The use of inactivated brain homogenate to determine the in vitro fraction unbound in brain for unstable compounds.

The use of IBH-5 decreased the kdeg values and increased the half-life of the compounds PNZ, TCP, Cpd I and Cpd II with kdeg values of 1.10 × 10-4 s- 1 (t1/2 = 115 min), 4 × 10-5 s-1 (t1/2 = 289 min), 4 × 10-5 s-1 (t1/2 = 289 min), and 3 × 10-5 s-1 (t1/2 = 385 min) respectively, compared to kdeg values of 1.25 × 10-2 s-1 (t1/2 = 0.9 min), 1.1 × 10-4 s-1 (t1/2 = 105 min), 1.0 × 10-3 s-1 (t1/2 = 11.5 min) and 4.5 × 10-4 s-1 (t1/2 = 26 min) in FBHThe use of lower temperature (4 °C) for the determination of fu,brain in this study is not successful due to the instability of the compounds during longer equilibration times required at lower temperatures.The fu,brain values for a set of 15 CNS drugs determined in FBH and IBH-5 using HT-dialysis were similar and are consistent with the literature values. The use of IBH-5 led to the determination of fu,brain for unstable compounds that could not be determined by other methods.The use of IBH-5 is an easy and convenient method to determine the fu,brain of unstable compounds in FBH during drug discovery and development.

Methoxsalen as an in vitro phenotyping tool in comparison with 1-aminobenzotriazole.

The objective is to evaluate methoxsalen as an in vitro phenotyping tool in comparison to ABT as a nonspecific inactivator of P450 mediated metabolism. The reversible inhibition of methoxsalen and ABT against the P450, FMO, AO, MAO-A and -B, enzymes were evaluated using standard marker probe reactions. The time-dependent inhibition of P450 enzymes was evaluated in human liver microsomes. CES1 activities were determined by monitoring the depletion of known substrate, the clopidogrel. The metabolism of P450 substrates in the presence and absence of methoxsalen or ABT was evaluated in human liver microsomes. Methoxsalen is a direct inhibitor and inhibited the activities (>90%) of all enzymes at a concentration of 300 µM except for CYP2C9. Methoxsalen is also a potent time-dependent inhibitor of all P450 enzymes except for CYP2C19 (moderate) at a concentration of 300 µM. Methoxsalen inhibited the metabolism of P450 substrates in the pre-incubation mode. ABT is a potent TDI of several P450 except for CYP2C19 (47%) and CYP2C9 (27%). The results indicate that methoxsalen is a potent pan P450 inhibitor than ABT and can be a better tool in distinguishing P450 mediated metabolism form non-P450 metabolism in human liver microsomes.