In Vitro ADME Studies for Permeability, CYP, Protein Binding by PhDs at 20+ Year, FDA-Inspected Lab

Less than the folklore says. A standard method wants around 15 mg with reference standard, but discovery teams rarely have that, so we start from roughly 1.5 mg where the assay set allows, and design the panel around the material you actually possess. Purity and a known salt form matter more than quantity; tell us what exists and the scope works backward from there.

PAMPA is the fast, cheap, membrane-only screen for passive permeability, right for early triage. Caco-2 adds biology, transporters and efflux in a human intestinal line, the standard for gut absorption questions. MDCK-MDR1 isolates P-gp specifically, which is the assay when brain exposure or efflux liability is the question. Run in the direction your question needs; bidirectional only when the efflux ratio matters.

It is the ratio of basolateral-to-apical over apical-to-basolateral permeability. Near one means passive movement; meaningfully above one, by convention two or more, means a transporter is pumping your compound back out. For CNS programs that usually reads as the brain barrier winning; for oral drugs it can cap absorption. It also flags drug-interaction questions worth asking before an agency asks them.

Microsomes are the fast first look: CYP-driven oxidative metabolism, cheap and comparable across series. Hepatocytes carry the full enzyme complement including phase II conjugation and transporters, so they read closer to the liver’s truth and catch what microsomes miss. Many programs screen in microsomes and confirm leads in hepatocytes; if the two disagree, that disagreement names the metabolic route.

Inhibition asks whether your compound raises exposure of co-medications by blocking their clearance; it is the early, standard screen across the major isoforms, reversible and time-dependent. Induction asks the opposite, whether your compound lowers co-medication exposure by upregulating enzymes through AhR, CAR or PXR, and typically enters as a candidate approaches IND. Both are drug-interaction questions, and the filing will want both answered eventually.

Because only free drug works. A compound 99.5 percent bound has one tenth the working concentration of one 95 percent bound, at identical total exposure. Free fraction by rapid equilibrium dialysis, with blood-to-plasma partitioning where red cells take their share, is what turns a total concentration into the number pharmacology actually runs on.

Quite possibly, and it is one of the cheapest questions to settle. Ester hydrolysis and other matrix instability masquerade as rapid clearance and send teams chasing metabolic soft spots that do not exist. Plasma and whole blood stability run early here precisely so an artifact never gets engineered against.

Single assays, priced one at a time. The page is built around that: order the number that is missing, a half-life, an efflux ratio, a free fraction, without buying a panel you do not need. When a full candidate-profiling package is genuinely the right call, the DMPK page next door scopes it as a package with interpretation included.