Bioactivation of α,β-Unsaturated Carboxylic Acids Through Acyl Glucuronidation

Abstract

Following oral administration of [ 14 C]GDC-0810, an α,β-unsaturated carboxylic acid, to monkeys, unchanged parent and its acyl glucuronide metabolite, M6, were the major circulating drug-related components. In addition, greater than 50% of circulating radioactivity in plasma was found to be non-extractable 12 hours post-dose, suggesting possible covalent binding to plasma proteins. In the same study, one of the minor metabolites was a cysteine conjugate of M6 (M11) that was detected in plasma and excreta (urine and bile). The potential mechanism for the covalent binding to proteins was further investigated using in vitro methods. In incubations with GSH or cysteine (5mM), GSH-and cysteine-conjugates of M6 were identified, respectively. The cysteine reaction was efficient with a half-life of 58.6 min (k react = 0.04 1/M/sec). Loss of 176 Da (glucuronic acid) followed by 129 Da (glutamate) in mass fragmentation analysis of the GSH-adduct of M6 (M13) suggested the glucuronic acid moiety was not modified. The conjugation of N-glucuronide M4 with cysteine in buffer was >1000-fold slower than with M6. Incubations of GDC-0810, M4, or M6 with monkey or human liver microsomes in the presence of NADPH and GSH did not produce any oxidative GSH adducts, and the respective substrates were qualitatively recovered. In silico analysis quantified the inherent reactivity differences between the glucuronide and its acid precursor.Collectively, these results show that acyl glucuronidation of α,β-unsaturated carboxylic acids can activate the compound towards reactivity with GSH, cysteine, or other biologically occurring thiols, and should be considered during the course of drug discovery. Significance statement:Acyl glucuronidation of the α,β-unsaturated carboxylic acid in GDC-0810 activates the conjugated alkene towards nucleophilic addition by GSH or other reactive thiols. This is the first example that a bioactivation mechanism could lead to protein covalent binding to α,β-unsaturated carboxylic acid compounds.

Identification of a Discrete Diglucuronide of GDC-0810 in Human Plasma after Oral Administration

“…Discrete diglucuronides on two different types of functional groups were rare (Burkon 2008 and Springer 2019) (King 1991). (Mulder 2020) where it was called M1. Following a single oral dose of [ 14 C]GDC-0810 in rats, M6…”

The Importance of Tracking “Missing” Metabolites: How and Why?

“…117,118 In contrast, M2 and M4 were not detected in the rat and monkey mass balance study following oral administration of [ 14 C]GDC-0810. 119 Several in vitro studies with incubations of [ 14 C]GDC-0810 in human liver and intestinal microsomes and hepatocytes were conducted to further understand the formation of M2. The results showed that minor amounts of M2 were only detected in liver microsomes and hepatocytes.…”

Bioactivation and reactivity research advances – 2022 year in review‡

“…This is another example of drug bioactivation through a phase II conjugation reaction. Other examples of conjugation leading to bioactivation include the carboxylic glucuronidation of GDC-0810 activating a,b-double bond for nucleophilic conjugate addition (Mulder et al 2020), and the carboxylic glucuronidation of gemfibrozil and clopidogrel leading to bioactivation via CYP2C8 (Ma et al 2017). Gan et al summarized the bioactivation mechanisms that were catalyzed by non-P450 enzymes (Popovic et al 2010;Gan et al 2016).…”