In Vitro Bioactivation of Dihydrobenzoxathiin Selective Estrogen Receptor Modulators by Cytochrome P450 3A4 in Human Liver Microsomes:  Formation of Reactive Iminium and Quinone Type Metabolites

Abstract

Estrogens and selective estrogen receptor modulators (SERMs) are prescribed widely in the clinic to alleviate symptoms in postmenopausal women, and they are metabolized to reactive intermediates, which may elicit adverse effects. As part of our efforts to develop safer SERMs, in vitro covalent protein binding of (2S,3R)-(+)-3-(4-hydroxyphenyl)-2-[4-(2-piperidin-1-ylethoxy)phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol (I) was evaluated. Radioactivity from [3H]I became covalently bound to proteins in a fashion that was both time- and NADPH-dependent in human liver microsomes and reached a value of 1106 pmol equiv/mg protein following a 45 min incubation. At least three pathways are involved in the bioactivation of I, namely, oxidative cleavage of the dihydrobenzoxathiin moiety to give a hydroquinone/para-benzoquinone redox couple, hydroxylation at position 5 or 7 of the benzoxathiin moiety leading to an o-quinone intermediate, and metabolism of the piperidine ring to give an iminium ion. The latter reactive intermediate was identified as its bis-cyano adduct when human liver microsomal incubations were performed in the presence of sodium cyanide. Structural modification of I, including a replacement of the piperidine with a pyrrolidine group, led to (2S,3R)-(+)-3-(3-hydroxyphenyl)-2-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol (II), which did not form a reactive iminium ion. Following the incubation of II with human liver microsomes, covalent binding to proteins was reduced (461 pmol equiv/mg protein), the residual level of binding apparently due to the formation of a rearranged biphenyl quinone type metabolite. Studies with inhibitory antibodies and chemical inhibitors showed that P450 3A4 was the primary enzyme responsible for oxidative bioactivation of I and II in human liver microsomes. These studies thus demonstrated that gaining an understanding of bioactivation mechanisms may be exploited in terms of guiding structural modifications of drug candidates to minimize covalent protein binding and, hopefully, to lower the potential for drug-mediated adverse effects.

Assessing and minimizing time-dependent inhibition of cytochrome P450 3A in drug discovery: A case study with melanocortin-4 receptor agonists

“…Subsequent experiments were performed to identify the mechanism underlying the observed enzyme inhibition, which typically involves the formation of a reactive metabolite from the inhibitor. A generic approach is to use trapping agents such as glutathione or potassium cyanide to capture reactive intermediates formed during drug metabolism (Chen et al 2002;Zhang et al 2005). In the case of 1, a cyano adduct was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, but thiol conjugates were not detected.…”

Simultaneous Screening of Glutathione and Cyanide Adducts Using Precursor Ion and Neutral Loss Scans-Dependent Product Ion Spectral Acquisition and Data Mining Tools

“…The majority of reactive metab-olites can be categorized as soft electrophiles, including quinones, quinone-imines, iminoquinone methides, epoxides, arene oxides, and nitrenium ions, which can readily react with soft nucleophiles such as the sulfhydryl group in glutathione (GSH) and cysteine residue in proteins. The hard reactive metabolites, most often aldehydes and iminium species, are readily reactive to hard nucleophiles such as bases in DNA and lysine residues in proteins [2,3]. Direct detection of the reactive intermediates has proven to be almost impossible due to their transient nature.…”

Bioactivation Pathways of the Cannabinoid Receptor 1 Antagonist Rimonabant

“…Iminium ion metabolites are electrophilic and have, in some cases, been linked to covalent binding to proteins. Examples include phencyclidine (Ward et al, 1982), nicotine (Obach and Van Vunakis, 1988;Shigenaga et al, 1988), and a dihydrobenzoxathiin analog (Zhang et al, 2005b). These metabolites have been characterized both directly and via the corresponding ␣-cyanoamines that are formed in incubation mixtures containing potassium cyanide (KCN).…”