Development of an in Vitro Screening Model for the Biosynthesis of Acyl Glucuronide Metabolites and the Assessment of Their Reactivity toward Human Serum Albumin

Abstract

ABSTRACT:An in vitro screening model was developed to determine the reactivity of acyl glucuronide metabolites from carboxylic drugs. This assay is composed of two phases. The first is a phase of biosynthesis of acyl glucuronides by human liver microsomes (HLM). The second, during which acyl glucuronides are incubated with human serum albumin (HSA), consists of assessing the reactivity of acyl glucuronides toward HSA. Both phases are performed successively in the same experiment. This model was validated using eight carboxylic drugs that were well known for their reactivity, their extent of covalent binding, and their immunological potential. These products were representative of the scale of reactivity. Each compound was incubated with HLM at 400 M and metabolized into acyl glucuronide to different extents, ranging from 5.6% (tolmetin) to 89.4% (diclofenac). The first-order aglycone appearance rate constant and the extent of covalent binding to proteins were assayed during the incubation of acyl glucuronides formed with HSA for 24 h. Extensive isomerization phenomenon was observed for each acyl glucuronide between the two phases. An excellent correlation was observed (r 2 , 0.94) between the extent of drug covalent binding to albumin and the aglycone appearance constant weighted by the percentage of isomerization. This correlation represents an in vitro reactivity scale, which will be helpful in drug discovery support programs to predict the covalent binding potential of new chemical entities. This screening model will also allow the comparison of acyl glucuronide reactivity for related structure compounds.Many acidic drugs with carboxylic acid functions are metabolized to reactive acyl glucuronides. These metabolites are unstable at physiological pH and can result in free aglycone by hydrolysis and lead to positional isomers by acyl migration. Acyl migration involves the transfer of the acyl group from the position 1␤ to the C-2, C-3, or C-4 position of the glucuronic acid ring, which results in the formation of isomeric acyl glucuronides (Faed, 1984;Spahn-Langguth and Benet, 1992).Acyl-migrated glucuronide isomers were shown to bind covalently to proteins in vitro and in vivo causing potential toxicity (SpahnLangguth et al., 1996). The glucuronide-mediated toxicity depends on the covalent binding of acyl glucuronides to specific target proteins located in specific tissues. The toxicological mechanisms are still unknown (Park et al., 1987;Riley and Leeder, 1995;Dansette et al., 1998).However, data literature review provides much information regarding immunologically based and clinically relevant adverse reactions of several drugs that are probably related to the formation of highly reactive acyl glucuronides. These drugs include tolmetin, zomepirac, diclofenac, and diflunisal (Hasegawa et al

Predictability of Idiosyncratic Drug Toxicity Risk for Carboxylic Acid-Containing Drugs Based on the Chemical Stability of Acyl Glucuronide

“…After that, several groups determined half-lives of AGs in buffer, HSA solution, or human fresh plasma for evaluating reactivity (Sallustio et al, 1997;Boelsterli, 2002;Bolze et al, 2002). Based on these interesting findings, we hypothesized that the chemical stability of AGs correlates with the IDT risk because the half-lives of AGs formed from drugs that are reported to induce IDT tended to be shorter than those of safer drugs with no IDT reported.…”

Inhibition of Methotrexate Uptake <i>via</i> Organic Anion Transporters OAT1 and OAT3 by Glucuronides of Nonsteroidal Anti-inflammatory Drugs

“…For example, the percentage of glucuronide metabolites formed from diclofenac and ibuprofen in human liver microsomes was reported to be 89.4 and 53.3%, respectively. 19) The generated NSAIDs-Glu molecules are secreted into the blood along with bile. 20) Separately, NSAIDs taken into tubular cells are also converted into glucuronide conjugates mainly by uridine diphosphate (UDP)-glucuronosyl transferase (UGT) 2B7.…”

A New Rapid In Vitro Assay for Assessing Reactivity of Acyl Glucuronides

“…The acyl migration protocol was adapted from previously published methods (Bolze et al, 2002;Chen et al, 2007;Jinno et al, 2013). The initial mixture contained potassium phosphate buffer (100 mM, pH 7.4), pooled human liver microsome (2 mg/ml), MgCl 2 (1 mM), and test compound (100 mM).…”

“…Compound optimization early in drug discovery requires rapid assessment methods that do not depend on authentic standard, since its generation and purification is too timeconsuming for application to multiple compounds. Therefore, methods using acyl glucuronides produced by liver microsomes for determining the half-lives in buffer, HSA solution, or fresh plasma have been developed and previously documented (Bolze et al, 2002;Chen et al, 2007;Jinno et al, 2013).…”