Apparent kinetic parameters characterizing the N-demethylation and 14-(R)-hydroxylation of CLAR by insect cell (BTI-TN-5B1-4) microsomes containing cDNA-expressed CYP3A4 and NADPH-CYP reductase
| Parameter3-a | Insect Cell Microsomes | Human Liver Microsomes3-d | |
|---|---|---|---|
| −Cytochrome b5 3-b | + Cytochrome b5 3-c | ||
| Hydroxylation | |||
| KM | 18.3 ± 3.8 | 20.6 ± 4.4 | 48.7 ± 17.7 |
| V max | 290 ± 18 | 3642 ± 236 | 206 ± 76 |
| V max′ | 27.8 | 350 | — |
| V max′/M | 1.5 | 17 | — |
| V max/KM | — | — | 4.2 ± 0.21 |
| N-Demethylation | |||
| KM | 19.6 ± 4.2 | 63.9 ± 14.3 | 59.1 ± 24 |
| V max | 675 ± 43 | 4742 ± 472 | 189 ± 52 |
| V max′ | 64.8 | 455 | — |
| V max′/KM | 3.3 | 7.1 | — |
| V max/KM | — | — | 3.3 ± 0.53 |
↵3-a Apparent KM (μM) and Vmax were determined using PCNONLIN.Vmax was expressed as pmol/min/mg (human liver microsomes) and pmol/min/nmol CYP3A4 (cDNA-expressed CYP3A4).Vmax′ represents Vmaxnormalized with respect to the nominal levels of CYP3A4 in native human liver microsomes (96 pmol/mg) and is expressed as pmol/min/mg. Intrinsic clearance (Vmax/KM andVmax′/KM ) was expressed as μml/min/mg protein. KM andVmax data for cDNA-expressed CYP3A4 represent mean ± SE of the parameter estimate.
↵3-b Insect cell microsomes containing only cDNA-expressed CYP3A4 and NADPH-CYP reductase (0.3 units of cytochromec reductase activity per nmol CYP3A4).
↵3-c Insect cell microsomes containing cDNA-expressed CYP3A4, NADPH-CYP reductase (11 units of cytochromec reductase activity per nmol CYP3A4), and cytochromeb5. The molar ratio of cytochromeb5 to CYP3A4 was 2:1.
↵3-d For comparison, data obtained with native human microsomes (mean ± SD, N = 3) are shown. Data are taken from table 1.