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TOXICOLOGY

The Metabolism and Toxicity of Furosemide in the Wistar Rat and CD-1 Mouse: a Chemical and Biochemical Definition of the Toxicophore

The Drug Safety Research Group, Department of Pharmacology, University of Liverpool, Liverpool, United Kingdom (D.P.W., D.J.A., L.R., B.K.P.); Pharmacokinetics Dynamics and Metabolism (R.J., A.P., M.H., I.G.) and Sexual Health Chemistry (J.B.), Pfizer, Sandwich, United Kingdom; and Cyprotex, Nacclesfield, United Kingdom (P.J.B.)

Furosemide, a loop diuretic, causes hepatic necrosis in mice. Previous evidence suggested hepatotoxicity arises from metabolic bioactivation to a chemically reactive metabolite that binds to hepatic proteins. To define the nature of the toxic metabolite, we examined the relationship between furosemide metabolism in CD-1 mice and Wistar rats. Furosemide (1.21 mmol/kg) was shown to cause toxicity in mice, but not rats, at 24 h, without resulting in glutathione depletion. In vivo covalent binding to hepatic protein was 6-fold higher in the mouse (1.57 ± 0.98 nmol equivalent bound/mg protein) than rat (0.26 ± 0.13 nmol equivalent bound/mg protein). In vivo covalent binding to mouse hepatic protein was reduced 14-fold by a predose of the cytochrome P450 (P450) inhibitor, 1-aminobenzotriazole (ABT; 0.11 ± 0.04 nmol equivalent bound/mg protein), which also reduced hepatotoxicity. Administration of [¹⁴C]furosemide to bile duct-cannulated rats demonstrated turnover to glutathione conjugate (8.8 ± 2.8%), -ketocarboxylic acid metabolite (22.1 ± 3.3%), N-dealkylated metabolite (21.1 ± 2.9%), and furosemide glucuronide (12.8 ± 1.8%). Furosemide-glutathione conjugate was not observed in bile from mice dosed with [¹⁴C]furosemide. The novel -ketocarboxylic acid, identified by nuclear magnetic resonance spectroscopy, indicates bioactivation of the furan ring. Formation of -ketocarboxylic acid was P450-dependent. In mouse liver microsomes, a -ketoenal furosemide metabolite was trapped, forming an N-acetylcysteine/N-acetyl lysine furosemide adduct. Furosemide (1 mM, 6 h) became irreversibly bound to primary mouse and rat hepatocytes, 0.73 ± 0.1 and 2.44 ± 0.3 nmol equivalent bound/mg protein, respectively, which was significantly reduced in the presence of ABT, 0.11 ± 0.03 and 0.21 ± 0.1 nmol equivalent bound/mg protein, respectively. Furan rings are part of new chemical entities, and mechanisms underlying species differences in toxicity are important to understand to decrease the drug attrition rate.

Address correspondence to: Dr. Dominic Williams, Drug Safety Research Group, Department of Pharmacology and Therapeutics, University of Liverpool, Sherrington Buildings, Ashton Street, Liverpool, Merseyside L69 3GE, UK. E-mail: dom{at}liv.ac.uk

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