Hyunmi Kim

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.

Identification of New CYP2C19 Variants Exhibiting Decreased Enzyme Activity in the Metabolism of S-Mephenytoin and Omeprazole

Although many cases of interindividual variation in the metabolism of CYP2C19 drugs are explained by the CYP2C19*2, *3, and *17, a wide range of metabolic variation still occurs in people who do not carry these genetic variants. The objectives of this study were to identify new genetic variants and to characterize functional consequences of these variants in metabolism of CYP2C19 substrates. In total, 21 single-nucleotide polymorphisms including three new coding variants, V394M, E405K, and D256N, were identified by direct DNA sequencing in 50 randomly selected subjects and in individuals who exhibited an outlier phenotype response in the omeprazole study. Recombinant proteins produced from the coding variants V394M, E405K, and D256N were prepared by using an Escherichia coli expression system and purified. Metabolism of S-mephenytoin and omeprazole by V394M was comparable with that of the wild-type protein. E405K showed a moderate decrease in metabolism of the substrates. However, D256N exhibited a significantly decreased activity in S-mephenytoin metabolism, resulting in 50 and 76% decreases in Vmax and intrinsic clearance, respectively, compared with the wild type. This variant also exhibited a significant decrease in omeprazole metabolism in vivo. CYP2C19 D256N and E405K were assigned as CYP2C19*26 and *2D, respectively, by the Cytochrome P450 Nomenclature Committee. In summary, this report characterizes the allele frequency and haplotype distribution of CYP2C19 in a Korean population and provides functional analysis of new coding variants of the CYP2C19 gene. Our findings suggest that individuals carrying CYP2C19*26 would have lower activity for metabolizing CYP2C19 substrate drugs.

Cytochrome P450 2B6 Catalyzes the Formation of Pharmacologically Active Sibutramine (N-{1-[1-(4-chlorophenyl)cyclobutyl]-3-methylbutyl}-N,N-dimethylamine) Metabolites in Human Liver Microsomes

We identified cytochrome P450 (P450) isozymes that are involved in the formation of two active sibutramine (N-{1-[1-(4-chlorophenyl)-cyclobutyl]-3-methylbutyl}-N,N-dimethylamine) metabolites, M1 (N-{1-[1-(4-chlorophenyl)cyclobutyl]-3-methylbutyl}-N-methylamine) and M2 (1-[1-(4-chlorophenyl)cyclobutyl]-3-methylbutylamine), in humans using a combination chemical inhibition, correlation analyses in human liver microsomes (HLMs), and activity assays using recombinant P450s. Mechanism-based CYP2B6 inhibitors (i.e., clopidogrel, ticlopidine, and triethylenethiophoramide) significantly inhibited the formation of M1 from sibutramine and M2 from M1, respectively; in contrast, no effect was observed when using potent inhibitors of eight P450 isozymes (CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A). In addition, the formations of M1 from sibutramine (r = 0.694, p = 0.0029) and M2 from M1 (r = 0.834, p < 0.0001) were strongly correlated with CYP2B6-catalyzed bupropion hydroxylation in 16 different HLM panels. Furthermore, recombinant CYP2B6 catalyzed M1 and/or M2 formation at the highest rate among 10 P450s. Although recombinant CYP2C19, 3A4, and 3A5 also catalyzed, to a less extent, M1 formation at high substrate concentrations (>5 μM), those contributions might be minor considering usual concentrations of sibutramine and M1 in the clinical setting. The kinetics of M1 and/or M2 formation from sibutramine in HLMs were fitted by a two-enzyme model, and the mean apparent Km value (4.79 μM) for high-affinity component was similar to that observed in recombinant CYP2B6 (8.02 μM). In conclusion, CYP2B6 is the primary catalyst for the formation of sibutramine two active metabolites, which may suggest that pharmacogenetics and drug interactions of sibutramine in relation to CYP2B6 activity should be considered in the pharmacotherapy of sibutramine.Department of Clinical Pharmacology and Clinical Trial Center, Inje University Busan Paik Hospital, Busan [S.K.B., J.-H.S., J.-G.S.]; Department of Pharmacology and PharmacoGenomics Research Center, Inje University College of Medicine, Busan, [S.C., K.-A.S., H. K., M.-J.K., J.-H.S., K.-H.L., J.-G.S. ], South Korea Pharmacogenetics Research Institute, Xiang-Ya School of Medicine, Central South University, ChangSha, Hunan, [S.C., H.-H.Z.], China DMD Fast Forward. Published on May 12, 2008 as doi:10.1124/dmd.108.020727

The Contributions of Cytochromes P450 3A4 and 3A5 to the Metabolism of the Phosphodiesterase Type 5 Inhibitors Sildenafil, Udenafil, and Vardenafil

The role of the genetically polymorphic CYP3A5 in the metabolism of CYP3A substrates is unclear. We investigated the contributions of the CYP3A4 and CYP3A5 isoforms to the metabolism of the phosphodiesterase type 5 inhibitors (PDE5Is) sildenafil, udenafil, and vardenafil. In vitro incubation studies of sildenafil N-demethylation, udenafil N-dealkylation, and vardenafil N-deethylation were conducted using recombinant CYP3A enzymes and 15 human liver microsome (HLM) preparations with predetermined CYP3A5 genotypes. Recombinant CYP3A4 and CYP3A5 both produced N-desalkyl metabolites of sildenafil, udenafil, and vardenafil. The catalytic efficiency (Clint = Vmax/apparent Km) of the rCYP3A5 isoform for vardenafil N-deethylation was about 3.2-fold that of rCYP3A4, whereas the intrinsic clearance rates for N-dealkylation of both sildenafil and udenafil were similar between rCYP3A5 and rCYP3A4. The metabolite formation activity was higher in HLMs heterozygous for the CYP3A5*3 allele (n = 9) than in HLMs homozygous for CYP3A5*3 (n = 6). These findings suggest that CYP3A5 and CYP3A4 play a significant role in the metabolism of PDE5Is. The genetic polymorphism of CYP3A5 may contribute to interindividual variability in the disposition of PDE5Is, especially vardenafil. Further in vivo studies are needed to confirm the effects of CYP3A5 genotypes on the pharmacokinetics of PDE5Is.

Simple and accurate quantitative analysis of 20 anti-tuberculosis drugs in human plasma using liquid chromatography-electrospray ionization-tandem mass spectrometry.

A simple and accurate liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method for the quantitation of 20 anti-tuberculosis (anti-TB) drugs in human plasma, was developed as a tool for therapeutic drug monitoring. Two protein precipitation methods were adopted; one using methanol containing 0.13N HCl, for precipitation of amikacin, kanamycin, streptomycin and pyrazinamide, and the other using acetonitrile, for precipitation of preamoxicillin, ciprofloxacin, clarithromycin, clofazimine, cycloserine, ethambutol, ethionamide, isoniazid, levofloxacin, linezolid, moxifloxacin, p-aminosalicylic acid (PAS), prothionamide, rifabutin, rifampin and roxithromycin. Separation was performed either on an HILIC silica column or a reversed-phase dC18 column, with a gradient elution. Detection was carried out in multiple reaction-monitoring (MRM) mode. The calibration curves were linear over a 50-fold concentration range, with correlation coefficients (r) greater than 0.9969 for all anti-TB drugs. The intra- and inter-day precision was less than 14.3%, and the accuracy ranged between 84.8 and 113.0%. The developed method was successfully applied to the identification and quantitation of anti-TB drugs in patients with multi-drug resistant TB.

Pharmacogenetics meets metabolomics: Discovery of tryptophan as a new endogenous OCT2 substrate related to metformin disposition

Genetic polymorphisms of the organic cation transporter 2 (OCT2), encoded by SLC22A2, have been investigated in association with metformin disposition. A functional decrease in transport function has been shown to be associated with the OCT2 variants. Using metabolomics, our study aims at a comprehensive monitoring of primary metabolite changes in order to understand biochemical alteration associated with OCT2 polymorphisms and discovery of potential endogenous metabolites related to the genetic variation of OCT2. Using GC-TOF MS based metabolite profiling, clear clustering of samples was observed in Partial Least Square Discriminant Analysis, showing that metabolic profiles were linked to the genetic variants of OCT2. Tryptophan and uridine presented the most significant alteration in SLC22A2-808TT homozygous and the SLC22A2-808G>T heterozygous variants relative to the reference. Particularly tryptophan showed gene-dose effects of transporter activity according to OCT2 genotypes and the greatest linear association with the pharmacokinetic parameters (Clrenal, Clsec, Cl/F/kg, and Vd/F/kg) of metformin. An inhibition assay demonstrated the inhibitory effect of tryptophan on the uptake of 1-methyl-4-phenyl pyrinidium in a concentration dependent manner and subsequent uptake experiment revealed differential tryptophan-uptake rate in the oocytes expressing OCT2 reference and variant (808G>T). Our results collectively indicate tryptophan can serve as one of the endogenous substrate for the OCT2 as well as a biomarker candidate indicating the variability of the transport activity of OCT2.

Cytochrome P450 2B6 (CYP2B6) Catalyzes the Formation of Pharmacologically Active Sibutramine Metabolites in Human Liver Microsomes

We identified cytochrome P450 (P450) isozymes that are involved in the formation of two active sibutramine (N-{1-[1-(4-chlorophenyl)-cyclobutyl]-3-methylbutyl}-N,N-dimethylamine) metabolites, M1 (N-{1-[1-(4-chlorophenyl)cyclobutyl]-3-methylbutyl}-N-methylamine) and M2 (1-[1-(4-chlorophenyl)cyclobutyl]-3-methylbutylamine), in humans using a combination chemical inhibition, correlation analyses in human liver microsomes (HLMs), and activity assays using recombinant P450s. Mechanism-based CYP2B6 inhibitors (i.e., clopidogrel, ticlopidine, and triethylenethiophoramide) significantly inhibited the formation of M1 from sibutramine and M2 from M1, respectively; in contrast, no effect was observed when using potent inhibitors of eight P450 isozymes (CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A). In addition, the formations of M1 from sibutramine (r = 0.694, p = 0.0029) and M2 from M1 (r = 0.834, p < 0.0001) were strongly correlated with CYP2B6-catalyzed bupropion hydroxylation in 16 different HLM panels. Furthermore, recombinant CYP2B6 catalyzed M1 and/or M2 formation at the highest rate among 10 P450s. Although recombinant CYP2C19, 3A4, and 3A5 also catalyzed, to a less extent, M1 formation at high substrate concentrations (>5 μM), those contributions might be minor considering usual concentrations of sibutramine and M1 in the clinical setting. The kinetics of M1 and/or M2 formation from sibutramine in HLMs were fitted by a two-enzyme model, and the mean apparent Km value (4.79 μM) for high-affinity component was similar to that observed in recombinant CYP2B6 (8.02 μM). In conclusion, CYP2B6 is the primary catalyst for the formation of sibutramine two active metabolites, which may suggest that pharmacogenetics and drug interactions of sibutramine in relation to CYP2B6 activity should be considered in the pharmacotherapy of sibutramine.Department of Clinical Pharmacology and Clinical Trial Center, Inje University Busan Paik Hospital, Busan [S.K.B., J.-H.S., J.-G.S.]; Department of Pharmacology and PharmacoGenomics Research Center, Inje University College of Medicine, Busan, [S.C., K.-A.S., H. K., M.-J.K., J.-H.S., K.-H.L., J.-G.S. ], South Korea Pharmacogenetics Research Institute, Xiang-Ya School of Medicine, Central South University, ChangSha, Hunan, [S.C., H.-H.Z.], China DMD Fast Forward. Published on May 12, 2008 as doi:10.1124/dmd.108.020727

Identification and characterization of potent CYP2B6 inhibitors in Woohwangcheongsimwon suspension, an herbal preparation used in the treatment and prevention of apoplexy in Korea and China

Woohwangcheongsimwon is a traditional medicine for treating hypertension, arteriosclerosis, coma, and stroke in China and Korea. To assess potential interactions of herb and drug metabolism, commercially available Woohwangcheongsimwon suspensions were examined for their potential to inhibit the activity of nine human cytochrome P450 enzymes. The Woohwangcheongsimwon suspensions showed strong inhibition of CYP2B6 activity. To identify individual constituents with inhibitory activity, the suspension was partitioned using hexane, ethyl acetate, and dichloromethane, and each fraction was tested for its inhibitory effect on CYP2B6-catalyzed bupropion hydroxylation. The hexane fraction possessed inhibitory activity, and gas chromatography/mass spectrometry analysis identified borneol and isoborneol as major constituents of the hexane fraction. These two terpenoids moderately inhibited CYP2B6-catalyzed bupropion hydroxylase activity in a competitive manner, with Ki values of 9.5 and 5.9 μM, respectively, as well as efavirenz 8-hydroxylase activity, with Ki values of 22 and 26 μM, respectively. Additionally, reconstituted mixtures of borneol and isoborneol, at the same concentrations as in the Woohwangcheongsimwon suspension, had comparable potency in inhibiting bupropion hydroxylation. These in vitro data indicate that Woohwangcheongsimwon preparations contain constituents that can potently inhibit the activity of CYP2B6 and suggest that these preparations should be examined for potential pharmacokinetic drug interactions in vivo.

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.

The monoterpenoids citral and geraniol are moderate inhibitors of CYP2B6 hydroxylase activity

Monoterpenes are found in the volatile essence of flowers, plants oils, and herbal medicines. Some are commonly used as food additives and fragrance components, and many are found in cosmetics, soaps, cleaning products, disinfectants, preservatives, and medicines. We have recently discovered a moderate inhibitory effect of borneol and isoborneol toward CYP2B6-catalyzed bupropion hydroxylase activity. Based on that result, we expanded our study to evaluate the inhibitory effects of 22 monoterpenoids on CYP2B6 activity in vitro. Among the monoterpenoids screened, borneol, camphor, cineole, isoborneol, menthol, and perillaldehyde showed slight inhibition of CYP2B6-catalyzed bupropion hydroxylation, displaying greater than 50% inhibition at 50muM. Citral and geraniol strongly inhibited CYP2B6 hydroxylase activity in a competitive manner, with K(i) values of 6.8 and 10.3muM, respectively, which are higher than the K(i) (1.8muM) of the well-known CYP2B6-selective inhibitor thio-TEPA. These in vitro data indicate that high amounts of these two monoterpenoids might interact with drugs that are metabolized by CYP2B6. The in vivo pharmacokinetics of these compounds should be examined to determine whether the inhibition of CYP2B6 activity by monoterpenoids has clinical relevance.