Kinetic parameters of EM-652 glucuronidation by microsomal proteins from stably expressed human UGT1A1, UGT1A3, UGT1A9, and monkey monUGT1A01 and monUGT1A09.
| Km(app.) | Vmax | NormalizedVmax | Vmax/Km | ||
|---|---|---|---|---|---|
| μM | pmol/min/mg of protein | μl/min/mg of protein | |||
| Human | |||||
| UGT1A1 | EM-652-4′-G | 168 ± 45 | 14.5 ± 4.0 | 14.5 | 0.08 |
| EM-652-7-G | 63 ± 8 | 59.5 ± 7.4 | 59.5 | 0.95 | |
| UGT1A3 | EM-652-4′-G | 36 ± 3 | 33.3 ± 3.0 | 47.6 | 1.31 |
| EM-652-7-G | 19 ± 4 | 21.1 ± 4.4 | 30.2 | 1.58 | |
| UGT1A9 | EM-652-4′-G | 172 ± 31 | 52.2 ± 9.4 | 3.0 | 0.01 |
| EM-652-7-G | 64 ± 23 | 30.8 ± 8.3 | 1.8 | 0.03 | |
| Monkey | |||||
| monUGT1A01 | EM-652-4′-G | 302 ± 36 | 16.6 ± 2.0 | 16.6 | 0.05 |
| EM-652-7-G | 233 ± 58 | 27.7 ± 6.9 | 27.7 | 0.12 | |
| monUGT1A09 | EM-652-4′-G | 170 ± 59 | 39.8 ± 14.0 | 13.2 | 0.08 |
| EM-652-7-G | 69 ± 7 | 166.6 ± 18.3 | 55.5 | 0.80 |
Kinetic studies were performed for 3 h using microsomal proteins from HEK293 cells stably expressing human and monkey UGT1A proteins (40 mg) in the presence of UDPGA (2 mM) and with EM-652 concentrations ranging from 25 to 1000 mM. Values obtained by double reciprocal plots (Lineweaver-Burk) represent the mean of two independent experiments ± S.D. EM-652-4′-G) and EM-652-7-glucuronide (EM-652-7-G) formation was quantified using LC/MS-MS. V max values were normalized for the variable level of UGT1A protein expression in stably expressing HEK293 cells (reference value determined for expression in UGT1A1-and monUGT1A01-HEK293 cells). Each normalizedV max value was used to determine the glucuronidation efficiency (ratioV max/K m).