Bioequivalence and Bioavailability Forum

Dear All,


Thanks in advance for any help in this problem which I do not know how to deal with!

We have done a cross-over study in 24 subjects to evaluate the effect of co-administration of drug B on the bioavailability of A. The purpose of the study was to hopefully demonstrate that B intake does not affect exposure to A, and thus no efficacy studies will be required for development of a combination product.
The subjects received A + placebo and A + B in randomized order for several weeks, with a wash-out of several weeks between the treatment periods. The combination A+B is hereafter called the test T and A + placebo the reference R. Samples were drawn on Day 1 of each period; troughs to evaluate attainment of steady state and complete profiles after the last dose of each period. We have gone through a lot of effort to ensure medication intake and believe that the study was properly designed.

Initially one group of 10 subjects and another group of 14 were to be enrolled, as 24 subjects in one go was not feasible. Treatment order was randomized and blocked for these two groups. But eventually due to recruitment problems one group of 8 subjects and one group of 16 subjects was enrolled, three weeks apart. In the group of 8 subjects 5 got the order R-T; 2 got the order T-R and one (T-R) dropped out. In the group of 16 subjects 7 got the order R-T; 7 got the order T-R and two (T-R) dropped out. So in all, 12 got the order R-T and 9 the order T-R (three T-R's dropped out, unrelated to the treatment).

The ratio T to R for AUCtau on the last dosing day (n=21) was 0.89, 90% CI 0.85 to 0.94; similar values were obtained for Cmax. Although the 90%CI falls within 0.80-1.25, 1.0 is not included so I looked at the data in more detail, mostly plots and geometric means for the two treatment sequences, and this seemed to point at a sequence effect.

The group receiving sequence R-T (n=12) had a perfect geometric mean AUCtau ratio (T to R) of 1.01, minimum individual ratio 0.80, maximum was 1.26 (I am not making this up ().
The group receiving sequence T-R (n=9) had a geometric mean AUCtau ratio (T to R) of 0.78, minimum individual ratio 0.68, maximum was 0.98.

The low ratios for the group receiving T-R (n=9) appeared to be due to a raised AUCtau for the reference treatment in Period 2. Geometric mean AUCs for the test in Period 1 (n=9) or test in Period 2 (n=12) and for the reference in Period 1 (n=12) were virtually the same; the only AUC that was substantially higher was R in Period 2 (n=9). Then I looked at the Day 1 data and the troughs and all these profiles indicated a higher exposure in the nine subjects receiving the Reference in Period 2. The wash out was such that a carry-over effect was not to be expected, so we seem to be left with an unexplained sequence effect.

As I have not encountered this before, and am not much of a statistician, any help is appreciated. Do we need to go into all this detail in the study report or just stick to the fact that technically speaking the treatments are bioequivalent? Is there any way to find out by whatever statistical test what has been going on? How will regulatory agencies look upon such a study result? Should we go talk with them? Helmut Schuetz has already kindly offered suggestions and thought it would be interesting for all and suggested to put my problem on the forum.

One other explanation could be the following, far-fetched maybe as too many assumptions all need to be true at the same time:
1) there is a Period effect (which may for instance have to do with different food intake in Autumn and in Winter) leading to a higher exposure in Period 2 for both sequences;
2) co-administration of B does reduce the exposure to A, irrespective of sequence
3) exposure to A is intrinsically higher in the n=9 subjects than in the n=12 subjects due to inter-subject variability (CV's were appr. 30-50%).

Then the group R-T (n=12) has a raised exposure to A in Period 2 due to 'winter' and a reduced exposure due to B intake which levels out 'winter' and R and T come out the same (as they did).
The group T-R (n=9) has a raised exposure to A in Period 2 due to 'winter' which is not counteracted by B, so a high R, and a reduced exposure in Period 1 due to B (Test). However, since they had a higher intrinsic exposure to A to start with, their exposure to test in Period 1 comes out the same as for the n=12 in Period 2 and also the same as R for n=12 in Period 1.
Does this make any sense?

Thanks in advance for any thoughts or suggestions,

Frieda