Kinetic analysis of M1 formation rates in incubations of DB289 with HIM
Incubation mixtures contained 0.1 to 0.2 mg/ml HIM and DB289 ranging from 0.05 to 25 μM. Reactions were carried out at 37°C for 5 min for all the HIM or 3 min for HLM. M1 formation rates were determined by HPLC/MS/MS analyses of duplicate incubations as described under Materials and Methods. Apparent kinetic parameters were derived from the equations as described under Materials and Methods. Results for HIM are arranged according to their intrinsic clearance values [CL′int(1)]. For comparison, kinetic parameters for pooled HLM (Wang et al., 2006) are also included.
HIM | HLM Pooled | |||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 32 | 7 | 2 | 30 | 3 | 1 | 12 | 23A | 31 | Pooled | |||||||||||
| KMapp(1) (μM) | 0.6 | 1.3 | 0.7 | 0.6 | 1.3 | 0.8 | 2.3 | 2.4 | 1.4 | 0.7 | 0.5 | |||||||||
| Vmax(1) (nmol/min/mg protein) | 0.44 | 0.89 | 0.42 | 0.19 | 0.24 | 0.12 | 0.11 | 0.09 | 0.02 | 0.19 | 3.8 | |||||||||
| CL′int(1) (ml/min/mg protein) | 0.73 | 0.68 | 0.6 | 0.32 | 0.18 | 0.15 | 0.05 | 0.04 | 0.01 | 0.27 | 7.6 | |||||||||
| CL′int(2) (ml/min/mg protein) | N.D.a | N.D. | 0.01 | N.D. | N.D. | 0.002 | N.D. | 0.003 | N.D. | 0.01 | N.D. | |||||||||
| Protein concentration (mg/ml) | 0.1 | 0.2 | 0.1 | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 | 0.02 | |||||||||
↵ a N.D., not determined because the corresponding Eadie-Hofstee plot exhibited monophasic enzyme kinetics.