Bioequivalence and Bioavailability Forum

Dear Ricardo!

❝ Does bioavailability measure the amount of total drug at a given time or only measure the amount of unbound drug?

Since you are mentioning bound/unbound drug, I guess you are talking about plasma/serum. Biovailability is defined as a concentration [mass/volume], not an amount [mass]. OK, in urine we have amounts.

It's important to differentiate between bioavailability and bioequivalence. In developing a new drug protein binding is assessed by suitable methods (dialysis, ultracentifugation,…) in order to discover potential problems (e.g., PK interactions due to competitive binding). Mainly these studies are performed in vitro. Even in early development most PK studies (absolute PK, dose proportionality, etc.) are measuring total drug only. I have seen just a few in vivo studies, where samples were measured twice (conventional sample preparation and after dialysis). Apart from the technical problems, sensitivity issues arise. If a drug is to 99 % bound to proteins, the analytical method must be 100× more sensitive (see this post).

In bioequivalence always total drug is measured. The rationale behind: In BE we are interested in differences between formulations (i.e., everything which may influence liberation, in vivo dissolution, and absorption). Once the drug reaches the systemic circulation (becomes bioavailable), the drug-specific ratio bound/unbound can't be influenced by the formulation any more.
Theoretically an exotic excipient - contained in different amounts in the formulations and getting absorbed itself - may force the API from the protein. In such a case the total concentrations will be the same, but the free fraction will be different. There were some discussions about this topic in the past (example: valproic acid - narrow therapeutic index, saturable protein binding). Though it may be an issue when it comes to PK interactions, formulations are so similar when it comes to biopharmaceutics and no evidence of problems were reported in clinical practice, this topic was ‘closed’ by consenus.