S-Naproxen and desmethylnaproxen glucuronidation by human liver microsomes and recombinant human UDP-glucuronosyltransferases (UGT): role of UGT2B7 in the elimination of naproxen

Abstract

Aims: To characterize the kinetics of S-naproxen ('naproxen') acyl glucuronidation and desmethylnaproxen acyl and phenolic glucuronidation by human liver microsomes and identify the human UGT isoform(s) catalysing these reactions.

Methods: Naproxen and desmethylnaproxen glucuronidation were investigated using microsomes from six and five livers, respectively. Human recombinant UGTs were screened for activity towards naproxen and desmethylnaproxen. Where significant activity was observed, kinetic parameters were determined. Naproxen and desmethylnaproxen glucuronides were measured by separate high-performance liquid chromatography methods.

Results: Naproxen acyl glucuronidation by human liver microsomes followed biphasic kinetics. Mean apparent K(m) values (+/-SD, with 95% confidence interval in parentheses) for the high- and low-affinity components were 29 +/- 13 microm (16, 43) and 473 +/- 108 microm (359, 587), respectively. UGT 1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10 and 2B7 glucuronidated naproxen. UGT2B7 exhibited an apparent K(m) (72 microm) of the same order as the high-affinity human liver microsomal activity, which was inhibited by the UGT2B7 selective 'probe' fluconazole. Although data for desmethylnaproxen phenolic glucuronidation by human liver microsomes were generally adequately fitted to either the single- or two-enzyme Michaelis-Menten equation, model fitting was inconclusive for desmethylnaproxen acyl glucuronidation. UGT 1A1, 1A7, 1A9 and 1A10 catalysed both the phenolic and acyl glucuronidation of desmethylnaproxen, while UGT 1A3, 1A6 and 2B7 formed only the acyl glucuronide. Atypical glucuronidation kinetics were variably observed for naproxen and desmethylnaproxen glucuronidation by the recombinant UGTs.

Conclusion: UGT2B7 is responsible for human hepatic naproxen acyl glucuronidation, which is the primary elimination pathway for this drug.

Figure 1

Structures of naproxen and desmethylnaproxen, showing sites of glucuronidation

Figure 2

Representative Eadie–Hofstee plots for the conversion of naproxen to naproxen acyl glucuronide using the following enzyme sources: (A) human liver microsomes (H12); (B) UGT2B7; (C) UGT1A9; and (D) UGT1A10. Units of V/[S] are pmol glucuronide µm⁻¹ min⁻¹ mg⁻¹. Points show experimentally determined values. Curves are the computer-generated curves of best fit

Figure 3

Formation of naproxen acyl glucuronide by recombinant human UDP-glucuronosyltransferases at substrate (naproxen; NAP) concentrations of 100 (□) and 1000 µm (▪). Results represent the means of duplicate estimations

Figure 4

Representative Eadie–Hofstee plots for the conversion of desmethylnaproxen to desmethylnaproxen phenolic glucuronide using the following enzyme sources: (A) human liver microsomes (H12); (B) human liver microsomes (H29); and (C) UGT1A9. Units of V/[S] are pmol glucuronide µm⁻¹ min⁻¹ mg⁻¹. Points show experimentally determined values. Curves are the computer-generated curves of best fit

Figure 5

Representative Eadie–Hofstee plots for the conversion of desmethylnaproxen to desmethylnaproxen acyl glucuronide using the following enzyme sources: (A) human liver microsomes (H13); (B) UGT2B7; (C) UGT1A6; and (D) UGT1A9. Units of V/[S] are pmol glucuronide µm⁻¹ min⁻¹ mg⁻¹. Points show experimentally determined values. Curves are the computer-generated curves of best fit

Figure 6

Formation of desmethylnaproxen acyl glucuronide by recombinant human UDP-glucuronosyltransferases at substrate (desmethylnaproxen; DMN) concentrations of 500 (□) and 5000 µm (▪). Results represent the means of duplicate estimations

Figure 7

Formation of desmethylnaproxen phenolic glucuronide by recombinant human UDP-glucuronosyltransferases at substrate (desmethylnaproxen; DMN) concentrations of 500 (□) and 5000 µm (▪). Results represent the means of duplicate estimations