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Metabolic stability plays a major role in drug clearance, with the liver being the primary site for biotransformation of drugs. In early drug discovery, microsomes are typically the preferred system for measuring liver metabolism due to their relatively low cost, robustness and ease of use for high-throughput applications, low lot-to-lot variability due to pooling multiple donors, and wide availability from commercially reputable suppliers. Microsomes are prepared by homogenizing fresh or frozen livers with subsequent isolation of the endoplasmic reticulum subcellular fraction via centrifugation. A number of drug-metabolizing enzymes are located in the endoplasmic reticulum membrane, especially cytochrome P450 (CYP) and UDP-glucuronosyltransferases (UGT). Liver microsomes require the addition of either reduced nicotinamide adenine dinucleotide phosphate (NADPH) or uridine-diphosphate-glucuronic acid (UDPGA) to support the catalytic cycle of CYP and UGT enzymes, respectively.

Metabolic stability screening in microsomes warns of potential liabilities to compounds, provides useful information for establishing structure-metabolic stability relationships, and helps to identify compounds with favorable pharmacokinetic properties. This high-throughput method is based on measuring the disappearance of parent compound. Potential species-related differences in the metabolism can also be detected by using liver microsomes from different animal species and human.

Assay Description

Species
Mouse, Rat, Dog, Monkey, Human

Microsomal concentration
0.5 mg/mL or custom

Compound requirements
50 µl of 10 mM solution or 1-2 mg of dry matter

Test compound concentration
3 µM (0.1% DMSO) or custom

Cofactor
NADPH (1 mM) or NADPH (1 mM) + UDPGA (2 mM)

Time points
0, 5, 15, 30, 45, 60 minutes

Assay controls
Positive control compounds with known activity
Negative control without co-factor
Blank control with vehicle (0.1% DMSO)

Analysis method
LC-MS/MS with internal standard

Data delivery
Intrinsic clearance (µl/min/mg protein)
Half-life (minutes)

References

  1. Ackley D. C. et al.; Metabolic Stability Assessed by Liver Microsomes and Hepatocytes. Optimization in Drug Discovery, 2004, 151-162.
  2. Coe K. J. et al.; Metabolic Stability Assessed by Liver Microsomes and Hepatocytes. Optimization in Drug Discovery, 2013, 87-99.

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