| pA2 online © Copyright 2004 The British Pharmacological Society |
134P
University of Newcastle Winter Meeting December 2004 |
Correcting for auto-inhibition via mechanism-based inactivation when assessing in vitro drug metabolism data L.M.Van1, J.S. Yang1, A.M. Heydari1, J. Hargreaves2, L. Wade2, T. Coleman2 and A. Rostami-Hodjegan1. Drug Metabolism and Pharmacogenetic Group1, Academic Unit of Clinical Pharmacology. Royal Hallamshire Hospital, University of Sheffield, Sheffield, S10 2JF, UK. AstraZeneca2, Alderley Park, Macclesfield, Chesire, SK10 4TG, UK. |
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Print abstractDrugs behaving as mechanism-based inhibitors (MBI) are known to reduce the amount of active enzyme(s) in a concentration- and time-dependent fashion (Silverman, 1998). If the inhibited enzymes are involved in the metabolism of the inhibitor itself, this can lead to biased estimates of the in vitro kinetic parameters for the metabolism of the inhibitor (Km, Vmax, and Clint). This investigation aimed to evaluate the magnitude of such bias.
N-methyl-3,4-methylenedioxymethamphetamine (MDMA; commonly known as ecstasy) was used as a model MBI compound. Simulations were carried out to assess CYP2D6 inactivation by ecstasy during different incubation times and with different concentration of the drug. The inactivation parameters for CYP2D6 expressed in Saccharomyces cerevisiae (kinact = 0.29 min-1 and KI = 12.9 µM) and human liver microsomes (HLM; kinact = 0.26 min-1 and KI = 14.4 µM) were obtained from previous studies in our laboratory (Heydari et al, 2004). The values were used to correct the rate of MDMA metabolism in HLM (Km = 2.2 µM and Vmax = 0.223 nmol/mg/min; Kreth et al, 2000) and in microsomes prepared from yeast cells (Km = 1.72 µM and Vmax= 6.45 nmol/nmol P450/min; Tucker et al, 1994) via the CYP2D6 pathway, by accounting for the loss of active enzyme during incubation that was ignored by aforementioned researchers.
The values after correcting for the MBI effect were 2 to 11 fold different than those previously reported (Table 1 and Table 2). The results put emphasis on accounting for enzyme inactivation during the incubation. The latter could be elucidated by investigating linearity of the rate of metabolism with time.
Table 1: Adjusted kinetic values for MBI in HLM
Kinetic Parameter |
MDMA* Metabolism |
MDMA Metabolism with MBI Correction |
Vmax (nmol/mg/min) |
0.22 |
0.86 |
Km ( µM) |
2.20 |
12.86 |
Clint (ml/mg/min) |
0.10 |
0.07 |
* Values obtained from Kreth et al. (2000)
Table 2: Adjusted kinetic values for MBI in yeast
Kinetic Parameter |
MDMA** Metabolism |
MDMA Metabolism |
Vmax(nmol/nmol P450/min) |
6.45 |
27.37 |
Km(µM) |
1.72 |
11.65 |
Clint(ml/nmol P450/min) |
0.27 |
2.34 |
** Values obtained from Tucker et al. (1994)
Heydari et al. (2004) DMD. 32(11): 1213-1217.
Kreth et al. (2000) Biochem. Pharm. 59: 1563-1571.
Silverman RB. (1998) In mechanism-based enzyme inactivation: chemistry and
enzymology . Vol 1. CRC Press Inc. Florida: 3-30.
Tucker et al. (1994) Biochem. Pharm. 47: 1151-1156.
Acknowledgement: L.M. Van is funded by an AstraZeneca PhD studentship .