The incidence of polypharmacy-which can result in drug-drug interactions-has increased in recent years. Drug-metabolizing enzymes and drug transporters are important polypharmacy modulators. In this study, the effects of bosentan and rifampin on the expression and activities of organic anion-transporting peptide (OATP) and cytochrome P450 (CYP450) 2C9 and CYP3A4 were investigated in vitro. HEK293 cells and primary human hepatocytes overexpressing the target genes were treated with bosentan and various concentrations of rifampin, which decreased the uptake activities of OATP transporters in a dose-dependent manner. In primary human hepatocytes, CYP2C9 and CYP3A4 gene expression and activities decreased upon treatment with 20 μM bosentan+200 μM rifampin. Rifampin also reduced gene expression of OATP1B1, OATP1B3, and OATP2B1 transporter, and inhibited bosentan influx in human hepatocytes at increasing concentrations. These results confirm rifampin- and bosentan-induced interactions between OATP transporters and CYP450.
Cytochrome P-450 CYP2C9 ; Cytochrome P-450 CYP3A ; Cytochrome P-450 Enzyme System* ; Cytochromes* ; Gene Expression ; HEK293 Cells ; Hepatocytes* ; Humans* ; In Vitro Techniques ; Incidence ; Organic Anion Transporters ; Polypharmacy ; Rifampin*

<p>AIMTo identify the drug-metabolizing enzymes involved in the hydroxylation of the new anti-inflammatory and anodyne imrecoxib.p><p>METHODSImrecoxib was incubated with heterologous expression human cytochrome P450 (rCYPs) in vitro, and metabolites and remained parent drug were detected with liquid chromatography-multistage mass spectrometry. The contribution of 4 CYPs in the hydroxylation metabolism of imrecoxib was evaluated by total normalized rate (TNR) method.p><p>RESULTSImrecoxib is metabolized by CYP2C9, CYP2D6 and CYP3A4, with the rate of 62.5%, 21.1% and 16.4%, respectively.p><p>CONCLUSIONCYP2C9 is the major enzyme involved in imrecoxib hydroxylation metabolism.p>
Aryl Hydrocarbon Hydroxylases ; metabolism ; Cyclooxygenase 2 Inhibitors ; metabolism ; Cytochrome P-450 CYP2C9 ; Cytochrome P-450 CYP2D6 ; metabolism ; Cytochrome P-450 CYP3A ; Cytochrome P-450 Enzyme System ; metabolism ; Hydroxylation ; Pyrroles ; metabolism ; Spectrometry, Mass, Electrospray Ionization ; Sulfides ; metabolism

Rotundine (1 micromol L(-1)) was incubated with a panel of rCYP enzymes (1A2, 2C9, 2C19, 2D6 and 3A4) in vitro. The remained parent drug in incubates was quantitatively analyzed by an Agilent LC-MS. CYP2C19, 3A4 and 2D6 were identified to be the isoenzymes involved in the metabolism of rotundine. The individual contributions of CYP2C19, 3A4 and 2D6 to the rotundine metabolism were assessed using the method of total normalized rate to be 31.46%, 60.37% and 8.17%, respectively. The metabolites of rotundine in incubates were screened with ESI-MS at selected ion mode, and were further identified using MS2 spectra and precise molecular mass obtained from an Agilent LC/Q-TOF-MSMS, as well as MS(n) spectra of LC-iTrap-MS(n). The predominant metabolic pathway of rotundine in rCYP incubates was O-demethylation. A total 5 metabolites were identified including 4 isomerides of mono demethylated rotundine and one di-demethylated metabolite. The results also showed that CYP2C19, 2D6 and 3A4 mediated O-demethylation of methoxyl groups at different positions of rotundine. Furthermore, the ESI-MS cleavage patterns of rotundine and its metabolites were explored by using LC/Q-TOF-MSMS and LC/iTrap-MS(n) techniques.
Analgesics, Non-Narcotic ; metabolism ; Aryl Hydrocarbon Hydroxylases ; metabolism ; Berberine Alkaloids ; metabolism ; Chromatography, Liquid ; Cytochrome P-450 CYP1A2 ; metabolism ; Cytochrome P-450 CYP2C19 ; Cytochrome P-450 CYP2C9 ; Cytochrome P-450 CYP2D6 ; metabolism ; Cytochrome P-450 CYP3A ; metabolism ; Cytochrome P-450 Enzyme System ; metabolism ; Dopamine Antagonists ; metabolism ; Humans ; Isoenzymes ; metabolism ; Methylation ; Recombinant Proteins ; metabolism ; Spectrometry, Mass, Electrospray Ionization

Pharmacogenetics is a rapidly evolving field and the number of pharmacogenetic tests for clinical use is steadily increasing. However, incorrect or inadequate implementation of pharmacogenetic tests in clinical practice may result in a rise in medical costs and adverse outcomes in patients. This document suggests guidelines for the clinical application, interpretation, and reporting of pharmacogenetic test results based on a literature review and the collection of evidence-based expert opinions. The clinical laboratory practice guidelines encompass the clinical pharmacogenetic tests covered by public medical insurance in Korea. Technical, ethical, and regulatory issues related to clinical pharmacogenetic tests have also been addressed. In particular, this document comprises the following pharmacogenetic tests: CYP2C9 and VKORC1 for warfarin, CYP2C19 for clopidogrel, CYP2D6 for tricyclic antidepressants, codeine, tamoxifen, and atomoxetine, NAT2 for isoniazid, UGT1A1 for irinotecan, TPMT for thiopurines, EGFR for tyrosine kinase inhibitors, ERBB2 (HER2) for erb-b2 receptor tyrosine kinase 2-targeted therapy, and KRAS for anti-epidermal growth factor receptor drugs. These guidelines would help improve the usefulness of pharmacogenetic tests in routine clinical settings.
Antidepressive Agents, Tricyclic ; Atomoxetine Hydrochloride ; Clinical Laboratory Services ; Codeine ; Cytochrome P-450 CYP2C19 ; Cytochrome P-450 CYP2C9 ; Cytochrome P-450 CYP2D6 ; Expert Testimony ; Genetic Testing ; Humans ; Insurance ; Isoniazid ; Korea ; Pharmacogenetics ; Protein-Tyrosine Kinases ; Tamoxifen ; Warfarin

The metabolic characteristics of ligustrazin (TMPz) in liver microsomes were investigated in the present study. The reaction phenotyping of TMPz metabolism was also identified by in vitro assessment using recombinant human cytochrome P450 enzymes (CYP) and UDP glucuronosyltransferases (UGT). TMPz was incubated at 37 degrees C with human (HLM) and rat liver microsomes (RLM) in the presence of different co-factors. The metabolic stability and enzyme kinetics of TMPz were studied by determining its remaining concentrations with a LC-MS/MS method. TMPz was only metabolically eliminated in the microsomes with NADPH or NADPH+UDPGA. In the HLM and RLM with NADPH+UDPGA, t1/2, K(m) and V(max) of TMPz were 94.24 +/- 4.53 and 105.07 +/- 9.44 min, 22.74 +/- 1.89 and 33.09 +/- 2.74 micromol x L(-1), 253.50 +/- 10.06 and 190.40 +/- 8.35 nmol x min(-1) x mg(-1) (protein), respectively. TMPz showed a slightly higher metabolic rate in HLM than that in RLM. Its primary oxidative metabolites, 2-hydroxymethyl-3, 5, 6-trimethylpyrazine (HTMP), could undergo glucuronide conjugation. The CYP reaction phenotyping of TMPz metabolism was identified using a panel of recombinant CYP isoforms (rCYP) and specific CYP inhibitors in HLM. CYP1A2, 2C9 and 3A4 were found to be the major CYP isoforms involved in TMPz metabolism. Their individual contributions were assessed b) using the method of the total normalized rate to be 19.32%, 27.79% and 52.90%, respectively. It was observed that these CYP isoforms mediated the formation of HTMP in rCYP incubation. The UGT reaction phenotyping of HTMP glucuronidation was also investigated preliminarily by using a panel of 6 UGT isoforms (rUGT). UGT1A1, 1A4 and 1A6 were the predominant isoforms mediated the HTMP glucuronidation. The results above indicate that the metabolism of TMPz involves multiple enzymes mediated phase I and phase II reactions.
Animals ; Cytochrome P-450 CYP1A2 ; metabolism ; Cytochrome P-450 CYP2C9 ; metabolism ; Cytochrome P-450 CYP3A ; metabolism ; Cytochrome P-450 Enzyme Inhibitors ; Cytochrome P-450 Enzyme System ; metabolism ; Drug Interactions ; Glucuronosyltransferase ; metabolism ; Humans ; Ligusticum ; chemistry ; Microsomes, Liver ; enzymology ; NADP ; metabolism ; pharmacology ; Pyrazines ; metabolism ; pharmacokinetics ; Rats ; Uridine Diphosphate Glucuronic Acid ; metabolism ; pharmacology

OBJECTIVE To assess the influence of genetic polymorphisms and non-genetic factors on warfarin maintenance dose variations in order to provide guidance for personalized use of warfarin. METHODS Two hundred patients from outpatient and inpatient with stable international normalized ratio(INR) were recruited. Clinical data and blood samples were collected. Genotypes of 4 genes involved in warfarin metabolic pathways were determined with Sanger sequencing. Based on statistical analysis of warfarin maintenance dosage, a mathematical model was established. RESULTS Among non-genetic factors, the age and height have significant influence in warfarin dosage. The dosage is negatively correlated with age but positively correlated with height. The difference in dosage for between the 20-year-old group and 60-year-old group has reached 1.81 mg/day, and that for between the 140 cm in height and 180 cm in height groups has reached 1.06 mg/day. VKORC1 -1639G/A, CYP2C9 430C/T, CYP2C9 1075A/C and CYP4F2 V433M polymorphisms have significant influence on stable warfarin dosage. The dosage for patients with wild type and mutant genotypes has varied from 0.35 mg/day to 0.84 mg/day. CONCLUSION Non-genetic factors and genetic polymorphisms play important roles in personalized variations of warfarin maintenance dose. The establishment of mathematical models considering multiple factors is helpful in evaluating the safety and effectiveness of warfarin dosage.
Adult ; Aged ; Anticoagulants ; administration & dosage ; Cytochrome P-450 CYP2C9 ; genetics ; Cytochrome P-450 Enzyme System ; genetics ; Cytochrome P450 Family 4 ; Female ; Humans ; Male ; Middle Aged ; Polymorphism, Genetic ; Vitamin K Epoxide Reductases ; genetics ; Warfarin ; administration & dosage

Licorice root has been frequently used as antitode in traditional Chinese medicine. As the main active component of Licorice root, glycyrrhetic acid (GA) is mainly metabolized in liver. This study was designed to investigate the in vitro metabolism of GA by human liver microsomes (HLM) and human recombinant cytochrome P450 (CYP) isoforms. The results indicated that GA was metabolized mainly by CYP3A4. The K(m), V(max) and CL(int) of GA in HLM were 18.6 micromol x L(-1), 4.4 nmol x mg(-1) (protein) x min(-1) and 0.237 mL x mg(-1) (protein) x min(-1), respectively. At concentration up to 50 micromol x L(-1), GA inhibited CYP2C19, CYP2C9 and CYP3A4 enzyme activities with the inhibitory potencies up to 50%.
Aryl Hydrocarbon Hydroxylases ; antagonists & inhibitors ; metabolism ; Cytochrome P-450 CYP2C19 ; Cytochrome P-450 CYP2C9 ; Cytochrome P-450 CYP3A ; metabolism ; Cytochrome P-450 CYP3A Inhibitors ; Enzyme Inhibitors ; pharmacology ; Glycyrrhetinic Acid ; isolation & purification ; pharmacokinetics ; pharmacology ; Glycyrrhiza ; chemistry ; Humans ; Isoenzymes ; metabolism ; Microsomes, Liver ; enzymology ; metabolism ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry

<p>AIMTo investigate the influence of cytochrom P450 CYP2C9 polymorphism on the pharmacokinetics of tolbutamide.p><p>METHODSAn oligonucleotide microarray was designed and fabricated to genotype the CYP2C9 accurately and quickly. 137 healthy volunteers were genotyped with the array to investigate the frequency of CYP2C9 functional SNPs. Moreover, 1 homozygous mutant, 9 heterozygous and 10 wild-genotypes subjects in the assay were selected randomly and sequenced directly. After orally taking tolbutamide, blood samples and urine samples were collected, and their pharmacokinetics was studied with HPLC.p><p>RESULTSCYP2C9 *1/*3 were found in 9 of 137 volunteers, CYP2C9 *3/*3 in only one, others were all CYP2C9 *1/*1 wild types. CYP2C9 *2, CYP2C9 *4 and CYP2C9 *5 alleles were not detected. Direct sequencing of the purified PCR products of the heterozygotes, mutant homozygotes and ten wild type individuals gave a corresponding result to that genotyped by microarray. Pharmacokinetic outcome showed that the individuals with CYP2C9 *1/*3 or CYP2C9 *3/*3 had slower metabolic elimination of tolbutamide than those with CYP2C9 *1/*1.p><p>CONCLUSIONCYP2C9 genetic polymorphism has a significant influence on the pharmacokinetics of tolbutamide. Pharmacogenomic study will be helpful in guiding rational and individualized medication. Key words: tolbutamide; cytochrom P450 CYP2C9; allele; single nucleotide polymorphism; genotypingp>
Aryl Hydrocarbon Hydroxylases ; genetics ; Cytochrome P-450 CYP2C9 ; Genotype ; Heterozygote ; Homozygote ; Humans ; Oligonucleotide Array Sequence Analysis ; Polymorphism, Single Nucleotide ; Random Allocation ; Tolbutamide ; pharmacokinetics

The study aimed to establish a population pharmacokinetic/pharmacodynamic (PPK/PD) model of warfarin. PCR-RFLP technique was used to genotype the CYP2C9 and VKORC1 polymorphisms of 73 patients. RP-HPLC-UV method was used to determine the 190 plasma concentrations of warfarin. Application of NONMEM, the clinical information and 263 international normalized ratio (INR) monitoring data were used to investigate the effect of genetic, physiological, pathological factors, other medication on clearance and anticoagulant response. The final model of warfarin PPK/PD was described as follows: CL = θCL · (WT/60)θWT · θCYP · eηCL (if CYP2C9*1/*1, θCYP = 1; if *1/*3, θCYP = 0.708); EC50 = θEC50 · θVKOR · eηEC50 (if VKORC1- 1639AA, θVKOR = 1; if GA, θVKOR = 2.01; V = θV; K(E0) = θK(E0); Emax = θEmax; E0 = θE0 · eηE0. Among them, the body weight (WT), CYP2C9 and VKORC1 genotype had conspicuous effect on warfarin PK/PD parameters. The goodness diagnosis, Bootstrap, NPDE verification showed that the final model was stable, effective and predictable. It may provide a reference for opitimizing the dose regimen of warfarin.
Anticoagulants ; pharmacology ; Body Weight ; Cytochrome P-450 CYP2C9 ; genetics ; Genotype ; Humans ; International Normalized Ratio ; Nonlinear Dynamics ; Polymorphism, Genetic ; Vitamin K Epoxide Reductases ; genetics ; Warfarin ; pharmacokinetics

Humans ; Warfarin/therapeutic use* ; Cytochrome P-450 CYP2C9/genetics* ; Anticoagulants/therapeutic use* ; Genotype ; Heart Valves ; China ; Apolipoproteins E/genetics* ; Dose-Response Relationship, Drug ; Receptors, Calcitriol/genetics*