Tomoko Ishizuka

Identification of the Human Cytochrome P450 Enzymes Involved in the Two Oxidative Steps in the Bioactivation of Clopidogrel to Its Pharmacologically Active Metabolite

The aim of the current study is to identify the human cytochrome P450 (P450) isoforms involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite. In the in vitro experiments using cDNA-expressed human P450 isoforms, clopidogrel was metabolized to 2-oxo-clopidogrel, the immediate precursor of its pharmacologically active metabolite. CYP1A2, CYP2B6, and CYP2C19 catalyzed this reaction. In the same system using 2-oxo-clopidogrel as the substrate, detection of the active metabolite of clopidogrel required the addition of glutathione to the system. CYP2B6, CYP2C9, CYP2C19, and CYP3A4 contributed to the production of the active metabolite. Secondly, the contribution of each P450 involved in both oxidative steps was estimated by using enzyme kinetic parameters. The contribution of CYP1A2, CYP2B6, and CYP2C19 to the formation of 2-oxo-clopidogrel was 35.8, 19.4, and 44.9%, respectively. The contribution of CYP2B6, CYP2C9, CYP2C19, and CYP3A4 to the formation of the active metabolite was 32.9, 6.76, 20.6, and 39.8%, respectively. In the inhibition studies with antibodies and selective chemical inhibitors to P450s, the outcomes obtained by inhibition studies were consistent with the results of P450 contributions in each oxidative step. These studies showed that CYP2C19 contributed substantially to both oxidative steps required in the formation of clopidogrel active metabolite and that CYP3A4 contributed substantially to the second oxidative step. These results help explain the role of genetic polymorphism of CYP2C19 and also the effect of potent CYP3A inhibitors on the pharmacokinetics and pharmacodynamics of clopidogrel in humans and on clinical outcomes.

Human carboxymethylenebutenolidase as a bioactivating hydrolase of olmesartan medoxomil in liver and intestine

Olmesartan medoxomil (OM) is a prodrug type angiotensin II type 1 receptor antagonist widely prescribed as an antihypertensive agent. Herein, we describe the identification and characterization of the OM bioactivating enzyme that hydrolyzes the prodrug and converts to its pharmacologically active metabolite olmesartan in human liver and intestine. The protein was purified from human liver cytosol by successive column chromatography and was identified by mass spectrometry to be a carboxymethylenebutenolidase (CMBL) homolog. Human CMBL, whose endogenous function has still not been reported, is a human homolog of Pseudomonas dienelactone hydrolase involved in the bacterial halocatechol degradation pathway. The ubiquitous expression of human CMBL gene transcript in various tissues was observed. The recombinant human CMBL expressed in mammalian cells was clearly shown to activate OM. By comparing the enzyme kinetics and chemical inhibition properties between the recombinant protein and human tissue preparations, CMBL was demonstrated to be the primary OM bioactivating enzyme in the liver and intestine. The recombinant CMBL also converted other prodrugs having the same ester structure as OM, faropenem medoxomil and lenampicillin, to their active metabolites. CMBL exhibited a unique sensitivity to chemical inhibitors, thus, being distinguishable from other known esterases. Site-directed mutagenesis on the putative active residue Cys132 of the recombinant CMBL caused a drastic reduction of the OM-hydrolyzing activity. We report for the first time that CMBL serves as a key enzyme in the bioactivation of OM, hydrolyzing the ester bond of the prodrug type xenobiotics.

Distribution of KAI-9803, a Novel δ-Protein Kinase C Inhibitor, after Intravenous Administration to Rats

KAI-9803 is composed of a selective δ-protein kinase C (δPKC) inhibitor peptide derived from the δV1-1 portion of δPKC (termed “cargo peptide”), conjugated reversibly to the cell-penetrating peptide 11-amino acid, arginine-rich sequence of the HIV type 1 transactivator protein (TAT47–57; termed “carrier peptide”) via a disulfide bond. KAI-9803 administration at the end of ischemia has been found to reduce cardiac damage caused by ischemia-reperfusion in a rat model of acute myocardial infarction. In the study presented here, we examined the TAT47–57-mediated distribution of KAI-9803 in rats after a single intravenous bolus administration (1 mg/kg). 14C-KAI-9803 was rapidly delivered to many tissues, including the heart (1.21 μg eq/g tissue), while being quickly cleared from the systemic circulation. The microautoradiography analysis showed that 14C-KAI-9803 was effectively delivered into various cells, including cardiac myocytes and cardiac endothelial cells within 1 min after dosing. The tissue distribution of 125I-labeled KAI-9803 was compared to that of 125I-labeled cargo peptide; this comparison demonstrated that the distribution of KAI-9803 to tissues such as the liver, kidney, and heart was facilitated by the reversible conjugation to TAT47–57. In an in vitro cardiomyocyte study, the extent of 125I-KAI-9803 internalization was greater at 37°C than that at 4°C, whereas the internalization of the 125I-cargo peptide at 37°C was not observed, indicating that the uptake of 125I-KAI-9803 into the cardiomyocytes was mediated by the TAT47–57 carrier. Our studies demonstrated that after a single intravenous administration, KAI-9803 can be delivered into the target cells in the liver, kidney, and heart by a TAT47–57-mediated mechanism.

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

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 (PON1192QQ and PON1192RR) 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

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 in-vitro 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.

Stereoselectivity in Pharmacokinetics of Rivoglitazone, A Novel Peroxisome Proliferator-Activated Receptor γ Agonist, in Rats and Monkeys: Model-Based Pharmacokinetic Analysis and In Vitro-In Vivo Extrapolation Approach

Stereoselectivity in pharmacokinetics of rivoglitazone, a novel peroxisome proliferator-activated receptor γ agonist, in rats and monkeys was examined. The pharmacokinetic model involving chiral inversion explained well the plasma profiles of R-isomer and S-isomer after intravenous and oral administration of (R)-rivoglitazone or (S)-rivoglitazone to rats and monkeys. The high stereoselectivity was evaluated in chiral inversion clearance (R/S ratio: 7.92), metabolic clearance (5.78), and volume of distribution (4.04) in rats; however, these were low (1.73, 1.31, and 1.06) in monkeys. The stereoselectivity in chiral inversion was also observed in in vitro incubation studies in plasma, and the R/S ratio of chiral inversion showed high correlation with the R/S ratio of plasma unbound fraction. The metabolic clearance of the primary five metabolic pathways of rivoglitazone was evaluated from an in vitro-in vivo extrapolation approach using rat and monkey liver microsomes. The high stereoselectivity in metabolic clearance in rat was evaluated (R/S ratio: 5.78), which was assumed to be because of the stereoselectivity in plasma unbound fraction, on the contrary, that in monkeys exhibited low stereoselectivity (0.774). Thus, the stereoselectivity in plasma unbound fraction was estimated to be a major determinant of stereoselectivity in pharmacokinetics of rivoglitazone in rats and monkeys.

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

Olmesartan medoxomil (OM) is a prodrug-type angiotensin II type 1 receptor antagonist. OM is rapidly converted into its active metabolite olmesartan by multiple hydrolases in humans, and we recently identified carboxymethylenebutenolidase homolog (CMBL) as one of the OM bioactivating hydrolases. In the present study, we further investigated the interindividual variability of mRNA and protein expression of CMBL and OM-hydrolase activity using 40 individual human liver and 30 intestinal specimens. In the intestinal samples, OM-hydrolase activity strongly correlated with the CMBL protein expression, clearly indicating that CMBL is a major contributor to the prodrug bioactivation in human intestine. The protein and activity were highly distributed in the proximal region (duodenum and jejunum) and decreased to the distal region of the intestine. Although there was high interindividual variability (16-fold) in both the protein and activity in the intestinal segments from the duodenum to colon, the interindividual variability in the duodenum and jejunum was relatively small (3.0- and 2.4-fold, respectively). In the liver samples, the interindividual variability in the protein and activity was 4.1- and 6.8-fold, respectively. No sex differences in the protein and activity were shown in the human liver or intestine. A genetically engineered Y155C mutant of CMBL, which was caused by a single nucleotide polymorphism rs35489000, showed significantly lower OM-hydrolase activity than the wild-type protein although no minor allele was genotyped in the 40 individual liver specimens.

In vivo multiple metabolic pathways for a novel G protein-coupled receptor 119 agonist DS-8500a in rats: involvement of the 1,2,4-oxadiazole ring-opening reductive reaction in livers under anaerobic conditions

Abstract A 1,2,4-oxadiazole ring-containing compound DS-8500a was developed as a novel G protein-coupled receptor 119 agonist. In vivo metabolic fates of [14C]DS-8500a differently radiolabeled in the benzene ring or benzamide side carbon in rats were investigated. Differences in mass balances were observed, primarily because after the oxadiazole ring-opening and subsequent ring-cleavage small-molecule metabolites containing the benzene side were excreted in the urine, while those containing the benzamide side were excreted in the bile. DS-8500a was detected at trace levels in urine and bile, demonstrating extensive metabolism prior to urinary/biliary excretion. At least 16 metabolite structures were proposed in plasma, urine, and bile samples from rats treated with [14C]DS-8500a. Formation of a ring-opened metabolite (reduced DS-8500a) in hepatocytes of humans, monkeys, and rats was confirmed; however, it was not affected by typical inhibitors of cytochrome P450s, aldehyde oxidases, or carboxylesterases in human hepatocytes. Extensive formation of the ring-opened metabolite was observed in human liver microsomes fortified with an NADPH-generating system under anaerobic conditions. These results suggest an in vivo unique reductive metabolism of DS-8500a is mediated by human non-cytochrome P450 enzymes.