Identification of the major human hepatic and placental enzymes responsible for the biotransformation of glyburide

Abstract

One of the factors affecting the pharmacokinetics (PK) of a drug during pregnancy is the activity of hepatic and placental metabolizing enzymes. Recently, we reported on the biotransformation of glyburide by human hepatic and placental microsomes to six metabolites that are structurally identical between the two tissues. Two of the metabolites, 4-trans-(M1) and 3-cis-hydroxycyclohexyl glyburide (M2b), were previously identified in plasma and urine of patients treated with glyburide and are pharmacologically active. The aim of this investigation was to identify the major human hepatic and placental CYP450 isozymes responsible for the formation of each metabolite of glyburide. This was achieved by the use of chemical inhibitors selective for individual CYP isozymes and antibodies raised against them. The identification was confirmed by the kinetic constants for the biotransformation of glyburide by cDNA-expressed enzymes. The data revealed that the major hepatic isozymes responsible for the formation of each metabolite are as follows: CYP3A4 (ethylene-hydroxylated glyburide (M5), 3-trans-(M3) and 2-trans-(M4) cyclohexyl glyburide); CYP2C9 (M1, M2a (4-cis-) and M2b); CYP2C8 (M1 and M2b); and CYP2C19 (M2a). Human placental microsomal CYP19/aromatase was the major isozyme responsible for the biotransformation of glyburide to predominantly M5. The formation of significant amounts of M5 by CYP19 in the placenta could render this metabolite more accessible to the fetal circulation. The multiplicity of enzymes biotransforming glyburide and the metabolites formed underscores the potential for its drug interactions in vivo.

Figure 1

Histograms representing the rates of formation of glyburide metabolites by pools of (A) human hepatic microsomes and (B) human placental microsomes. Each histogram represents the mean of data obtained from 18 experiments ± standard error of the mean (S.E.M.).

Figure 2

Histograms representing the effect of inhibitors selective for specific CYP isozymes on the metabolism of glyburide by the pool of human hepatic microsomes. Each inhibitor is followed by the corresponding CYP isoform: ketoconazole, KT, and troleandomycin, TR (CYP3A4); sulfaphenazole, SF (CYP2C9); lansoprazole, LP (CYP2C19). The amount of each metabolite formed in a presence of the inhibitor is expressed as a percent of that in the control (absence of inhibitor). Each histogram represents the mean ± S.E.M. of two experiments.

Figure 3

The effect of antibodies against human CYP 3A4, 2C8, and 2C9 on the formation of each metabolite and their total by the pool of human hepatic microsomes. The amounts of metabolites are represented as described in Fig. 2. Each histogram is the mean of the data from three experiments ± S.E.M. * Not statistically significant (P>0.05).

Figure 4

The effect of inhibitors selective for specific CYP isozymes on the metabolism of glyburide by pooled term human placental microsomes. The inhibitors and their concentrations (in parentheses) are as follows: 4-hydroxy-androstendione, 4-OHA (10 μM); α-naphthoflavone, α-NP (0.05 μM); troleandomycin, TR (50 μM); sulfaphenazole, SF (10 μM); lansoprazole, LP (10 μM); quinidine, QN (5 μM); and 4-methylpyrazole, 4-MP (25 μM). Data presented are the mean of two experiments ± S.E.M.

Figure 5

Biotransformation of glyburide by cDNA-expressed CYP19. The rate of the M5 formation exhibits monophasic saturation kinetics of a typical experiment.

Figure 6

Histograms illustrating the rates (V_max ) for the formation of the glyburide metabolites by cDNA-expressed CYP isozymes. Each histogram represents the mean of three experiments.