Pooled human liver microsomes

Table 9.2 Summary of enzyme kinetic parameters and inhibitor potencies for 11 human CYP activities in pooled human liver microsomes [117]. 

Table 7 Inhibition of CYP Enzymes in Pooled Human Liver Microsomes by Direct Inhibitors (Positive Controls)... 

Figure 11 Irreversible inhibition of CYP2A6 by 8-methoxypsoralen at two concentrations with and without a dilution step. The overall design of the experiment is discussed in section II.C.7.b. Panels A and B show the effects of preincubating 8-methoxypsoralen (0.05 and 1.25 pM) for 30 minutes with NADPH-fortified human liver microsomes (0.0125 mg/mL) without a dilution prior to the incubation with substrate (coumarin). Panels B and D show the effects of preincubating 8-methoxypsoralen (0.05 and 1.25 pM) for 30 minutes with NADPH-fortified human liver microsomes (0.3125 mg/mL) with a 25-fold dilution prior to the incubation with substrate (coumarin). Panel E shows the effects of preincubating 8-methoxypsoralen (1.25 pM) for 30 minutes with pooled human liver microsomes (0.0125 mg/mL) in the absence of NADPH without a dilution step prior to the incubation with substrate (coumarin). Inhibition in the latter case is caused by inactivation of CYP2A6 during the substrate incubation step (5 minutes) because it occurs to the same extent in both the 0- and 30-minute preincubation samples.

Pooled human liver microsomes or individual human liver microsomal samples should be used for experiments designed to examine the effects of CYP-selective chemical inhibitors or selective inhibitory antibodies. 

Typical experimental procedures are as follows The test drug candidate is incubated with pooled human liver microsomes (e.g., 1 mg protein/mL) that were previously preincubated with ABT (1 or 2 mM) for 30 minutes at (37 1)°C in the presence of an NADPH-generating system. Incubations of the drug candidate in the absence of ABT serve as controls. For hepatocytes, suspensions of freshly isolated or cryopreserved hepatocytes (lx 106 cells/ mL) are preincubated with 100-pM ABT for 30 minutes in 0.25 mL of Krebs-Henseleit buffer or Waymouth s medium (without phenol red) supplemented with FBS (4.5%), insulin (5.6 pg/mL), glutamine (3.6 mM), sodium pyruvate (4.5 mM), and dexamethasone (0.9 pM) at the final concentrations indicated. After the preincubation, the drug candidate is added to the incubation and the rate of metabolism of the drug candidate is compared in hepatocytes or microsomes with and without ABT treatment. A marked difference in metabolism caused by ABT is evidence that CYP plays a prominent role in the metabolism of the drug candidate. 

Figure 18 Effect of nonionic detergent (Triton X-100) on benzydamine N-oxygenation (FMO) and N-demethylation (CYP) by human liver microsomes. Benzydamine (500 pM) was incubated with pooled human liver microsomes (1.0 mg protein/mL) in tricine buffer (50 mM, pH 8.5 at 37°C) with or without Triton X-100 [1% (v/v)]. Reactions were initiated by the addition of an NADPH-generating system and stopped after 10 minute by the addition of an equal volume (500 pL) of methanol. Precipitated protein was removed by centrifugation, and an aliquot (25 pL) of the supernatant fraction was analyzed by HPLC with fluorescence detection. Abbreviations FMO, flavin monooxygenase CYP, cytochrome P450.

An alternative approach to normalizing rates of drug metabolism by recombinant CYP enzymes is the application of a relative activity factor (RAF), in which the correction is not based on specific content but on specific activity, which requires a comparison of the rate of metabolism of a selective marker substrate by each recombinant CYP enzyme and human liver microsomes (75,194). The RAF is then multiplied by the observed rates of drug metabolism by each recombinant CYP enzyme before assessing the relative contribution of each enzyme to the metabolism of the drug. This approach has not been well validated. For example, it is not known whether the relative activity factor remains constant for several marker substrate reactions catalyzed by the same CYP enzyme. If the relative activity factor varies in a substrate-dependent manner, it would be difficult to know which RAF value to apply to the drug candidate under investigation. Another limitation of this approach is that the relative activity factor must be empirically determined for each lot of recombinant CYP enzyme (and preferably each batch of pooled human liver microsomes). 

Conducted with pooled human liver microsomes, which contains all the relevant CYP enzymes and which are generally used to establish initial rate conditions as well as Km and Vmax. 

TABLE 16.1 LC-MS/MS analyses of individual CYP marker substrates in pooled human liver microsomes. ... 

TABLE 16.4 ICso Values of selective chemical inhibitors for individual CYPs in pooled human liver microsomes. 

TABLE 4.2 ICso Values of Selective Chemical Inhibitors for Individual CYPs in Pooled Human Liver Microsomes... 

---