Involvement of Human CYP 1 A Isoenzymes in the Metabolism and Drug Interactions of Riluzole In Vitro 1

Cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) isoenzymes involved in riluzole oxidation and glucuronidation were characterized in (1) kinetic studies with human hepatic microsomes and isoenzyme-selective probes and (2) metabolic studies with genetically expressed human CYP isoenzymes from transfected B-lymphoblastoid and yeast cells. In vitro incubation of [C]riluzole (15 mM) with human hepatic microsomes and NADPH or UDPGA cofactors resulted in formation of N-hydroxyriluzole (Km 5 30 mM) or an unidentified glucuroconjugate (Km 5 118 mM). Human microsomal riluzole N-hydroxylation was most strongly inhibited by the CYP1A2 inhibitor a-naphthoflavone (IC50 5 0.42 mM). Human CYP1A2-expressing yeast microsomes generated N-hydroxyriluzole, whereas human CYP1A1-expressing yeast microsomes generated N-hydroxyriluzole, two additional hydroxylated derivatives and an O-dealkylated derivative. CYP1A2 was the only genetically expressed human P450 isoenzyme in B-lymphoblastoid microsomes to metabolize riluzole. Riluzole glucuronidation was inhibited most potently by propofol, a substrate for the human hepatic UGT HP4 (UGT1.8/9) isoenzyme. In vitro, human hepatic microsomal hydroxylation of riluzole (15 mM) was weakly inhibited by amitriptyline, diclofenac, diazepam, nicergoline, clomipramine, imipramine, quinine and enoxacin (IC50 ' 200–500 mM) and cimetidine (IC50 5 940 mM). Riluzole (1 and 10 mM) produced a weak, concentration-dependent inhibition of CYP1A2 activity and showed competitive inhibition of methoxyresorufin O-demethylase. Thus, riluzole is predominantly metabolized by CYP1A2 in human hepatic microsomes to N-hydroxyriluzole; extrahepatic CYP1A1 can also be responsible for the formation of several other metabolites. Direct glucuronidation is a relatively minor metabolic route. In vivo, riluzole is unlikely to exhibit significant pharmacokinetic drug interaction with coadministered drugs that undergo phase I metabolism. Riluzole [2-amino-6-(trifluoromethoxy)benzothiazole], a novel antiglutamate agent with neuroprotective properties in animal models of neurodegenerative disease (Doble, 1996), has been shown to prolong survival in patients with ALS (Bensimon et al., 1994; Lacomblez et al., 1996). After oral administration to humans, the drug is almost completely absorbed, undergoes limited first-pass metabolism and is excreted predominantly via the urine in the form of metabolites resulting from phase I and II metabolism. Characterization of the CYP isoenzymes responsible for the metabolism of riluzole is of importance in assessing the likelihood of pharmacokinetic variability due to genetic polymorphism and differential regulation and in identifying potential drug interactions. In the present study, the in vitro oxidative metabolism and glucuronidation of riluzole were investigated using human hepatic microsomes. Identity of the CYP isoenzymes involved in riluzole biotransformation was established using genetically expressed human CYP isoenzymes from transfected cell lines and yeast and isoenzyme-selective inhibitory probes. Similarly, pathways of hepatic microsomal glucuronidation of riluzole were investigated with known inhibitors/substrates of UGT isoenzymes. To identify potential metabolic drug interactions, the effects of known CYP substrates/inhibitors and frequently coadministered drugs on the hepatic microsomal oxidation of riluzole and, conversely, the effects of riluzole on specific human hepatic CYP-dependent drug metabolism reactions were determined. Materials and Methods