P18 enantioselective glucuronidation of lorazepam by ugt2b7 and ugt2b15: assignment of fractional metabolism and IMPACT of ugt2b15 genotype on lorazepam pharmacokinetics

Jian Lin , Pharmacokinetics, Dynamics, and Metabolism, Pfizer Worldwide Research & Development, Groton, CT
Mark Niosi , Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., Groton, CT
Kimberly Lapham , Pharmacokinetics, Dynamics and Metabolism, Pfizer, Inc., Groton, CT
Susanna Tse , Pfizer Inc., Groton, CT
Theunis C. Goosen , Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., Medicinal Sciences, Groton, CT
Lorazepam, a benzodiazepine with antianxiety, sedative and anticonvulsant effects is primarily cleared by UDP-glucuronosyltransferase (UGT)-mediated clearance. Previous in vitro studies in human liver microsomes (HLM) have characterized enantioselective lorazepam glucuronidation primarily involving UGT2B4, UGT2B7 and UGT2B15. Clinically, the UGT2B15*2/*2 polymorphism reduced systemic lorazepam clearance by 42% compared to UGT2B15 wild-type subjects while UGT2B7 polymorphism had no significant impact. The objectives of this study were to evaluate enantioselective lorazepam glucuronidation in HLM in the presence of isoform-selective UGT inhibitors, UGT2B15 genotyped HLM and recombinantly expressed UGTs. In addition, a physiologically-based pharmacokinetic (PBPK) model for lorazepam was developed with in vitro and clinical input parameters in Simcyp to evaluate UGT2B15*2/*2 polymorphic metabolism. Enzyme kinetic characterization of lorazepam glucuronidation and HLM chemical inhibition experiments were conducted in the presence of 2% bovine serum albumin (BSA) since BSA is known to impact in vitro glucuronidation rates. R-lorazepam and S-lorazepam were glucuronidated by multiple hepatic UGTs including UGTs 1A1, 1A3, 1A9, 2B4, 2B7, 2B15, and 2B17 as well as primarily extrahepatic UGTs 1A7 and 1A8. R-lorazepam and S-lorazepam glucuronidation in HLM with 2% BSA were best described by weak substrate inhibition kinetics with apparent unbound substrate concentration at half-maximal velocity (Km,u) of 10.6 and 14.9 μM, respectively, and maximal velocity (Vmax) values of 33.9 and 40.7 pmol/mg/mg protein, respectively. The fraction unbound (fu,bsa) due to nonspecific binding to 2% BSA was 0.14. Accordingly, unbound in vitro intrinsic clearance (CLint,u) of R-lorazepam and S-lorazepam were comparable at 3.2 and 2.7 μL/min/mg, respectively. Applying the well-stirred model for metabolic clearance (CL), the predicted systemic clearance for lorazepam was 0.53 mL/min/kg, which is within approximately 2-fold of the observed CL of 1.1 mL/min/kg. Chemical inhibition experiments with β-phenyllongifolol-2 incubated at the respective 90% inhibition concentrations (IC90) for UGT2B7 (0.3 μM) and UGT2B15 (44 μM) were performed to delineate their relative contributions to lorazepam glucuronidation. Lorazepam glucuronidation was almost completely inhibited by β-phenyllongifolol-2 (44 μM), indicating that UGT2B7 and UGT2B15 are the primary UGTs involved in hepatic glucuronidation. UGT1A1 (atazanavir 10 μM) and UGT1A9 (digoxin 10 μM) selective chemical inhibition experiments indicated a negligible contribution to lorazepam glucuronidation. R-lorazepam fractional metabolism (fm) by UGT 2B7 (fm= 0.64) was somewhat selective compared to UGT2B15 (fm= 0.32) while S-lorazepam glucuronidation was higher by UGT2B15 (fm= 0.61) compared to UGT 2B7 (fm= 0.34). The overall in vitro fm UGT2B7 was 0.50 versus fm UGT2B15 of 0.45. Lorazepam CLint in UGT2B15*2/*2 genotyped HLM (1.64 μL/min/mg) was reduced significantly (62%) compared to wild-type UGT2B15*1/*1 HLM (4.37 μL/min/mg). Incorporation of in vitro kinetic parameters in the PBPK model predicted a 33% reduction in lorazepam CL in UGT2B15 poor metabolizer subjects, comparable to the 42% reduction clinically observed. In conclusion, lorazepam hepatic glucuronidation was primarily and comparably mediated by UGT2B7 and UGT2B15.