Elsevier

Metabolism

Volume 55, Issue 6, June 2006, Pages 711-721
Metabolism

N-Glucuronidation of the antiepileptic drug retigabine: results from studies with human volunteers, heterologously expressed human UGTs, human liver, kidney, and liver microsomal membranes of Crigler-Najjar type II

https://doi.org/10.1016/j.metabol.2006.01.006Get rights and content

Abstract

Retigabine (D-23129), an N-2-amino-4-(4-fluorobenzylamino)phenylcarbamine acid ethyl ester, is a novel antiepileptic drug which is currently in phase II clinical development. This drug undergoes N-glucuronidation. We aimed to identify the principal enzymes involved in the N-glucuronidation pathway of retigabine and compared our findings with those obtained from human liver (a pool of 30 donors) and kidney microsomes (a pool of 3 donors) and with results from a human absorption, distribution, metabolism, and excretion study upon administration of 200 μCi of [14C]-D-23129. Essentially, microsomal assays with UGT1A1 produced only one of the 2 N-glucuronides, whereas UGT1A9 is capable of forming both N-glucuronides. The rates of metabolism for UGT1A9, human liver microsomes, and UGT1A1 were 200, 100, and 100 pmol N-glucuronide per minute per milligram of protein, respectively. At the 50 μmol/L uridine diphosphate glucoronic acid (UDPGA) concentration, UGT1A4 also catalyzed the N-glucuronidation of retigabine, the rates being approximately 5 and 6 pmol/(min·mg protein). With UGT1A9, the production of metabolites 1 and 2 proceeded at a Km of 38 ± 25 and 45 ± 15 μmol/L, whereas the Km for retigabine N-glucuronidation by human liver microsomal fractions was 145  ± 39 μmol/L. Furthermore, a Vmax of 1.2 ± 0.3 (nmol/[min·mg protein]) was estimated for human liver microsomes (4 individual donors).

We investigated the potential for drug-drug interaction using the antiepileptic drugs valproic acid, lamotrigine, the tricyclic antidepressant imipramine, and the anesthetic propofol. These are commonly used medications and are extensively glucuronidated. No potential for drug-drug interactions was found at clinically relevant concentrations (when assayed with human liver microsomes or UGT1A9 enzyme preparations).

Notably, the biosynthesis of retigabine–N-glucuronides was not inhibited in human liver microsomal assays in the presence of 330 μmol/L bilirubin, and glucuronidation of retigabine was also observed with microsomal preparations from human kidney and Crigler-Najjar type II liver. This suggests that lack of a particular UDP-glucuronosyltransferase (UGT) isoform (eg, UGT1A1 in kidney) or functional loss of an entire UGT1A gene does not completely abolish disposal of the drug. Finally, chromatographic separations of extracts from microsomal assays and human urine of volunteers receiving a single dose of 14C-retigabine provided clear evidence for the presence of the 2 N-glucuronides known to be produced by UGT1A9. We therefore suggest N-glucuronidation of retigabine to be of importance in the metabolic clearance of this drug.

Introduction

Retigabine (D-23129), an N-2-amino-4-(4-fluorobenzylamino)phenylcarbamine acid ethyl ester (see Fig. 1), is a new antiepileptic drug which is in clinical phase II development. It exerts potent anticonvulsant effects in a wide variety of animal models of epileptic seizures including models with electrical and chemical induction of convulsion and in genetic models of epilepsy [1], [2], [3], [4]. Unlike standard anticonvulsants, retigabine is more active in the amygdala kindling model, which is believed to be a predictive model of complex partial seizures in humans, when compared with models of generalized seizures like the maximal electroshock seizure threshold test. Importantly, the drug exerts antiepileptogenic effects in low doses and no dependence liability could be observed during chronic treatment.

It was shown that retigabine interacts with specific potassium M channels and particularly the KCNQ2/3 potassium channel heteromultimere, which are involved in the control of the excitability of neuronal cells [5], [6]. Thus, retigabine can be classified as M-channel agonist, and its mode of action targets novel and powerful cellular endpoints for the treatment of epilepsy. This particular mode of action may be termed as selective neuronal potassium channel opener, as suggested by Kornhuber et al [7]. Furthermore, retigabine potentiated γ-aminobutyric acid–induced currents in rat cortical neurons in a concentration-dependent manner. These effects were seen at concentrations of 10 μmol/L, whereas the potassium channel opening effects can be seen at concentrations as low as 0.1 μmol/L [1], [8].

The metabolism of retigabine is dominated by N-glucuronidation [9]. The main biotransformation pathway in rat involves N-dealkylation and N-glucuronidation of the unchanged parent compound, with N-glucuronides being the major metabolites in plasma and bile [9]. In rats, the pharmacokinetics of retigabine was linear at pharmacodynamically effective doses [10].

In dogs, the metabolism of retigabine is less complex with high levels of systemically available N-glucuronides. In contrast to rats, neither acetylation nor N-dealkylation products could be identified in plasma or urine samples [9].

In vitro investigations with fresh human liver slices and human liver microsomes are suggestive for the production of N-glucuronides with little or no evidence for cytochrome P450 monooxygenase–facilitated metabolism of retigabine [9].

In view of its extensive glucuronidation, we wished to investigate the contribution of individual UDP-glucuronosyltransferase (UGT) isozymes using cloned human UGTs expressed in V79 cell lines.

We further compared findings from individual enzyme preparations with those obtained from human liver and kidney microsomes (which lack UGT1A1) and with results from a human absorption, distribution, metabolism, and excretion study upon administration of 200 mg of [14C]-labeled retigabine.

We finally investigated the biotransformation of retigabine with liver tissue from a single pediatric patient diagnosed with Crigler-Najjar type II syndrome. This patient had complete functional loss of the entire UGT1A gene and received liver transplantation.

Assuming competitive enzyme kinetics, we addressed the issue of drug-drug interactions of commonly prescribed and/or coadministered antiepileptic drugs including valproic acid and lamotrigine, as well as the tricyclic antidepressant imipramine and the anesthetic propofol. Particularly, these drugs are also extensively glucuronidated.

Section snippets

Chemicals

Retigabine (D-23129) was synthesized by ASTA Medica (Dresden, Germany) and was of more than 99% purity (batch no. 9805102, Certificate of Analysis FAS/980137).

14C-Retigabine with a specific activity of 333 MBq/mmol (= 9 mCi/mmol) was synthesized by Amersham (Little Chalfont, Buckinghamshire, England) and was of 98.5% purity as determined by thin layer chromatography using a cyclohexane/acetone/0.88 ammonia (7:6:1) solvent system.

Unless otherwise stated, all other reagents were of highest purity

Studies with human volunteers

The metabolism of retigabine in humans was investigated after a single oral administration of a 200-mg dose of [14C]-retigabine to 6 healthy male subjects (study 3065A1-108-US). Plasma, feces, and urine samples were collected before and up to 240 hours after dose administration and were analyzed by HPLC/tandem mass spectroscopy (HPLC/MS/MS) as detailed in Material and Methods. Total radioactivity was measured by liquid scintillation counting.

The mean recovery of radioactivity was 97.9% at 240

Discussion

Retigabine is the first selective neuronal potassium channel opener which is currently under advanced clinical development for the treatment of epilepsy. We show UGT1A1 and UGT1A9 to be important in the metabolism of retigabine, which resulted in 2 distinct N-glucuronides. In addition, UGT1A1 catalyzed the formation of one of the N-glucuronides, whereas UGT1A9 is capable of producing both glucuronides at approximately similar rates, for example, 0.15 vs 0.18 nmol/(min·mg protein).

In the plasma

Acknowledgment

The supply of UGT isoforms by Prof Brian Burchell and the support of Dr Brian Ethell in radio-HPLC assays are gratefully acknowledged.

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