Paraoxonase 1 as a Major Bioactivating Hydrolase for Olmesartan Medoxomil in Human Blood Circulation: Molecular Identification and Contribution to Plasma Metabolism

Abstract

ABSTRACT:Olmesartan medoxomil (OM) is a prodrug-type angiotensin II type 1 receptor antagonist. The OM-hydrolyzing enzyme responsible for prodrug bioactivation was purified from human plasma through successive column chromatography and was molecularly identified through N-terminal amino acid sequencing, which resulted in a sequence of 20 amino acids identical to that of human paraoxonase 1 (PON1). Two recombinant allozymes of human PON1 (PON1 192QQ and PON1 192RR ) were constructed and were clearly demonstrated to hydrolyze OM; hydrolysis by the latter allozyme was slightly faster than that by the former. In addition, we evaluated the contribution of PON1 to OM bioactivation in human plasma. Enzyme kinetic studies demonstrated that OM was hydrolyzed more effectively by the recombinant PON1 proteins than by purified albumin. The OM-hydrolyzing activities of the recombinant PON1 proteins and diluted plasma were greatly reduced in the absence of calcium ions. Immunoprecipitation with anti-PON1 IgG completely abolished the OM-hydrolyzing activity in human plasma, whereas the activity was partially inhibited with anti-albumin IgG. The distribution pattern of the OM-hydrolyzing activity in human serum lipoprotein fractions and lipoprotein-deficient serum was examined and showed that most of the OM-hydrolyzing activity was located in the high-density lipoprotein fraction, with which PON1 is closely associated. In conclusion, we identified PON1 as the OM-bioactivating hydrolase in human plasma on a molecular basis and demonstrated that PON1, but not albumin, plays a major role in OM bioactivation in human plasma.

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

“…of triplicate determinations. reported that the bioactivation of OM predominantly proceeds via intestinal CMBL in humans and that hepatic CMBL and plasma paraoxonase 1 are additionally involved (Ishizuka et al, 2010(Ishizuka et al, , 2012. On the contrary, present in vitro studies demonstrated that the contribution of intestinal enzymes to the CC bioactivation is relatively small and that hepatic CES1 is mainly involved.…”

“…1, the prodrug moieties of OM and AM are identical, where the medoxomil moiety links to the carboxylic acid of the drug via an ester bond (Laeis et al, 2001;Kawaguchi et al, 2013), while the CC is a cyclohexyloxycarbonyloxy ethyl ester of the carboxylic acid of candesartan (Nishikawa et al, 1997). We recently reported that a novel human hydrolase, carboxymethylenebutenolidase homolog (CMBL), is responsible for the OM bioactivation (Ishizuka et al, 2010(Ishizuka et al, , 2012; however, responsible enzymes for CC-and AM-bioactivations have not been clearly identified thus far.…”

Interindividual Variability of Carboxymethylenebutenolidase Homolog, a Novel Olmesartan Medoxomil Hydrolase, in the Human Liver and Intestine

“…Our computer simulation with the previously reported method (Ishizuka et al, 2012) estimated the F g value of the prodrug OM to be only 20%, even with the lowest activity (0.814 ng/min/mg protein) among 13 cytosolic fractions of the duodenum and jejunum in the present in vitro study. Notably, a clear positive correlation was observed between OM-hydrolase activity and the CMBL protein expression level among the 13 samples as well as in all 30 samples from the duodenum to colon (unpublished data).…”

“…The intestine is considered to be the first site of exposure of orally administered prodrug OM to be converted to its active moiety olmesartan by metabolic bioactivation. According to the computer-simulated intestinal first-pass availability (F g ) of the prodrug OM (Ishizuka et al, 2012), the majority of the prodrug is converted to active olmesartan in the intestinal epithelial cells. In fact, active olmesartan is reported to be the only species in human blood circulation .…”

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

“…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).…”