In-June Cha

Effects of Cyp2c19 and Cyp2c9 genetic polymorphisms on the disposition of and blood glucose lowering response to tolbutamide in humans

Several recent in-vitro data have revealed that CYP2C19, in addition to CYP2C9, is also involved in the 4-methylhydroxylation of tolbutamide. We evaluated the relative contribution of CYP2C9 and CYP2C19 genetic polymorphisms on the disposition of blood glucose lowering response to tolbutamide in normal healthy Korean subjects in order to reappraise tolbutamide as a selective in-vivo probe substrate of CYP2C9 activity. A single oral dose of tolbutamide (500 mg) or placebo was administered to 18 subjects in a single-blind, randomized, crossover study with a 2-week washout period. Twelve subjects (of whom six were CYP2C19 extensive metabolizer (EM) and six were CYP2C19 poor metabolizer (PM) genotype) were of the homozygous wild-type CYP2C9*1 genotype; the other six subjects were of the CYP2C9*1/*3 and CYP2C19 EM genotype. Pharmacokinetic parameters were estimated from plasma and urine concentrations of tolbutamide and 4-hydroxytolbutamide. Serum glucose concentrations were measured before and after oral intake of 100 g dextrose. In subjects heterozygous for the CYP2C9*3 allele, C(max) and AUC of tolbutamide were significantly greater and the plasma half-life significantly longer than those in homozygous CYP2C9*1 subjects. No pharmacokinetic differences were found between CYP2C19 EM and PM genotype subjects. The estimated AUC of the increase in serum glucose after oral intake of 100 g dextrose was 2.7-fold higher in subjects with the wild-type CYP2C9 genotype than in those with CYP2C9*1/*3, but CYP2C19 genetic polymorphism did not alter the blood glucose lowering effect of tolbutamide. The plasma AUC of 4-hydroxytolbutamide and the ratio of 4-hydroxytolbutamide/tolbutamide did not differ significantly between CYP2C19 PM and EM genotype subjects, while these parameters were about twice as high in subjects with the wild-type CYP2C9 genotype than in heterozygous CYP2C9*3 subjects (P < 0.05). Our results strongly suggest that the disposition and hypoglycemic effect of tolbutamide are affected mainly by CYP2C9 genetic polymorphism, but not by CYP2C19 polymorphism. The in-vivo contribution of CYP2C19 to tolbutamide 4-methylhydroxylation appears to be minor in humans. This suggests that, at least in vivo, tolbutamide remains a selective probe for measuring CYP2C9 activity in humans.

Inhibitory effects of fruit juices on CYP3A activity

There have been very limited reports on the effects of commercial fruit juices on human CYP3A activity. Therefore, the inhibitory effects of readily available commercial fruit juices on midazolam 1′-hydroxylase activity, a marker of CYP3A, were evaluated in pooled human liver microsomes. The fruit juices investigated were black raspberry, black mulberry, plum, and wild grape. White grapefruit, pomegranate, and orange juice were used as positive and negative controls. The black mulberry juice showed the most potent inhibition of CYP3A except for grapefruit juice. The inhibition depended on the amount of a fruit juice added to the incubation mixture. The inhibitory potential of human CYP3A was in the order: grapefruit > black mulberry > wild grape > pomegranate > black raspberry. The IC50 values of all fruit juices tested were reduced after preincubation with microsomes in the presence of the NADPH-generating system, suggesting that a mechanism-based inhibitory component was present in these fruit juices, as in the case of grapefruit. The results suggest that, like grapefruit juice, commercial fruit juices also have the potential to inhibit CYP3A-catalzyed midazolam 1′-hydroxylation. Therefore, in vivo studies investigating the interactions between fruit juices such as black mulberry and wild grape and CYP3A substrates are necessary to determine whether inhibition of CYP3A activity by fruit juices is clinically relevant.

Development of the inje cocktail for high-throughput evaluation of five human cytochrome P450 isoforms in vivo

To develop and validate an in vivo cocktail method for high‐throughput phenotyping of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A, 12 healthy subjects received five probe drugs alone or simultaneously. The in vivo phenotyping index of CYP2C9, the ratio of 8 h urine concentration of losartan to its metabolite after a single administration of losartan, was not significantly different from that obtained using the five‐drug cocktail. Similarly, the ratios of [omeprazole]/[5‐hydroxyomeprazole] (CYP2C19) and [paraxanthine]/[caffeine] (CYP1A2) in 4 h plasma samples and the log ratio of [dextromethorphan]/[dextrorphan] (CYP2D6) in 8 h urine samples and the 4 h plasma concentrations of midazolam (CYP3A) after single administration or well‐established three‐drug cocktail of caffeine, omeprazole, and dextromethorphan were not significantly different from those after the new five‐drug cocktail. In conclusion, the new five‐drug cocktail regimen, named the “Inje cocktail,” can be used as a tool to phenotype in vivo enzyme activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A with only 4 h blood sampling and 8 h urine collection following simultaneous administration of the five probe drugs.

Relationship of paroxetine disposition to metoprolol metabolic ratio and CYP2D6*10 genotype of Korean subjects

To evaluate the relationship between the metabolic ratio (MR) of metoprolol, CYP2D6*10B genotype, and the disposition of paroxetine in Korean subjects.

Diclofenac attenuates Wnt/β-catenin signaling in colon cancer cells by activation of NF-κB

The dysregulation of Wnt/β‐catenin signaling and subsequent upregulation of β‐catenin response transcription (CRT) occur frequently in colon cancer cells. Non‐steroidal anti‐inflammatory drugs (NSAIDs) can repress CRT in colorectal cancer, but little is known about the mechanism of action. We show that the NSAID diclofenac inhibits Wnt/β‐catenin signaling without altering the level of β‐catenin protein and reduces the expression of β‐catenin/TCF‐dependent genes. Diclofenac induced the degradation of IκBα, which increased free nuclear factor κB (NF‐κB) in cells. Also, the ectopic expression of p65, which is a component of NF‐κB, suppressed CRT. Our findings suggest that diclofenac inhibits Wnt/β‐catenin signaling via the activation of NF‐κB in colon cancer cells.

Inhibition of human cytochrome P450 isoforms and NADPH‐CYP reductase in vitro by 15 herbal medicines, including Epimedii herba

Objective:  We evaluated the potential of 15 herbal medicines (HMs), commonly used in Korea, to inhibit the catalytic activities of several cytochrome P450 (CYP) isoforms and microsomal NADPH‐CYP reductase.

Rapid simple high-performance liquid chromatographic determination of paroxetine in human plasma

A rapid, simple method for the measurement of paroxetine in human plasma by reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection is described. This method includes only one-step extraction of paroxetine and dibucaine, an internal standard, with chloroform. Their recoveries were around 90%. The mobile phase, 10 mM phosphate buffer-acetonitrile (40:60, v/v) was eluted isocratically. Between- and within-day coefficients of variation were in the range of 1.9-9.4% and 2.3-13.3%, respectively. The detection limit was 0.2 ng/ml. The method we describe can be easily applied to the measurement of plasma paroxetine concentration for pharmacokinetic studies as well as for therapeutic drug monitoring in patients taking paroxetine.

Genetic variations and haplotypes of UDP-glucuronosyltransferase 1A locus in a Korean population.

Glucuronidation catalyzed by UDP-glucuronosyltransferases (UGTs) is one of the most important mechanisms for host defense against xenobiotics and endobiotics. Although genetic polymorphisms of several UGT1A isoforms have been reported separately, the haplotypes in all functional exons have not been identified, and little information is available regarding single nucleotide polymorphisms in Koreans. We analyzed genetic polymorphisms in all functional exons of the UGT1A locus by direct sequencing of genomic DNA from 50 healthy Korean subjects, and their haplotypes were inferred from genotype data using an expectation-maximization algorithm. We identified 67 polymorphisms, including three novel single nucleotide polymorphisms, 233C>T in UGT1A1 (T78M), 292C>T in 1A4 (Q98Stop), and 701T>C in 1A7 (I234T). Two amino acid substitutions, 1A4 Q98Stop and 1A7 I234T, were each associated with a decrease of enzymatic activity, whereas UGT1A1 T78M had no significant influence on catalytic function. The frequencies of the known variants in Koreans differed significantly from those reported in other ethnic groups. Haplotype analysis was performed within the polymorphisms in each UGT1A isoform as well as across the isoforms. Based on strong linkage disequilibrium within UGT1A7, between 1A5 and 1A4, and within 1A3, the complex was divided into three blocks, Block 7, Block 5/4, and Block 3. The haplotypes for each block were subsequently determined, revealing a profile that differed from those of other ethnic groups. These results suggest that genetic polymorphisms of the UGT1A locus differ between Koreans and other ethnic populations. Such differences should be considered in pharmacogenetic studies.

Effect of rifampin on the plasma concentration and the clinical effect of haloperidol concomitantly administered to schizophrenic patients.

We assessed the changes of plasma haloperidol concentrations and clinical responses repeatedly up to 4 weeks after coadministration or discontinuation of rifampin in 12 schizophrenic patients taking haloperidol alone (group I) and 5 patients taking haloperidol and antituberculotic drugs (group II). After coadministration of rifampin in group I, daily trough haloperidol concentrations rapidly decreased and reached 63% of baseline level by day 3, 41.3% by day 7, and 30% by day 28. On the other hand, after discontinuation of rifampin in group II, plasma haloperidol concentration increased to 140.7% of baseline level by day 3, 228.7% by day 7, and 329% by day 28. In this study, a 30% or greater change in the clinical rating scale was considered a positive clinical response of the drug interaction. Using this criterion, 50% of the group I subjects responded according to the Brief Psychiatric Rating Scale (BPRS) total score, and 25% responded according to the BPRS subscale for psychiatric symptoms. No positive responses were observed in group II patients. These results strongly suggest that rifampin interacts with the clinical effects as well as the plasma concentrations of coadministered haloperidol, and careful monitoring should be considered when coadministration or discontinuation of rifampin is needed in a schizophrenic patient taking haloperidol.

Characterization of Benidipine and Its Enantiomers' Metabolism by Human Liver Cytochrome P450 Enzymes

Benidipine is a dihydropyridine calcium antagonist that has been used clinically as an antihypertensive and antianginal agent. It is used clinically as a racemate, containing the (-)-α and (+)-α isomers of benidipine. This study was performed to elucidate the metabolism of benidipine and its enantiomers in human liver microsomes (HLMs) and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of benidipine. Human liver microsomal incubation of benidipine in the presence of NADPH resulted in the formation of two metabolites, N-desbenzylbenidipine and dehydrobenidipine. The intrinsic clearance (CLint) of the formation of N-desbenzylbenidipine and dehydrobenidipine metabolites from (-)-α isomer was similar to those from the (+)-α isomer (1.9 ± 0.1 versus 2.3 ± 2.3 μl/min/pmol P450 and 0.5 ± 0.2 versus 0.6 ± 0.6 μl/min/pmol P450, respectively). Correlation analysis between the known P450 enzyme activities and the rate of the formation of benidipine metabolites in the 15 HLMs showed that benidipine metabolism is correlated with CYP3A activity. The P450 isoform-selective inhibition study in liver microsomes and the incubation study of cDNA-expressed enzymes also showed that theN-debenzylation and dehydrogenation of benidipine are mainly mediated by CYP3A4 and CYP3A5. The total CLint values of CYP3A4-mediated metabolite formation from (-)-α isomer were similar to those from (+)-α isomer (17.7 versus 14.4 μl/min/pmol P450, respectively). The total CLint values of CYP3A5-mediated metabolite formation from (-)-α isomer were also similar to those from (+)-α isomer (8.3 versus 11.0 μl/min/pmol P450, respectively). These findings suggest that CYP3A4 and CYP3A5 isoforms are major enzymes contributing to the disposition of benidipine, but stereoselective disposition of benidipine in vivo may be influenced not by stereoselective metabolism but by other factors.