Ji-Hong Shon

Genetic variants of the organic cation transporter 2 influence the disposition of metformin.

Genetic variants of the organic cation transporter 2 (protein, OCT2; gene, SLC22A2) were evaluated for their contribution to the variations in the pharmacokinetics of metformin, especially to its renal elimination. Genetic variants of SLC22A2 (c.596C>T, c.602C>T, and c.808G>T) showed significant differences in metformin pharmacokinetics when compared with the reference genotype, with higher peak plasma concentration (Cmax) and area under the curve (AUC) and lower renal clearance (Clrenal), thereby suggesting that a decrease in transport function associated with the SLC22A2 variants results in reduced Clrenal of metformin and consequently leads to increased plasma concentrations.

The effect of SLCO1B1*15 on the disposition of pravastatin and pitavastatin is substrate dependent: the contribution of transporting activity changes by SLCO1B1*15.

Objective This study was addressed to understand the underlying mechanism of the substrate-dependent effect of genetic variation in SLCO1B1, which encodes OATP1B1 (organic anion transporting polypeptide) transporter, on the disposition of two OATP1B1 substrates, pravastatin and pitavastatin, in relation to their transport activities. Methods The uptake of pravastatin, pitavastatin, and fluvastatin was measured in oocytes overexpressing SLCO1B1*1a and SLCO1B1*15 to compare the alterations of in-vitro transporting activity. After 40-mg pravastatin or 4-mg pitavastatin was administered to 11 healthy volunteers with homozygous genotypes of SLCO1B1*1a/*1a and SLCO1B1*15/*15, the pharmacokinetic parameters of pravastatin and pitavastatin were compared among participants with SLCO1B1*1a/*1a and SLCO1B1*15/*15 genotypes. Results The uptake of pravastatin and pitavastatin in SLCO1B1*15 overexpressing oocytes was decreased compared with that in SLCO1B1*15, but no change occurred with fluvastatin. The fold change of in-vitro intrinsic clearance (Clint) for pitavastatin in SLCO1B1*15 compared with SLCO1B1*1a was larger than that of pravastatin (P<0.0001). The clearance (Cl/F) of pitavastatin was decreased to a greater degree in participant with SLCO1B1*15/*15 compared with that of pravastatin in vivo (P<0.01), consistent with in-vitro study. As a result, Cmax and area under the plasma concentration–time curve of these nonmetabolized substrates were increased by SLCO1B1*15 variant. The greater decrease in the transport activity for pitavastatin in SLCO1B1*15 variant compared with SLCO1B1*1a was, however, associated with the greater effect on the pharmacokinetics of pitavastatin compared with pravastatin in relation to the SLCO1B1 genetic polymorphism. Conclusion This study suggests that substrate dependency in the consequences of the SLCO1B1*15 variant could modulate the effect of SLCO1B1 polymorphism on the disposition of pitavastatin and pravastatin.

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.

Effect of itraconazole on the pharmacokinetics and pharmacodynamics of fexofenadine in relation to the MDR1 genetic polymorphism

Our objective was to evaluate the effect of itraconazole, a P‐glycoprotein inhibitor, on the pharmacokinetics and pharmacodynamics of fexofenadine, a P‐glycoprotein substrate, in relation to the multidrug resistance 1 gene (MDR1) G2677T/C3435T haplotype.

Characterization of Ebastine, Hydroxyebastine, and Carebastine Metabolism by Human Liver Microsomes and Expressed Cytochrome P450 Enzymes: Major Roles for CYP2J2 and CYP3A

Ebastine undergoes extensive metabolism to form desalkylebastine and hydroxyebastine. Hydroxyebastine is subsequently metabolized to carebastine. Although CYP3A4 and CYP2J2 have been implicated in ebastine N-dealkylation and hydroxylation, the enzyme catalyzing the subsequent metabolic steps (conversion of hydroxyebastine to desalkylebastine and carebastine) have not been identified. Therefore, we used human liver microsomes (HLMs) and expressed cytochromes P450 (P450s) to characterize the metabolism of ebastine and that of its metabolites, hydroxyebastine and carebastine. In HLMs, ebastine was metabolized to desalkyl-, hydroxy-, and carebastine; hydroxyebastine to desalkyl- and carebastine; and carebastine to desalkylebastine. Of the 11 cDNA-expressed P450s, CYP3A4 was the main enzyme catalyzing the N-dealkylation of ebastine, hydroxyebastine, and carebastine to desalkylebastine [intrinsic clearance (CLint) = 0.44, 1.05, and 0.16 μl/min/pmol P450, respectively]. Ebastine and hydroxyebastine were also dealkylated to desalkylebastine to some extent by CYP3A5. Ebastine hydroxylation to hydroxyebastine is mainly mediated by CYP2J2 (0.45 μl/min/pmol P450; 22.5- and 7.5-fold higher than that for CYP3A4 and CYP3A5, respectively), whereas CYP2J2 and CYP3A4 contributed to the formation of carebastine from hydroxyebastine. These findings were supported by chemical inhibition and kinetic analysis studies in human liver microsomes. The CLint of hydroxyebastine was much higher than that of ebastine and carebastine, and carebastine was metabolically more stable than ebastine and hydroxyebastine. In conclusion, our data for the first time, to our knowledge, suggest that both CYP2J2 and CYP3A play important roles in ebastine sequential metabolism: dealkylation of ebastine and its metabolites is mainly catalyzed by CYP3A4, whereas the hydroxylation reactions are preferentially catalyzed by CYP2J2. The present data will be very useful to understand the pharmacokinetics and drug interaction of ebastine in vivo.

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.

Pharmacokinetic and Pharmacodynamic Evaluation of a Novel Proton Pump Inhibitor, YH1885, in Healthy Volunteers

To evaluate the pharmacokinetic and pharmacodynamic characteristics of YH1885, a novel proton pump inhibitor, a single‐blind, randomized, placebo‐controlled, dose‐rising, parallel‐group study was conducted in 46 healthy volunteers. The volunteers were randomly allocated to single dose groups of 60 mg, 100 mg, 150 mg, 200 mg, and 300 mg (6 subjects per dose, including 2 placebos) or to multiple‐dose groups of 150 mg and 300 mg (once‐daily dosing for 7 days; 8 subjects per dose, including 2 placebos). The multiple‐dose study was conducted separately after the single‐dose study. YH1885 was administered orally after overnight fasting. Serial blood samples, urine samples, and pharmacodynamic measurements were taken. Drug concentrations in plasma and urine were determined by liquid chromatography/mass spectrometry (LC/MS). Pharmacodynamic changes were evaluated by ambulatory intragastric pH monitoring and by serial measurements of serum gastrin concentrations. Assessments of safety and tolerability also were made. Plasma concentrations of YH1885 reached peak levels 1.3 to 2.5 hours after single‐dose administration and then declined monoexponentially with a terminal half‐life (t1/2) of 2.2 to 2.4 hours in dosage groups up to 200 mg in the single‐dose study. YH1885 showed linear pharmacokinetic characteristics, and little accumulation occurred after multiple administrations. The parent drug was not detected in urine. Dose‐related pharmacological effects were obvious for dose groups of 150 mg and higher in the single‐dose study. The mean intragastric pH and the percentage of time at pH > 4 were significantly increased. The onset of drug effect was rapid, and maximal effects were observed on the first day of administration during multiple dosing. Serum gastrin levels also showed rapid increases during dosing but with a weak dose‐effect relationship. Neither serious nor dose‐limiting adverse effects were observed. YH1885 was found to be safe and well tolerated and effectively inhibited acid secretion with dose‐dependent increases in intragastric pH. The acid‐suppressing efficacy of YH1885 needs to be further evaluated in patients with gastric acid‐related diseases.

LC–MS/MS for the simultaneous analysis of arachidonic acid and 32 related metabolites in human plasma: Basal plasma concentrations and aspirin-induced changes of eicosanoids

Eicosanoids play an important role in various biological responses and can be used as biomarkers for specific diseases. Therefore, we developed a highly selective, sensitive, and robust liquid chromatography-tandem mass spectrometric method to measure arachidonic acid and its 32 metabolites in human plasma. Sample preparation involved solid phase extraction, which efficiently removed sources of interference present in human plasma. Chromatographic separation was performed using a Luna C(8)-column with 0.5mM ammonium formate buffer and acetonitrile as the mobile phase under gradient conditions. Detection was performed using tandem mass spectrometry equipped with an electrospray ionization interface in negative ion mode. The matrix did not affect the reproducibility and reliability of the assay. All analytes showed good linearity over the investigated concentration range (r>0.997). The validated lower limit of quantitation for the analytes ranged from 10 to 400pg/mL. Intra- and inter-day precision (RDS%) over the concentration ranges for all eicosanoids were within 16.8%, and accuracy ranged between 88.1 and 108.2%. This assay was suitable for the determination of basal plasma levels of eicosanoids and the evaluation of effect of aspirin on eicosanoid plasma levels in healthy subjects.