Gohachiro Miyamoto

Metabolism of a New Positive Inotropic Agent, 3,4-Dihydro-6-[4-(3,4-Dimethoxybenzoyl)-1-Piperazinyl]-2(1H)-Quinolinone (Opc-8212) in the Rat, Mouse, Dog, Monkey and Human

1. After OPC-8212 was orally given to rats, mice, dogs, monkeys and humans, its metabolites were identified by n.m.r. and mass spectrometry, and their concentrations in the plasma, urine and faeces of these species were measured by high-performance liquid chromatography (h.p.l.c.). 2. Hydrolysis of the amide group, oxidation and cleavage of the piperazine ring, O-demethylation of the methoxy group, and conjugation were proposed as metabolic pathways of OPC-8212. 3. In rats, mice and monkeys given OPC-8212 orally, metabolites M-1 to M-6 were detected in the plasma, urine and faeces, while M-1, -4, -5 and M-6 were detected in dogs, and M-1, M-3, M-4, M-5 and M-6 were detected in humans. 4. Conjugates of metabolites M-6 and M-7, with glucuronic acid and sulphuric acid, were observed in the urine of rats and humans.

The Metabolism of a Bronchodilator Procaterol HCl in the Rat in vitro and in vivo

1. The metabolism of the bronchodilator, 5-(1-hydroxy-2-isopropylamino-butyl)-8-hydroxycarbostyril hydrochloride hemihydrate (procaterol HCl), has been studied in vitro and in vivo after oral and intravenous administration to rats. 2. The recovery of [14 C] procaterol HCl and its metabolites in 72 h was about 42% each in urine and faeces for an oral dose (30 mg/kg) and about 53% in urine and 33% in faeces for an intravenous dose (30 mg/kg). 3. Six metabolites in rat excreta were identified as procaterol glucuronide, 5-(2-amino-1-hydroxybutyl)-8-hydroxycarbostyril (desisopropylprocaterol), 5-formyl-8-hydroxycarbostyril (5-formyl-8-HCS), 8-hydroxycarbostyril (8-HCS), procaterol sulphate and unchanged procaterol. 4. In experiments in vitro, procaterol HCl was metabolized into desisopropylprocaterol, 5-formyl-8-HCS, and their conjugates, by rat liver 9000 g supernatant fraction, but not by preparations of kidney, lung and small intestine. Conjugation of procaterol HCl with glucuronic acid occurred in liver and small intestine preparations.

Involvement of cytochrome P450 in the metabolism of rebamipide by the human liver.

1. The metabolism of rebamipide, a gastroprotective agent, was investigated using human liver microsomes and cDNA-expressed human cytochrome P450 systems. 2. 6-Hydroxy and 8-hydroxyrebamipide were produced by human cytochrome P450 enzyme(s), and 8-hydroxylation was the major metabolic pathway. K m and V max for 8-hydroxylation were 1.35 ± 0.20 mM and 0.32 ± 0.34 nmol min -1 mg protein -1, respectively (mean SD, n = 6). Kinetic analysis showed that the 8-hydroxylation reaction consisted of a single component. 3. 8-Hydroxylation was inhibited by the addition of CYP3A4 antibodies as well as troleandomycin, a specific inhibitor of CYP3A4. Furthermore, the metabolism of rebamipide in human liver microsomes was compatible with that in a human cDNA-expressed CYP3A4 system, but not for other human P450 expression systems. It is therefore suggested that the hydroxylation of rebamipide only involves CYP3A4. 4. Rebampide showed no inhibitory effect on CYP1A2-, 2C9-, 2C19-, 2D6-, 2E1- and 3A4-catalysed metabolism. In addition, the metabolic contribution by CYP3A4 was considered to be slight for the overall elimination of rebamipide in man. It is therefore considered that drug interactions with cytochrome P450 enzymes are not involved in either the metabolism of rebamipide or the metabolism of other drugs concomitantly administered with rebamipide.

Proteases Involved in the Metabolic Degradation of Human Interleukin-1beta by Rat Kidney Lysosomes

The in vitro metabolic degradation of human interleukin (IL)-1beta was studied using lysates of rat kidney lysosomes, and proteases involved in the degradation were identified. In the study of IL-1beta degradation, fluorescein isothiocyanate (FITC)-labeled IL-1beta was used as a substrate. The maximal degradation of IL-1beta occurred at pH 3.0, and the reaction was proportional to the lysosomal protein concentration and time of incubation. The degradation was stimulated by the addition of L-cysteine. The reaction was not inhibited by phenylmethanesulfonyl fluoride or EDTA, indicating that serine proteases or metalloproteases do not play a major role in the degradation process. N-Ethylmaleimide, leupeptin and E-64, inhibitors of thiol protease, inhibited the degradation of IL-1beta, by 59%-70%. Pepstatin A, an inhibitor of carboxyl protease, inhibited the degradation by 58%. Combinations of thiol and carboxyl protease inhibitors nearly completely inhibited the degradation. Bio-Gel P-10 gel filtration chromatography of in vitro reactants confirmed the ability of lysosomal proteases to degrade IL-1beta and revealed four to five peaks of degradation products. Taken together, these results indicate that thiol protease and carboxyl protease play an important role in the IL-1beta degradation process by kidney lysosomes. Leupeptin and E-64 dose dependently inhibited both cathepsin B and cathepsin L activities, and pepstatin A strongly inhibited cathepsin D activity in rat kidney lysosomes. The present results suggest that cathepsin B, cathepsin L, and cathepsin D in kidney lysosomes are involved in the metabolic degradation of human IL-1beta.

Metabolism of 1-(3,4-dichlorobenzyl)-5- octylbiguanide in the dog

1. The biotransformation of 1-(3,4-dichlorobenzyl)-5-octylbiguanide (OPB-2045), a new potent biguanide antiseptic, was investigated in male beagle dogs. Urinary and faecal excretion of unchanged compound and metabolites were studied following a single subcutaneous injection of 14C-labeled compound at a dose of 1 mg/kg. 2. Four urinary metabolites were structually identified using synthetic standards and/or spectral data as 3,4-dichlorobenzoic acid, 6-[5-(3,4-dichlorobenzyl)-1-biguanidino] hexanoic acid (DM-210), 4-[5-(3,4-dichlorobenzl)-1-biguanidino] butanoic acid (DM-212) and 5-[5-(3,4-dichlorobenzyl)-1-biguanidino] pentanoic acid (DM-213). 3. The predominant radioactive substances in the excreta were DM-213 and DM-210 at 26.1% and 25.5%, respectively, of the dose. No unchanged compound was detected in the urine, and in the faeces it was only 2% of the dose.

Stereoselective high-performance liquid chromatographic assay for the determination of OPC-18790 enantiomers in human plasma and urine

A high-performance liquid chromatographic assay method for the quantification of OPC-18790 enantiomers in human plasma and urine is described. A human plasma or urine was extracted with organic solvent under alkaline conditions following the addition of internal standard. The enantiomers and internal standard were then derivatized by reaction with the chiral reagent GITC (2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate), followed by octadecylsilica chromatographic separation of the diastereomeric products. The mobile phase consisted of acetonitrile-water (41:59). The fluorescence of the eluate was monitored at 355/405 nm. The lowest quantification limit of each enantiomer was 10 ng/ml in plasma and 0.1 micrograms/ml in urine. Both intra- and inter-day coefficients of variation were below 10%. The assay is sensitive, specific and applicable for stereoselective pharmacokinetic studies in human.

Oxidative one-carbon cleavage of the octyl side chain of olanexidine, a novel antimicrobial agent, in dog liver microsomes

1. The oxidative one-carbon cleavage reaction in the octyl side chain of olanexidine [l-(3,4-dichlorobenzyl)-5-octylbiguanide], a new potent biguanide antiseptic, was characterized in dog liver microsomes. 2. Olanexidine was initially biotransformed to a monohydroxylated metabolite, 8-[5- (3,4-dichlorobenzyl)-l-biguanidino]-2-octanol (DM-215), and DM-215 was subsequently oxidized to the diol derivative, 8-[5-(3,4-dichlorobenzyl)-l-biguanidino]-l,2-octandiol (DM-220). DM-220 was further biotransformed to 2-hydroxy aldehyde derivative, 2-hydroxy carboxylic acid derivative, and an oxidative C-l –C-2 bond cleavage metabolite, 7-[5-(3,4-dichlorobenzyl)-l-biguanidino] heptanoic acid [DM-223 (C7), a seven-carbon chain derivative], after incubation with dog liver microsomes. 3. DM-223 formation required NADPH as a cofactor and was inhibited by quinidine and quinine, relatively selective inhibitors of CYP2D subfamilies in dogs. 4. The results suggest that the one-carbon fragment of the octyl side chain of olanexidine could be removed by the oxidative C-C bond cleavage with the possible involvement of cytochrome P450 systems such as CYP2D subfamily. This oxidative C-C bond cleavage reaction by cytochrome P450s could play an important role in the removal of one-carbon fragment of other drugs or endogenous compounds containing aliphatic chains.

The Pharmacokinetics of Toborinone in Subjects with Congestive Heart Failure and Concomitant Renal Impairment and/or Concomitant Hepatic Impairment

The pharmacokinetics of toborinone was studied in subjects with congestive heart failure (CHF) and concomitant renal and/or hepatic disease. At the time of admission, subjects were grouped based on estimated creatinine clearance and serum bilirubin. Glomerular filtration rate was assessed using iothalamate clearance. Hepatic function was assessed using the caffeine metabolism test and indocyanine green clearance. No significant differences were observed in mean toborinone pharmacokinetic parameters among the four study groups. Positive correlations were observed between toborinone clearance and the measured indices of renal and hepatic function: creatinine clearance, iothalamate renal clearance, paraxanthine/caffeine ratio, and indocyanine green clearance. Toborinone clearance decreased with decreasing creatinine clearance, decreasing glomerular filtration rate, decreasing demethylation metabolic activity, and decreasing hepatic blood flow, although no significant differences were observed in any mean toborinone pharmacokinetic parameters evaluated among the four study groups.

The effects of concomitant administration of theophylline and toborinone on the pharmacokinetics of both compounds in poor and extensive metabolizers via CYP2D6.

This study investigated the effects of the concomitant administration of theophylline and toborinone on the pharmacokinetics of both compounds in poor and extensive metabolizers via CYP2D6. In period 1, a single dose of 3.5 mg/kg theophylline was administered orally. In period 2, a single dose of 1.0 μg/kg/min toborinone was infused over 6 hours. In period 3, 3.5 mg/kg theophylline was coadministered with 1.0 μg/kg/min toborinone. Serial blood and pooled urine samples were collected before and after toborinone administration for the quantification of toborinone and its metabolites in plasma and urine. Serial blood samples were collected before and after theophylline administration for the quantification of theophylline and its metabolites in plasma. No significant differences were observed in toborinone pharmacokinetics between poor and extensive metabolizers via CYP2D6. Toborinone coadministration with theophylline did not result in a substantive effect on the disposition of theophylline and vice versa.

High-performance liquid chromatography-electrospray tandem mass spectrometry for the measurement of 1-(3,4-dichlorobenzyl)-5-octylbiguanide in human serum

1-(3,4-dichlorobenzyl)-5-octylbiguanide (OPB-2045) is a new biguanide antimicrobial agent currently in clinical use as a topical bactericidal antiseptic. A method combining high-performance liquid chromatography (HPLC) with electrospray ionization (triple and quadruple stage) tandem mass spectrometry (ESI-MS/MS) was developed to quantify OPB-2045 in human serum. Solid phase extraction was performed on 0.2 ml of sample to ensure a high level of sensitivity before HPLC-ESI-MS/MS analysis. The limit of quantitation for the method was set at 0.05 ng/ml. Intra-assay and interassay precision were less than 13.7%, with a deviation from the expected value of no greater than 10.5% at a concentration range of 0.05 ng/ml to 5 ng/ml. Decomposition of OPB-2045 in human serum did not occur after storage for 15 months at -20 degrees C, even after three repetitions of freezing and thawing. Application of this method was demonstrated in a pharmacokinetic study of OPB-2045 in healthy patient subjects after a single topical application of 5 g/L preparation of its liquid formulation.