Inhibition Assays
➦ To determine the inhibitory potential of a test item the intracellular accumulation of the labeled probe substrate is performed in the absence or presence of increasing concentrations of the test item.
Determining IC₅₀ or Kᵢ after pre-incubation with the investigational drug is recommended, as some inhibitors exhibit enhanced inhibitory potency under these conditions, in line with ICH M12 guidelines for OATP1B1 and OATP1B3.
Experience has shown that a two-step procedure is recommendable:
First Step
Two point Inhibition: Inhibition of transporter-mediated uptake of a probe substrate by two different concentrations of a test item.
Example: Test item identified as an inhibitor showed a significant inhibitory effect.
Second Step
Determination of IC50 value: Concentration dependent inhibition of a probe substrate by a test item and determination of the half-maximal inhibition value (IC50).
Alternative Second Step
Determination of the inhibition character (Ki value): Dixon Plot analysis to determine competitive or non-competitive inhibition.
