Reagent Selection and Reaction Conditions

The development of biological tools to support DDI studies has paralleled the development of bioanalytical techniques. To better understand in vitro-in vivo (IVIV) correlations, the effects of differences in enzyme preparations and incubation conditions must be understood. Differences between enzyme preparations include nonspecific binding, the ratio of accessory proteins (cytochrome b5 and reductase) to CYPs and genetic variability differences in incubation conditions include buffer strength, the presence of inorganic cations and solvent effects. Understanding how biology influences enzymatic activity is crucial to accurate and consistent prediction of the inhibition potential. 

The key factor in understanding in vitro D DI data is to determine how the apparent kinetic measurements match the unbound concentration of an inhibitor in the 

Enzyme preparation Ratio of P450 reductase/CYP2C9 Ratio of cytochrome 1 5/CYP2C9 

The architecture of various CYPs may accommodate entities of different shapes [139]. Several CYPs, particularly CYP3A4 [140,141] and CYP2C9 [142], may exhibit atypical (non-Michaelis-Menten) kinetics such as heterotropic activation, homotropic activation, substrate inhibition and partial inhibition, all in a substrate-effector-dependent manner [143]. Several hypotheses have been proposed to account for the observation of atypical kinetics, including simultaneous occupancy of the CYP active site by two substrates (or one substrate and one effector simultaneously) [144] and allosteric changes in CYP architecture due to binding of an effector [145,146]. Along 

Partial inhibition occurs when an inhibitor partially inhibits the turnover of a substrate, but is not able to fully inhibit substrate turnover even at saturating conditions [143], The most common explanation for partial inhibition is simultaneous occupancy of the CYP active site by both the effector and substrate probe in 

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