
3. improvement in skill and technique of those researchers taking the measurements.
The following is a listing of various crossover designs with some, all, or none of the properties.
It would be a good idea to go through each of these designs and diagram out what these would look like, the
degree to which they are uniform and/or balanced. Make sure you see how these principles come into play!
15.7 - Statistical Precision

Now that we have examined statistical biases that can arise in crossover designs, we next examine statistical
precision.
During the design phase of a trial, the question may arise as to which crossover design provides the best
precision. For our purposes, we label one design as more precise than another if it yields a smaller variance
for the estimated treatment mean difference.
Although a comparison of treatment means may be the primary interest of the experimenter, there may be
other circumstances that affect the choice of an appropriate design. For example, later we will compare
designs with respect to which designs are best for estimating and comparing variances.
At the moment, however, we focus on differences in estimated treatment means in two-period, two-treatment
designs.
The two-period, two-treatment designs we consider here are the 2 2 crossover design AB|BA in [Design 1],
Balaam's design AB|BA|AA|BB in [Design 6], and the two-period parallel design AA|BB.
In order for the resources to be equitable across designs, we assume that the total sample size, n, is a positive
integer divisible by 4. Then:
1. n patients will be randomized to each sequence in the AB|BA design

2. n patients will be randomized to each sequence in the AA|BB design, and
3. n patients will be randomized to each sequence in the AB|BA|AA|BB design.
Because the designs we are considering involve repeated measurements on patients, the statistical modeling
must account for between-patient variability and within-patient variability.
Between-patient variability accounts for the dispersion in measurements from one patient to another. Within-
patient variability accounts for the dispersion in measurements from one time point to another within a
patient. Within-patient variability tends to be smaller than between-patient variability.
The variance components we model are as follows:
1. WAA = between-patient variance for treatment A;

2. WBB = between-patient variance for treatment B;
3. WAB = between-patient covariance between treatments A and B;
4. AA = within-patient variance for treatment A;

5. BB = within-patient variance for treatment B.
The following table provides expressions for the variance of the estimated treatment mean difference for
each of the two-period, two-treatment designs:
Design Variance
Crossover 2/n = {1.0(WAA + WBB) - 2.0(WAB) + (AA + BB)}/n
Balaam 2/n = {1.5(WAA + WBB) - 1.0(WAB) + (AA + BB)}/n
Parallel 2/n = {2.0(WAA + WBB) - 0.0(WAB) + (AA + BB)}/n
Under most circumstances, WAB will be positive, so we assume this is so for the sake of comparison. Not
surprisingly, the 2 2 crossover design yields the smallest variance for the estimated treatment mean
difference, followed by Balaam's design and then the parallel design.
The investigator needs to consider other design issues, however, prior to selecting the 2 2 crossover. In
particular, if there is any concern over the possibility of differential first-order carryover effects, then the 2
2 crossover is not recommended. In this situation the parallel design would be a better choice than the 2 2
crossover design. Balaam's design is strongly balanced so that the treatment difference is not aliased with
differential first-order carryover effects, so it also is a better choice than the 2 2 crossover design.
With respect to a sample size calculation, the total sample size, n, required for a two-sided, significance
level test with 100(1 - )% statistical power and effect size A - B is:
\[n=(z_{1-\alpha/2}+z_{1-\beta})^2 \sigma2/(\mu_A -\mu_B)^2 \]
Suppose that an investigator wants to conduct a two-period trial but is not sure whether to invoke a parallel
design, a crossover design, or Balaam's design. He wants to use a 0.05 significance level test with 90%
statistical power for detecting the effect size of A - B= 10. From published results, the investigator assumes
that:
WAA = WBB = WAB = 400, and
AA = BB = 100
The sample sizes for the three different designs are as follows:
Parallel n = 190
Balaam n = 105
Crossover n = 21
The crossover design yields a much smaller sample size because the within-patient variances are one-fourth
that of the inter-patient variances (which is not unusual).
Another issue in selecting a design is whether the experimenter wishes to compare the within-patient
variances AA and BB.
For the 2 2 crossover design, the within-patient variances can be estimated by imposing restrictions on the
between-patient variances and covariances. The resultant estimators of AA and BB, however, may lack
precision and be unstable. Hence, the 2 2 crossover design is not recommended when comparing AA and
BB is an objective.
The parallel design provides optimal estimation of the within-unit variances because it has n patients who
can provide data in estimating each of AA and BB, whereas Balaam's design has n patients who can
provide data in estimating each of AA and BB. Again, Balaam's design is a compromise between the 2 2
crossover design and the parallel design.
15.8 - Analysis - Continuous Outcome

The statistical analysis of normally-distributed data from a 2 2 crossover trial, under the assumption that
the carryover effects are equal ( A = A = ), is relatively straightforward.
Remember the statistical model we assumed for continuous data from the 2 2 crossover trial:

Sequence AB A + + B + - + A
Sequence BA B - + A - - + B
For a patient in the AB sequence, the Period 1 vs. Period 2 difference has expectation AB = A - B + 2 -
.
For a patient in the BA sequence, the Period 1 vs. Period 2 difference has expectation BA = B - A + 2 -
.
Therefore, we construct these differences for every patient and compare the two sequences with respect to
these differences using a two-sample t test or a Wilcoxon rank sumtest. Thus, we are testing:
H0 : AB - BA = 0
The expression:
AB - BA = 2( A - B )
so testing H0 : AB - BA = 0, is equivalent to testing:
H0 : A - B = 0
To get a confidence interval for A - B , simply multiply each difference by prior to constructing the
confidence interval for the difference in population means for two independent samples.
SAS Example ( 16.1_-_2x2_crossover__contin.sas )
This is an example of an analysis of the data from a 2 2 crossover trial. The example is taken from
Example 3.1 from Senn's book (Senn S. Cross-over Trials in Clinical Research , Chichester, England: John
Wiley & Sons, 1993). The data set consists of 13 children enrolled in a trial to investigate the effects of two
bronchodilators, formoterol and salbutamol, in the treatment of asthma. The outcome variable is peak
expiratory flow rate (liters per minute) and was measured eight hours after treatment. There was a one-day
washout period between treatment periods.
The estimated treatment mean difference was 46.6 L/min in favor of formoterol (p = 0.0012) and the 95%
confidence interval for the treatment mean difference is (22.9, 70.3). The Wilcoxon rank sumtest also
indicated statistical significance between the treatment groups (p = 0.0276).
15.9 - Analysis - Binary Outcome

Suppose that the response from a crossover trial is binary and that there are no period effects. Then the
probabilities of response are:
Failure on Success on marginal

B B probabilities
Failure on A p00 p01 p0.
Success on A p10 p11 p1.

marginal p.0 p.1
probabilities
The probability of success on treatment A is p1. and the probability of success on treatment B is p.1 testing
the null hypothesis:
H0 : p1. - p.1 = 0
is the same as testing:
H0 : p1. - p.1 = (p10 + p11) - (p01 + p11) = p10 - p01 = 0
This indicates that only the patients who display a (1,0) or (0,1) response contribute to the treatment
comparison. For instance, if they failed on both, or were successful on both, there is no way to determine
which treatment is better. Therefore we will let:
Failure on B Success on B
Failure on A n00 n01
Success on A n10 n11
denote the frequency of responses from the study data instead of the probabilities listed above.
McNemar's test for this situation is as follows. Given the number of patients who displayed a treatment
preference, n10 + n01 , then n10 follows a binomial(p, n10 + n01) distribution and the null hypothesis reduces
to testing:
H0 : p = 0.5
i.e., we would expect a 50-50 split in the number of patients that would be successful with either treatment in
support of the null hypothesis, looking at only the cells where there was success with one treatment and
failure with the other. The data in cells for both success or failure with both treatment would be ignored.
SAS Example ( 16.2_-_2x2_crossover__binary.sas )
This is an example of an analysis of the data from a 2 2 crossover trial with a binary outcome of
failure/success. Fifty patients were randomized and the following results were observed:
Failure on B Success on B
Failure on A 21 15
Success on A 7 7
Thus, 22 patients displayed a treatment preference, of which 7 preferred A and 15 preferred B. McNemar's
test, however, indicated that this was not statistically significant (exact p = 0.1338).
A problem that can arise from the application of McNemar's test to the binary outcome from a 2 2
crossover trial can occur if there is non-negligible period effects. If that is the case, then the treatment
comparison should account for this. This is possible via logistic regression analysis.
The Rationale:
The probability of a 50-50 split between treatment A and treatment B preferences under the null hypothesis
is equivalent to the odds ratio for the treatment A preference to the treatment B preference being 1.0.
Because logistic regression analysis models the natural logarithm of the odds, testing whether there is a 50-
50 split between treatment A preference and treatment B preference is comparable to testing whether the
intercept term is null in a logistic regression analysis.
To account for the possible period effect in the 2 2 crossover trial, a term for period can be included in the
logistic regression analysis.
SAS Example ( 16.3_-_2x2_crossover__binary.sas )
Use the same data set from SAS Example 16.2 only now it is partitioned as to patients within the two
sequences:
Sequence AB Failure on B Success on B

Failure on A 10 7
Success on A 3 5
Sequence BA Failure on B Success on B

Failure on A 11 8
Success on A 4 2
The logistic regression analysis yielded a nonsignificant result for the treatment comparison (exact p =
0.2266). There is still no significant statistical difference to report.
15.10 - Analysis - Time-to-Event Outcome

You don't often see a cross-over design used in a time-to-event trial. If the event is death, the patient would
not be able to cross-over to a second treatment. Even when the event is treatment failure, this often implies
that patients must be watched closely and perhaps rescued with other medicines when event failure occurs.
When it is implemented, a time-to-event outcome within the context of a 2 2 crossover trial actually can
reduce to a binary outcome score of preference. Suppose that in a clinical trial, time to treatment failure is
determined for each patient when receiving treatment A and treatment B.
If the time to treatment failure on A equals that on B, then the patient is assigned a (0,0) score and
displays no preference.
If the time to treatment failure on A is less than that on B, then the patient is assigned a (0,1) score and
prefers B.
If the time to treatment failure on B is less than that on A, then the patient is assigned a (1,0) score and
prefers A.
If the patient does not experience treatment failure on either treatment, then the patient is assigned a
(1,1) score and displays no preference.
Hence, we can use the procedures which we implemented with binary outcomes.
15.11 - Analysis - More Complex Designs

The analysis of continuous, binary, and time-to-event outcome data from a design more complex than the 2
2 crossover is not as straightforward as that for the 2 2 crossover design.
With respect to a continuous outcome, the analysis involves a mixed-effects linear model (SAS PROC
MIXED) to account for the repeated measurements that yield period, sequence, and carryover effects and to
model the various sources of intra-patient and inter-patient variability.
With respect to a binary outcome, the analysis involves generalized estimating equations (SAS PROC
GENMOD) to account for the repeated measurements that yield period, sequence, and carryover effects and
to model the various sources of intra-patient and inter-patient variability.
In either case, with a design more complex that the 2 2 crossover, extensive modeling is required.
15.12 - Bioequivalence Trials

The objective of a bioequivalence trial is to determine whether test (T) and reference (R) formulations of a
pharmaceutical product are "equivalent" with respect to blood concentration time profiles.
Bioequivalence trials are of interest in two basic situations:
1. Company A demonstrates the safety and efficacy of a drug formulation, but wishes to market a more
convenient formulation, ( i.e., an injection vs a time-release capsule). This situation is less common.
2. Company B wishes to market a drug formulation similar to the approved formulation of Company A
with an expired patent. Company B has to prove that they can deliver the same amount of active drug
into the blood stream which the approved formula does.
Pharmaceutical scientists use crossover designs for such trials in order for each trial participant to yield a
profile for both formulations. The blood concentration time profile is a multivariate response and is a
surrogate measure of therapeutic response. The pharmaceutical company does not need to demonstrate the
safety and efficacy of the drug because that already has been established.
Are the reference and test blood concentration time profiles similar? The test formulation could be toxic if
it yields concentration levels higher than the reference formulation. On the other hand, the test formulation
could be ineffective if it yields concentration levels lower than the reference formulation.
Typically, pharmaceutical scientists summarize the rate and extent of drug absorption with summary
measurements of the blood concentration time profile, such as area under the curve (AUC), maximum
concentration (CMAX), etc. These summary measurements are subjected to statistical analysis (not the
profiles) and inferences are drawn as to whether or not the formulations are bioequivalent.
There are numerous definitions for what is meant by bioequivalence:
1. population bioequivalence - the formulations are equivalent with respect to their underlying
probability distributions. You want the see that the AUC or CMAX distributions would be similar.
2. average bioequivalence - the formulations are equivalent with respect to the means (medians) of their
probability distributions.
3. individual bioequivalence - the formulations are equivalent for a large proportion of individuals in the
population. i.e., how well do the AUC's and CMAX compare across patients?
Prescribability means that a patient is ready to embark on a treatment regimen for the first time, so that either
the reference or test formulations can be chosen. Switchability means that a patient, who already has
established a regimen on either the reference or test formulation, can switch to the other formulation without
any noticeable change in efficacy and safety.
Prescribability requires that the test and reference formulations are population bioequivalent, whereas
switchability requires that the test and reference formulations have individual bioequivalence.
Currently, the USFDA only requires pharmaceutical companies to establish that the test and reference
formulations are average bioequivalent. It is felt that most consumers, however, assume bioequivalence
refers to individual bioequivalence, and that switching formulations does not lead to any health problems.
The hypothesis testing problem for assessing average bioequivalence is stated as:
H0 : { T / R 1 or T / R 2 } vs. H1 : {1 < T / R < 2 }
where T and R represent the population means for the test and reference formulations, respectively, and 1
and 2 are chosen constants.
The FDA recommended values are 1 = 0.80 and 2 = 1.25, ( i.e., the ratios 4/5 and 5/4), for responses such
as AUC and CMAX which typically follow lognormal distributions.
Thus, a logarithmic transformation typically is applied to the summary measure, the statistical analysis is
performed for the crossover experiment, and then the two one-sided testing approach or corresponding
confidence intervals are calculated for the purposes of investigating average bioequivalence.
SAS Example ( 16.4_-_bioequivalence.sas )
Test and reference formulations were studied in a bioequivalence trial that used a 2 2 crossover design.
There were 28 healthy volunteers, (instead of patients with disease), who were randomized (14 each to the
TR and RT sequences). AUC and CMAX were measured and transformed via the natural logarithm.
The analysis yielded the following results:
AUC CMAX
Est for loge ( R / T) 0.0893 -0.104
Est for R / T 1.09 0.90
95% CI for loge ( R / T) (-0.113, 0.294) (-0.289, 0.080)
95% CI for R / T (0.89, 1.34) (0.75, 1.08)
Neither 95% confidence interval lies within (0.80, 1.25) specified by the USFDA, therefore bioequivalence
cannot be concluded in this example and the USFDA would not allow this company to market their generic
drug. Both CMAX and AUC are used because they summarize the desired equivalence.
15.13 - Summary
In this lesson, among other things, we learned:
Distinguish between situations where a crossover design would or would not be advantageous.
Use the following terms appropriately: first-order carryover, sequence, period, washout, aliased effect.
State why an adequate washout period is essential between periods of a crossover study in terms of
aliased effects.
Evaluate a crossover design as to its uniformity and balance and state the implications of these
characteristics.
Understand and modify SAS programs for analysis of data from 2x2 crossover trials with continuous
or binary data.
Provide an approach to analysis of event time data from a crossover study.
Distinguish between population bioequivalence, average bioequivalence and individual
bioequivalence.
Relate the different types of bioequivalence to prescribability and switchability.
Let's put what we have learned to use by completing the following homework assignment:
Homework
Look for homework assignment and the dropbox in the folder for this week in ANGEL.

Lesson 15 - Crossover Designs

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Lesson 15 - Crossover Designs

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19/07/2017 Lesson 15: Crossover Designs

Lesson 15: Crossover Designs

Learning objectives & outcomes

Upon completion of this lesson, you should be able to do the following:

15.1 - Overview of the Crossover Designs

We express this particular design as AB|BA or diagram it as:

[Design 1] Period 1 Period 2

Examples of 3-period, 2-treatment crossover designs are:

[Design 2] Period 1 Period 2 Period 3

[Design 3] Period 1 Period 2 Period 3

Examples of 3-period, 3-treatment crossover designs are

[Design 4] Period 1 Period 2 Period 3

[Design 5] Period 1 Period 2 Period 3

Some designs even incorporate non-crossover sequences such as Balaam's design:

[Design 6] Period 1 Period 2

What can we do about this carryover effect?

How long of a wash out period should there be?

15.3 - Definitions with a Crossover Design

A crossover design is labeled as:

[Design 7] Period 1 Period 2 Period 3 Period 4

[Design 8] Period 1 Period 2 Period 3 Period 4

Think About It!

Strongly Balanced Designs

[Design 9] Period 1 Period 2 Period 3 Period 4 Period 5

Uniform and Strongly Balanced Design

[Design 10] Period 1 Period 2 Period 3 Period 4

15.4 - Statistical Bias

[Design 11] Period 1 Period 2

\[\hat{\mu}_A=\frac{1}{2}\left( \bar{Y}_{AB, 1}+ \bar{Y}_{BA, 2}\right) \text{ and }

The mathematical expectations of these estimates are as follows: [13]

\(E(\hat{\mu}_A)=\frac{1}{2}\left( \mu_A+\nu+\rho+\mu_A-\nu-\rho+ \lambda_B

[14] Period 1 Period 2 Period 3

\[\hat{\mu}_A=\frac{1}{3}\left( \bar{Y}_{ABB, 1}+ \bar{Y}_{BAA, 2}+ \bar{Y}_{BAA, 3}\right) \text{

The mathematical expectations of these estimates are solved to be: [16]

15.5 - Higher-order Carryover Effects

[Design 17] Period 1 Period 2 Period 3

Summary of Impacts of Design Types

15.6 - Implementation Overview

1. increased patient comfort in later periods with trial processes;

15.7 - Statistical Precision

1. n patients will be randomized to each sequence in the AB|BA design

The variance components we model are as follows:

1. WAA = between-patient variance for treatment A;

4. AA = within-patient variance for treatment A;

Balaam 2/n = {1.5(WAA + WBB) - 1.0(WAB) + (AA + BB)}/n

Parallel 2/n = {2.0(WAA + WBB) - 0.0(WAB) + (AA + BB)}/n

\[n=(z_{1-\alpha/2}+z_{1-\beta})^2 \sigma2/(\mu_A -\mu_B)^2 \]

WAA = WBB = WAB = 400, and

15.8 - Analysis - Continuous Outcome

[Design 11] Period 1 Period 2

so testing H0 : AB - BA = 0, is equivalent to testing:

SAS Example ( 16.1_-_2x2_crossover__contin.sas )

15.9 - Analysis - Binary Outcome

Failure on Success on marginal

Success on A p10 p11 p1.

is the same as testing:

H0 : p1. - p.1 = (p10 + p11) - (p01 + p11) = p10 - p01 = 0

Success on A n10 n11

SAS Example ( 16.2_-_2x2_crossover__binary.sas )

SAS Example ( 16.3_-_2x2_crossover__binary.sas )

Sequence AB Failure on B Success on B

Sequence BA Failure on B Success on B

15.10 - Analysis - Time-to-Event Outcome