Different Hydrolases Involved in Bioactivation of Prodrug-Type Angiotensin Receptor Blockers: Carboxymethylenebutenolidase and Carboxylesterase 1

Abstract

Olmesartan medoxomil (OM) is a prodrug-type angiotensin II type 1 receptor blocker (ARB). We recently identified carboxymethylenebutenolidase homolog (CMBL) as the responsible enzyme for OM bioactivation in humans. In the present study, we compared the bioactivating properties of OM with those of other prodrug-type ARBs, candesartan cilexetil (CC) and azilsartan medoxomil (AM), by focusing on interspecies differences and tissue specificity. In invitro experiments with pooled tissue subcellular fractions of mice, rats, monkeys, dogs, and humans, substantial OM-hydrolase activities were observed in cytosols of the liver, intestine, and kidney in all the species tested except for dog intestine, which showed negligible activity, whereas lung cytosols showed relatively low activities compared with the other tissues. AM-hydrolase activities were well correlated with the OM-hydrolase activities. In contrast, liver microsomes exhibited the highest CC-hydrolase activity among various tissue subcellular fractions in all the species tested. As a result of Western blot analysis with the tissue subcellular fractions, the band intensities stained with anti-human CMBL and carboxylesterase 1 (CES1) antibodies well reflected OM-and AM-hydrolase activities and CC-hydrolase activity, respectively, in animals and humans. Recombinant human CMBL and CES1 showed significant AM-and CC-hydrolase activities, respectively, whereas CC hydrolysis was hardly catalyzed with recombinant carboxylesterase 2 (CES2). In conclusion, OM is bioactivated mainly via intestinal and additionally hepatic CMBL not only in humans but also in mice, rats, and monkeys, while CC is bioactivated via hepatic CES1 rather than intestinal enzymes, including CES2. AM is a substrate for CMBL.

Hepatic, Intestinal, Renal, and Plasma Hydrolysis of Prodrugs in Human, Cynomolgus Monkey, Dog, and Rat: Implications for In Vitro–In Vivo Extrapolation of Clearance of Prodrugs

“…The metabolic activities for CES1 substrates were not detected in human intestine, in contrast to CES2 and CMBL substrates ( Fig. 2B; Table 4); the findings were generally in agreement with expression data reported for these enzymes in human intestine (Hosokawa, 2008;Ishizuka et al, 2013). The scaled human CL int,IS9 values were lower than the CL int,LS9 values for CES2 and CMBL substrates (Fig.…”

“…The latter is most likely the reason for observed hydrolysis of CES1 substrate temocapril in rat IS9 (Table 4), the opposite of the CES expression reported in this species. In the case of olmesartan medoxomil, the lowest CL int obtained in dog IS9 supported the minimal CMBL expression levels reported in dog intestine relative to other species (Ishizuka et al, 2013). There was no consistency in differences seen in animal intestinal hydrolysis CL int for CES2 and CMBL substrates relative to human IS9.…”

“…In addition, significant species differences were observed in CES expression in plasma, with high levels reported for rat, but not human, monkey, or dog plasma (Li et al, 2005;Berry et al, 2009). CMBL is highly expressed in the liver, intestine, and kidney across different species except in the dog intestine (Ishizuka et al, 2013).…”

“…CMBL is a relatively novel hydrolysis enzyme, and any potential overlap in its substrates with CES is not well characterized. Cellular localization of these enzymes differs, as CES are present both in the membrane and cytosol (Satoh and Hosokawa, 1998;Fujiyama et al, 2010) whereas CMBL is a predominantly cytosolic enzyme (Ishizuka et al, 2013), emphasizing the need for a careful choice of in vitro system for adequate characterization of hydrolysis. In addition, paraoxonase 1 (PON1), predominantly located in plasma (Bahar et al, 2012), hydrolyzes lactones and aromatic carboxylic acid esters (Fukami and Yokoi, 2012;Ishizuka et al, 2012).…”

Human Intestinal Raf Kinase Inhibitor Protein (RKIP) Catalyzes Prasugrel as a Bioactivation Hydrolase

“…The expression vector of hCE2 fused with carboxyl terminal FLAG tag was prepared as described (Ishizuka et al, 2013). The expression vector of the N-terminal FLAGtagged enhanced green fluorescent protein (GFP) was kindly provided by Dr. Keisuke Fukuchi from Daiichi Sankyo Co., Ltd. Human RKIP was cloned from cDNA library of human 293F cells, and the expression vector was constructed with N-terminal FLAG tag under cytomegalovirus promoter as described (Kubota et al, 2015).…”