Specificity of in vitro Covalent Binding of Tienilic Acid Metabolites to Human Liver Microsomes in Relationship to the Type of Hepatotoxicity: Comparison with Two Directly Hepatotoxic Drugs

Abstract

In order to better understand the first steps leading to drug-induced immunoallergic hepatitis, we studied the target of anti-LKM2 autoantibodies appearing in tienilic acid-induced hepatitis, and the target of tienilic acid-reactive metabolites. It was identified as cytochrome P450 2C9, (P450 2C9): indeed, anti-LKM2 specifically recognized P450 2C9, but none of the other P450s tested (including other 2C subfamily members, 2C8 and 2C18). Tienilic acid-reactive metabolite(s) specifically bound to P450 2C9, and experiments with yeast expressing active isolated P450s showed that P450 2C9 was responsible for tienilic acid-reactive metabolite(s) production. Results of qualitative and quantitative covalent binding of tienilic acid metabolite(s) to human liver microsomes were then compared to those obtained with two drugs leading to direct toxic hepatitis, namely, acetaminophen and chloroform. Kinetic constants (Km and Vmax) were measured, and the covalent binding profile of the metabolites to human liver microsomal proteins was studied. Tienilic acid had both the lowest Km and the highest covalent binding rate at pharmacological doses. For acetaminophen and chloroform, several microsomal proteins were covalently bound, while covalent binding was highly specific for tienilic acid and dihydralazine, another drug leading to immunoallergic hepatitis. Although low numbers of drugs were tested, these results led us to think that there may exist a relationship between the specificity of covalent binding and the type of hepatotoxicity.

In Vitro Metabolism of Rivaroxaban, an Oral, Direct Factor Xa Inhibitor, in Liver Microsomes and Hepatocytes of Rats, Dogs, and Humans

“…After incubation with [ 14 C]rivaroxaban, recovery of 14 C-activity was very high (Ͼ95%; in most cases approximately 100%), indicating no detectable covalent protein binding caused by bioactivation in vitro, as has been reported for other thiophene-containing compounds (Lecoeur et al, 1994;Valadon et al, 1996). This high recovery of 14 C-activity also indicates that structure elucidation comprised all relevant metabolites.…”

A Zone Classification System for Risk Assessment of Idiosyncratic Drug Toxicity Using Daily Dose and Covalent Binding

“…Estimation of covalent binding to cellular macromolecules by using radiolabeled drugs is a direct and reliable method. There are several examples of reactive metabolites forming covalent bonds with IDT-causing drugs, such as tienilic acid, acetaminophen, and clozapine (Lecoeur et al, 1994;Hinson et al, 1995;Gardner et al, 1998). Evans et al (2004) proposed a threshold level of 50 pmol/mg protein as a screening criterion of covalent binding to human liver microsomes (HLMs) in vitro and rat liver in vivo.…”

CYP2D6-Mediated Mexiletine-Protein Adduct Formation

“…12) Dihydralazine is suggested to form adducts with CYP1A2, which is also recognized by autoantibodies from patients with dihydralazine-induced hepatitis. 13) Though we could not yet identify the protein covalently bound with mexiletine, similarity in the molecular weight (52 kDa) suggested that the mexiletine-bound protein might be human CYP2D6 itself.…”