Bioequivalence and Bioavailability Forum

Dear Imran!

❝ In Bioequivalence study, if formulation (Treatment) effect is significant (since ANOVA p-value is less than 0.05) for both Cmax and AUC and individually for C_max or AUC, what we can conclude from this significant formulation effect.

❝ In case of significant formulation effect is that mean number of subject used in the study were more than required.

Simple answer: Yes.
But: Your sample size estimation relied on a couple of uncertainties (CV_intra, ∆, …); you should not bother about a significant treatment effect (i.e., 100% not included in confidence interval) unless your drug is a NTID and you have to to deal with Danish Regulators.

❝ Similarly, what we can conclude from period and sequence significant effect.

Bioequivalenve assessment is not influenced by a period effect.
As an example you may use this data, which gives:
┌───────────┬─────────┬──────────┬────────────┬─────────────────┐
│   Effect  │ p-Value │ Estimate │ Confidence │     Interval    │
├───────────┼─────────┼──────────┼────────────┼─────────────────┤
│ Period    │  0.7856 │   98.4%  │     95%    │  87.4% - 110.8% │
│ Sequence  │  0.3239 │  115.4%  │     95%    │  86.0% - 154.8% │
│ Treatment │  0.5429 │   96.5%  │     90%    │  87.5% - 106.5% │
└───────────┴─────────┴──────────┴────────────┴─────────────────┘

If we multiply all values of the second period by 1.25 (i.e., adding a “true” period effect of 25%), we get a highly significant period effect (100% not within the CI); but sequence and treatment effects (and their CIs as well) stay exactly the same:
┌───────────┬─────────┬──────────┬────────────┬─────────────────┐
│   Effect  │ p-Value │ Estimate │ Confidence │     Interval    │
├───────────┼─────────┼──────────┼────────────┼─────────────────┤
│ Period    │  0.0015 │  123.0%  |     95%    │ 109.3% - 138.5% │
│ Sequence  │  0.3239 │  115.4%  │     95%    │  86.0% - 154.8% │
│ Treatment │  0.5429 │   96.5%  │     90%    │  87.5% - 106.5% │
└───────────┴─────────┴──────────┴────────────┴─────────────────┘

Sequence effects in a 2×2 cross-over study are a different cup of tea, and worth an entire lecture
As an entry point have a look at:

• http://www.fda.gov/cder/guidance/3616fnl.pdf FDA's guideline, Section VII. B.
  
• D'Angelo G, Potvin D and J Turgeon
  Carry-Over Effects in Bioequivalence Studies
  J Biopharm Stat 11, 35-43 (2001)

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Edit: Link corrected for FDA’s new site. [Helmut]