Bioequivalence and Bioavailability Forum

Dear Imran!

❝ What will be significance of Sequence effect (P value less than 0.05)

Are you meaning: “What will be the clinical relevance of a statistical significant effect…”?

Testing for a sequence effect in a 2×2 cross-over study (Grizzle 1965¹) is statistically flawed and therefore simply futile (Freeman 1989²).

❝ … in the Primary variables (Cmax, AUCt and AUCinf) in the estimation of bioequivalence.

Nothing? OK, to be more precise, if:

• the study was performed in healthy subjects,
  
• the drug is not an endogenous entity, and
  
• an adequate washout were maintained (no pre-dose concentrations in period II).

As an entry point have a look at the FDA’s guidance, Section VII.B.

❝ What justification or rational can be given if sequence effects are found in AUCs and the study is bioequivalent.

To quote one of the “fathers” of statistical BE testing (Westlake 1988³):

Note that the carryover effect is, essentially, the sequence effect, which can be tested against the sum of squares within sequence. If this carryover effect exists, then it confounds the test on formulations. […] My own experience with a large number of comparative bioavailability trials has led me to believe that significant carryover effects (at the 0.05 level) tend to occur in about 5% of the trials; in other words, I believe that carryover effects do not normally exist.

(my emphasis)

In a large metastudy significant effects were found in about ~10% of 2×2 studies (n=324), and ~5% of 96 6×3 studies (D’Angelo et al. 2001⁴), which was actually the level of the tests.

Since no valid statistical method to correct for a true sequence effect exists, and if the conditions of the study design given above hold, even testing for a sequence effect should be abandoned and put into the statistical “trash can” (my personal interpretation of Senn 2002⁵).

For an overview you may also see one of my presentations and a later article.

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1. Grizzle JE. The two-period change over design and its use in clinical trials. Biometrics. 1965;21(2):467–80. doi:10.2307/2528104
   
2. Freeman P. The performance of the two-stage analysis of two-treatment, two-period cross-over trials. Stat Med. 1989;8(12):1421–32. doi:10.1002/sim.4780081202
   
3. Westlake WJ. Bioavailability and Bioequivalence of Pharmaceutical Formulation. In: Peace KE, editor. Biopharmaceutical Statistics for Drug Development. New York: Marcel Dekker; 1988. p. 336–7.
   
4. D’Angelo G, Potvin D, Turgeon J. Carry-Over Effects in Bioequivalence Studies. J Biopharm Stat. 2001;11(1–2):35–43. doi:10.1081/BIP-100104196
   
5. Senn S. Cross-over Trials in Clinical Research. Chichester: Wiley; 2002. p. 35–88.