CHARACTERIZATION OF AFLOQUALONE N-GLUCURONIDATION: SPECIES DIFFERENCES AND IDENTIFICATION OF HUMAN UDP-GLUCURONOSYLTRANSFERASE ISOFORM(S)

Abstract

ABSTRACT:Afloqualone (AFQ) is one of the centrally acting muscle relaxants. AFQ N-glucuronide is the most abundant metabolite in human urine when administered orally, whereas it was not detected in the urine when administered to rats, dogs, and monkeys. Species differences in AFQ N-glucuronidation were investigated with liver microsomes obtained from humans and experimental animals. The kinetics of AFQ N-glucuronidation in human liver microsomes showed a typical Michaelis-Menten plot. The K m and V max values accounted for 2019 ؎ 85.9 M and 871.2 ؎ 17.9 pmol/min/mg protein, respectively. The V max and intrinsic clearance (CL int ) values of AFQ N-glucuronidation in human liver were approximately 4-to 10-fold and 2-to 4-fold higher than those in rat, dog, and monkey, respectively. Among 12 recombinant human UDP-glucuronosyltransferase (UGT) isoforms, both UGT1A4 and UGT1A3 exhibited high AFQ N-glucuronosyltransferase activities. The K m value of AFQ N-glucuronidation in recombinant UGT1A4 microsomes was very close to that in human liver microsomes. The formation of AFQ N-glucuronidation by human liver, jejunum, and recombinant UGT1A4 microsomes was effectively inhibited by trifluoperazine, a known specific substrate for UGT1A4. The AFQ N-glucuronidation activities in seven human liver microsomes were significantly correlated with trifluoperazine N-glucuronidation activities (r 2 ‫؍‬ 0.798, p < 0.01). In contrast, the K m value of AFQ N-glucuronidation in recombinant UGT1A3 microsomes was relatively close to that in human jejunum microsomes. These results demonstrate that AFQ N-glucuronidation in human is mainly catalyzed by UGT1A4 in the liver and by UGT1A3, as well as UGT1A4 in the intestine.Afloqualone (AFQ; Fig. 1), 6-amino-2-fluoromethyl-3-(o-tolyl)-4-(3H)-quinazolinone, is one of the centrally acting muscle relaxants (Tani et al., 1979;Ochiai and Ishida, 1981). It has been reported that AFQ is extensively metabolized in humans (Furuuchi et al., 1983), rats (Otsuka et al., 1983b), dogs, and monkeys (Otsuka et al., 1983a), and there are species differences in the composition of urinary metabolites. The major metabolic pathways of AFQ consist of acetylation of the aromatic amino group, followed by hydroxylation at the methyl carbon of either the acetyl or 2Ј-methyl residue, whereas the N-acetylated metabolites have not been found in dogs. In addition, it has been reported that when AFQ is administered orally to humans, rats, dogs, and monkeys, the parent AFQ and its metabolites are excreted into urine (human, 20% of dose; experimental animals, 50 -70% of dose), in which AFQ N-glucuronide (Fig. 1) is the most abundant metabolite (8% of dose) in human urine (Furuuchi et al., 1983; Otsuka et al., 1983a,b). Although this type of conjugation seems common to primary aromatic amines, the N-glucuronide of AFQ has not been detected in rats, dogs, and monkeys. The N-glucuronidation of AFQ plays an important role in the metabolism of AFQ in humans.Glucuronidation is one of the most common phase II biotransformation reacti...

Absorption, Metabolism, and Excretion of [14C]Imidafenacin, a New Compound for Treatment of Overactive Bladder, After Oral Administration to Healthy Male Subjects

“…The metabolic pathway of imidafenacin for N-glucuronidation, however, was uncommon in rats and dogs, and rats have the unique metabolic pathway for the oxidation on the phenyl moiety, generating M-8 and its related metabolites (Sato et al, unpublished observations). Species differences for N-glucuronidation were also observed in afloqualone in common with imidafenacin, indicating high N-glucuronidation activity in humans compared with activity in experimental animals except rabbits (Green and Tephly, 1998;Kaji and Kume, 2005).…”

Regioselective Glucuronidation of Tanshinone IIa after Quinone Reduction: Identification of Human UDP-Glucuronosyltransferases, Species Differences, and Interaction Potential

“…UGT2B7 shows extreme regioselectivity in favoring M1 formation. Although the regioselectivity of glucuronidation for many endogenous and exogenous compounds (Kaji and Kume, 2005a;Bowalgaha et al, 2007;Kaivosaari et al, 2008) has been reported, the underlying mechanism is still difficult to address because the crystal structures of UGTs are largely unknown (Miley et al, 2007). The CL int (M1 ϩ M2) of UGT1A9 was approximately 8-fold higher than that in the UGT2B7 system, suggesting the predominant contribution from UGT1A9 in catalyzing the reduced TSA glucuronidation.…”

Phase Ii Drug Metabolizing Enzymes

“…Species differences in the glucuronidation of Beviramat, an anti-HIV drug candidate, have been demonstrated in human, rat, mouse, dog and marmoset liver microsomes 24 . Species differences between human, rats, dogs and monkeys in the N-glucuronidation of the muscle relaxant Afloqualone were described by Kaji and Kume 40 . Some studies have demonstrated that cats have remarkably low hepatic levels of UGT1A6.…”