In Vitro Screens for Clearance

The metabolic stability of compounds can be assayed in a high-throughput or semi-high-throughput screening system using recombinant enzymes, human liver microsomes, or human hepatocytes.8 The use of mass spectrometry provides near-universal detectors for many of these in vitro systems, and separation systems are continually evolving to allow more and more direct introduction of sample. The choice of reagent governs the breadth of metabolic processes examined. The recombinant enzyme, normally CYP3A4, obviously only studies reactions performed by that enzyme. However some 60-70% of all drugs are cleared predominantly by CYP3A4, so screening against this isoenzyme will provide useful SAR. Microsomal systems with the appropriate cofactors provide a comprehensive screening reagent for oxidative metabolism by the liver. The cytosolic oxidation systems are absent such as aldehyde oxidase but these generally play a minor role. In most cases, screening is run using the microsomes fortified with NADPH (via a regenerating system) to study P450 metabolism (and flavin monooxygenases). Broad screening for metabolic stability is best accomplished with hepatocytes, which provide a system containing all the enzyme systems: oxidative, conjugative, and hydrolytic. In terms of ease of use the hierarchy is reversed as hepatocyte systems are more difficult to obtain, difficult to cryopreserve, and generally show limited linearity against cell concentration. This means, in terms of measuring stability, hepatocytes have a lower dynamic range. Human systems also suffer from the inter-subject variability of the donors and even when pooled into fairly large batches show differences in metabolic rate across batches. Screening results are normally percentage remaining or described by disappearance half-life. This is convertible into intrinsic clearance using appropriate scaling, a parameter that has direct pharmacokinetic significance in terms of in vivo pharmacokinetics. Intrinsic clearance (CLi) relates to hepatic extraction (E) and hence systemic clearance CLs and hepatic first-pass effect (F):

where /u is fraction unbound in blood and QH is liver blood flow.

Bioavailability is also dependent on the fraction absorbed through the gut (permeability and gut metabolism, partially estimated by screens in Section above).

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