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1.
Drug Metab Dispos ; 40(2): 374-80, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22086979

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.


Subject(s)
Angiotensin II Type 2 Receptor Blockers/metabolism , Antihypertensive Agents/metabolism , Aryldialkylphosphatase/metabolism , Imidazoles/metabolism , Prodrugs/metabolism , Tetrazoles/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Aryldialkylphosphatase/chemistry , Aryldialkylphosphatase/genetics , Aryldialkylphosphatase/isolation & purification , Calcium/metabolism , Humans , Hydrolysis , Isoenzymes/blood , Isoenzymes/chemistry , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Kinetics , Lipoproteins, HDL/metabolism , Mutant Proteins/blood , Mutant Proteins/chemistry , Mutant Proteins/isolation & purification , Mutant Proteins/metabolism , Olmesartan Medoxomil , Plasma/enzymology , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Analysis, Protein , Species Specificity , Substrate Specificity
2.
J Biol Chem ; 285(16): 11892-902, 2010 Apr 16.
Article in English | MEDLINE | ID: mdl-20177059

ABSTRACT

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 Cys(132) 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.


Subject(s)
Carboxylic Ester Hydrolases/metabolism , Imidazoles/pharmacokinetics , Intestines/enzymology , Liver/enzymology , Prodrugs/pharmacokinetics , Tetrazoles/pharmacokinetics , Amino Acid Sequence , Angiotensin II Type 1 Receptor Blockers/pharmacokinetics , Biotransformation , Carboxylic Ester Hydrolases/antagonists & inhibitors , Carboxylic Ester Hydrolases/genetics , Carboxylic Ester Hydrolases/isolation & purification , Cell Line , Cytosol/enzymology , DNA Primers/genetics , Enzyme Inhibitors/pharmacology , Female , Humans , Imidazoles/chemistry , In Vitro Techniques , Kinetics , Male , Molecular Sequence Data , Mutagenesis, Site-Directed , Olmesartan Medoxomil , Pregnancy , Prodrugs/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Tetrazoles/chemistry , Tissue Distribution
3.
Eur J Pharmacol ; 512(2-3): 239-46, 2005 Apr 11.
Article in English | MEDLINE | ID: mdl-15840410

ABSTRACT

A close relationship between the renin-angiotensin system and the pathophysiology of diabetic retinopathy has been suggested, several angiotensin II type 1 receptor (angiotensin AT1 receptor) antagonists being effective in animal models. Therefore, we examined the efficacy of an angiotensin AT1 receptor antagonist, olmesartan medoxomil (CS-866), in animal retinopathy models. In diabetic stroke-prone spontaneously hypertensive (SHRSP) rats, 4-week treatment with CS-866 prevented the elongation of oscillatory potential peaks dose-dependently which almost normalized at 3 mg/kg/day. Next, in oxygen-induced retinopathy mice, CS-866 at 1 mg/kg significantly prevented the retinal neovascularization. In these animal models, plasma concentrations of CS-866 were comparable to the in vitro IC50 value of the angiotensin AT1 receptor. In summary, our data demonstrated that CS-866 was effective in early and late stage retinopathy models through the inhibition of the angiotensin AT1 receptor. These findings suggest the possibility of CS-866 as a therapeutic agent for diabetic retinopathy.


Subject(s)
Angiotensin II Type 1 Receptor Blockers/pharmacology , Diabetic Retinopathy/prevention & control , Imidazoles/pharmacology , Tetrazoles/pharmacology , Angiotensin II Type 1 Receptor Blockers/pharmacokinetics , Animals , Area Under Curve , Benzimidazoles/pharmacokinetics , Benzimidazoles/pharmacology , Biphenyl Compounds/pharmacokinetics , Biphenyl Compounds/pharmacology , Blood Glucose/metabolism , Blood Pressure/drug effects , Body Weight/drug effects , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/physiopathology , Diabetic Retinopathy/blood , Diabetic Retinopathy/physiopathology , Disease Models, Animal , Dose-Response Relationship, Drug , Electroretinography , Glycated Hemoglobin/metabolism , Heart Rate/drug effects , Hypertension/complications , Hypertension/physiopathology , Hypoxia/physiopathology , Imidazoles/pharmacokinetics , Male , Olmesartan Medoxomil , Oxygen/pharmacology , Rats , Rats, Inbred SHR , Retinal Neovascularization/metabolism , Retinal Neovascularization/physiopathology , Retinal Neovascularization/prevention & control , Tetrazoles/pharmacokinetics
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