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In vitro characterization of the inhibitory effects of ketoconazole on metabolic activities of cytochrome P-450 in canine hepatic microsomes

Masanori KurohaDepartment of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwaicho, Fuchu, Tokyo 183-8509, Japan.

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Yoji KuzeDepartment of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwaicho, Fuchu, Tokyo 183-8509, Japan.

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Minoru ShimodaDepartment of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwaicho, Fuchu, Tokyo 183-8509, Japan.

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Eiichi KokueDepartment of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwaicho, Fuchu, Tokyo 183-8509, Japan.

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Abstract

Objective—To evaluate the inhibitory potency of ketoconazole (KTZ) on the metabolic activities of isozymes of cytochrome P-450 (CYP) in dogs.

Animals—4 healthy 1-year-old male Beagles.

Procedure—Hepatic microsomes were harvested from 4 dogs after euthanasia. To investigate the effects of KTZ on CYP metabolic activities, 7- ethoxyresorufin, tolbutamide, bufuralol, and midazolam hydrochloride were used as specific substrates for CYP1A1/2, CYP2C21, CYP2D15, and CYP3A12, respectively. The concentrations of metabolites formed by CYP were measured by high-performance liquid chromatography, except for the resorufin concentrations that were measured by a fluorometric method. The reaction velocity-substrate concentration data were analyzed to obtain kinetic variables, including maximum reaction velocity, Michaelis-Menten constant, and inhibitory constant (Ki).

Results—KTZ competitively inhibited 7-ethoxyresorufin O-deethylation and midazolam 4-hydroxylation; it noncompetitively inhibited tolbutamide methylhydroxylation. Bufuralol 1'-hydroxylation was inhibited slightly by KTZ. The mean Ki values of KTZ were 10.6 ± 6.0, 17.0 ± 2.5, and 0.180 ± 0.131 µM for 7-ethoxyresorufin O-deethylation, tolbutamide methylhydroxylation, and midazolam 4-hydroxylation, respectively.

Conclusion and Clinical Relevance—In dogs, KTZ at a therapeutic dose may change the pharmacokinetics of CYP3A12 substrates as a result of inhibition of their biotransformation. Furthermore, no influence of KTZ on the pharmacokinetics of CYP1A1/2, CYP2C21, and CYP2D15 substrates are likely. In clinical practice, adverse drug effects may develop when KTZ is administered concomitantly with a drug that is primarily metabolized by CYP3A12. (Am J Vet Res 2002;63:900–905)

Abstract

Objective—To evaluate the inhibitory potency of ketoconazole (KTZ) on the metabolic activities of isozymes of cytochrome P-450 (CYP) in dogs.

Animals—4 healthy 1-year-old male Beagles.

Procedure—Hepatic microsomes were harvested from 4 dogs after euthanasia. To investigate the effects of KTZ on CYP metabolic activities, 7- ethoxyresorufin, tolbutamide, bufuralol, and midazolam hydrochloride were used as specific substrates for CYP1A1/2, CYP2C21, CYP2D15, and CYP3A12, respectively. The concentrations of metabolites formed by CYP were measured by high-performance liquid chromatography, except for the resorufin concentrations that were measured by a fluorometric method. The reaction velocity-substrate concentration data were analyzed to obtain kinetic variables, including maximum reaction velocity, Michaelis-Menten constant, and inhibitory constant (Ki).

Results—KTZ competitively inhibited 7-ethoxyresorufin O-deethylation and midazolam 4-hydroxylation; it noncompetitively inhibited tolbutamide methylhydroxylation. Bufuralol 1'-hydroxylation was inhibited slightly by KTZ. The mean Ki values of KTZ were 10.6 ± 6.0, 17.0 ± 2.5, and 0.180 ± 0.131 µM for 7-ethoxyresorufin O-deethylation, tolbutamide methylhydroxylation, and midazolam 4-hydroxylation, respectively.

Conclusion and Clinical Relevance—In dogs, KTZ at a therapeutic dose may change the pharmacokinetics of CYP3A12 substrates as a result of inhibition of their biotransformation. Furthermore, no influence of KTZ on the pharmacokinetics of CYP1A1/2, CYP2C21, and CYP2D15 substrates are likely. In clinical practice, adverse drug effects may develop when KTZ is administered concomitantly with a drug that is primarily metabolized by CYP3A12. (Am J Vet Res 2002;63:900–905)