Bioequivalence and Bioavailability Forum

Dear Detlew!

❝ Seems there is an ambiguity in the EMA guidance. On page 15 of 27 under the heading Carry-over effects it is stated:

❝ "... If there are any subjects for whom the pre-dose concentration is greater than 5 percent of the Cmax value for the subject in that period ... ...

❝ This approach does not apply to endogenous drugs."

❝

❝ It is not quite clear for me if they talk there about the original concentrations only. Common sense would tell us yes.

❝ But it's not always common sense what characterizes the guideline .

Right. My common-sense-interpretion is: “This approach [= excluding subjects with measured pre-dose concentrations of >5% before baseline-correction] does not apply to endogenous drugs.”

See also this bright sentence (p.11):

“In bioequivalence studies with endogenous substances, it cannot be directly assessed whether carryover has occurred, so extra care should be taken to ensure that the washout period is of an adequate duration.”

It can’t?
OK, directly = uncorrected; IMHO this implies that it can be done indirectly = corrected (see also the FDA’s guidance).

Example:
Averaged pre-dose 30 ng/mL, C_max of endogenous profile 200 ng/mL, theoretical C_max after dosing 100 ng/mL. Note that the pre-dose is already 15% of the blank profile’s C_max. Following the naïve/savage assumption¹ that the shape of the blank profile is similar to the (theoretical) one after administration, we measure a C_max of 300 ng/mL and estimate the pre-dose with 0 and C_max with 270 ng/mL (simple correction); following the logic of excluding subjects with uncorrected pre-dose values of >5% C_max we would have to exclude many (all?) subjects, since 30 ng/mL = 10% of 200+100 ng/mL. Thus it does not make (common-)sense to deal with uncorrected data.
Let’s go further: Theoretical C_max of the reference 100 ng/mL and of the test 90 ng/mL (T/R 90%). We measure 300 ng/mL (R) / 290 ng/mL (T) and estimate C_max 270 ng/mL (R) / 260 ng/mL (T). Our T/R ratio will be 96.3% instead of the correct 90%: the method is not sensitive to detect true differences between formulations. In such a case it is mandatory to subtract a complete profile.

Another pitfall: We have to test the most sensitive dose.² With endogenous compounds we may experience nasty feedback-loops! The highest dose may downregulate the system or rocket it to a nonlinear state…

---

1. Endogenous compounds can be quite complicated: (sometimes distorted) sinusoidals and/or flip-flop PK depending on prandial state.
   
2. GL, p.11: “If a separation in exposure following administration of different doses of a particular endogenous substance has not been previously established this should be demonstrated, either in a pilot study or as part of the pivotal bioequivalence study using different doses of the reference formulation, in order to ensure that the dose used for the bioequivalence comparison is sensitive to detect potential differences between formulations.”

---

Not for an initiate like you, but the archive.
Green line: sinusoidal baseline + release of endogenous compound triggered by food (depending on caloric content) at 4, 9, and 13 h post dose (C_max 200, t_max 5.7 h). Thin black lines show the model’s components.
Red line: theoretical PK after administration (C_max 100, t_max 3 h, t_½ 5.78 h).
Blue line: measured total concentration (C_max 285, t_max 5.4 h, t_½ ??).

That’s nasty. The theoretical C_max is 50% of the baseline, the AUC 52%, and the t_max occurs 2.7 h earlier.

Now let’s see. Pre-doses are 30, the concentrations at -8 152 and at -16 166. Mean 94.4.

If we subtract only the pre-dose we get the pink line. Note the increasing profile after 23 h.
If we subtract the mean we get the gray line. Oh, a lag-time!
Only if we subtract the entire blank profile we get the ‘true’ values.
Note the bias in C_max and AUC, the shifted t_max, and the crazy t_½:
         -blank    -mean   -profile   theoret.
Cmax      255.2     190.8     100.0     100.0
tmax        5.4       5.4       3.0       3.0
AUCt     2522.3    1430.3    1108.8    1108.9
t½          0.14      0.13      5.77      5.78
AUC∞     2524.3    1430.3    1188.6    1188.8

Lesson learned: Never follow simple methods if you don’t understand the PK of the drug.