Characterization of fimasartan metabolites in human liver microsomes and human plasma.

1. The metabolites of fimasartan (FMS), a new angiotensin II receptor antagonist, were characterized in human liver microsomes (HLM) and human subjects. 2. We developed a method for a simultaneous quantitative and qualitative analysis using predictive multiple reaction monitoring information-dependent acquisition-enhanced product ion scanning. To characterize metabolic reactions, FMS metabolites were analyzed using quadrupole-time of flight mass spectrometer in full-scan mode. 3. The structures of metabolites were confirmed by comparison of chromatographic retention times and mass spectra with those of authentic metabolite standards. 4. In the cofactor-dependent microsomal metabolism study, the half-lives of FMS were 56.7, 247.9 and 53.3 min in the presence of NADPH, UDPGA and NADPH + UDPGA, respectively. 5. The main metabolic routes in HLM were S-oxidation, oxidative desulfuration, n-butyl hydroxylation and N-glucuronidation. 6. In humans orally administered with 120 mg FMS daily for 7 days, the prominent metabolites were FMS S-oxide and FMS N-glucuronide in the 0-8-h pooled plasma sample of each subject. 7. This study characterizes, for the first time, the metabolites of FMS in humans to provide information for its safe use in clinical medicine.

Ilaprazole, a new proton pump inhibitor, is primarily metabolized to ilaprazole sulfone by CYP3A4 and 3A5.

Ilaprazole is a new proton pump inhibitor, designed for treatment of gastric ulcers, and developed by Il-Yang Pharmaceutical Co (Seoul, Korea). It is extensively metabolised to the major metabolite ilaprazole sulfone. In the present study, several in vitro approaches were used to identify the cytochrome P450 (CYP) enzymes responsible for ilaprazole sulfone formation. Concentrations of ilaprazole sulfone were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Incubation of ilaprazole with cDNA-expressed recombinant CYPs indicated that CYP3A was the major enzyme that catalyses ilaprozole to ilaprazole sulfone. This reaction was inhibited significantly by ketoconazole, a CYP3A inhibitor, and azamulin, a mechanism-based inhibitor of CYP3A, while no substantial effect was observed using selective inhibitors for eight other P450s (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1). In addition, the formation of ilaprazole sulfone correlated well with CYP3A-catalysed testosterone 6ß-hydroxylation and midazolam 1'-hydroxylation in 20 different human liver microsome panels. The intrinsic clearance of the formation of ilaprazole sulfone by CYP3A4 was 16-fold higher than that by CYP3A5. Collectively, these results indicate that the formation of the major metabolite of ilaprazole, ilaprazole sulfone, is predominantly catalysed by CYP3A4/5.

In vitro metabolism and transport of the new dipeptidyl peptidase 4 inhibitors, KR66222 and KR66223.

We investigated the in vitro metabolism and transport of KR66222 and KR66223, new inhibitors of dipeptidyl peptidase (DPP) 4, using human liver microsomes (HLMs) and a Caco-2 cell monolayer. Human liver microsomal incubation of KR66222 in the presence of the NADPH-generating system resulted in the formation of two metabolites, identified as S-oxidation (KR68334) and hydrolysis (KR66223) products using liquid chromatography/tandem mass spectrometry. The formation of KR66223 via an esterase and the formation of KR68334 via CYP3A5 and CYP3A4 seem to be major factors in the in vitro metabolism of KR66222 using HLMs. Additionally, KR66222 had a significantly greater basal to apical transport rate (2.5-fold) than apical to basal transport in the Caco-2 cell monolayer, suggesting the involvement of an efflux transport system. Further studies using inhibitors of efflux transporters and P-glycoprotein (P-gp) overexpressed cells revealed that P-gp was involved in the basal to apical transport of KR66222. These findings suggest that KR66222 undergoes a significant first pass effect, which may serve to decrease the bioavailability of KR66222. The active metabolite, KR66223, was stable for 1 h at 37°C in pooled HLMs (98.9 ± 2.6% of control) and did not undergo P-gp-mediated efflux in Caco-2 cells. Apparent permeability of KR66223 (4.96 × 10(-6) cm/s) was comparable to that of KR66222 (4.08 × 10(-6) cm/s). In conclusion, considering pharmacokinetic variability and the intestinal first-pass effect caused by the involvement of CYP3A and P-gp, KR66223 seems to have better in vitro metabolism and permeability characteristics than KR66222.

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 K(i) values of 9.5 and 5.9 microM, respectively, as well as efavirenz 8-hydroxylase activity, with K(i) values of 22 and 26 microM, 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.

Effect of rifampin, an inducer of CYP3A and P-glycoprotein, on the pharmacokinetics of risperidone.

The authors studied the effect of rifampin, a dual inducer of CYP3A and P-glycoprotein, on the pharmacokinetics and pharmacodynamics of risperidone in humans. Ten healthy male subjects were treated daily for 7 days with 600 mg rifampin or with placebo. On day 6, a single dose of 1 mg risperidone was administered. Plasma risperidone and 9-hydroxyrisperidone concentrations were measured. Rifampin significantly decreased the mean area under the plasma concentration-time curve by 51% for risperidone, by 43% for 9-hydroxyrisperidone, and by 45% for the active moieties (risperidone + 9-hydroxyrisperidone). Rifampin also decreased the peak plasma concentration of risperidone by 38%, 9-hydroxyrisperidone by 46%, and the active moieties by 41%. The apparent oral clearance of risperidone approximately doubled after rifampin treatment. Thus, rifampin reduced the exposure to risperidone, probably because of a decrease in its bioavailability through the induction of CYP3A and probably P-glycoprotein.

Rifampin enhances cytochrome P450 (CYP) 2B6-mediated efavirenz 8-hydroxylation in healthy volunteers.

The effect of rifampin on the in vivo metabolism of the antiretroviral drug efavirenz was evaluated in healthy volunteers. In a cross-over placebo control trial, healthy subjects (n = 20) were administered a single 600 mg oral dose of efavirenz after pretreatment with placebo or rifampin (600 mg/day for 10 days). Plasma and urine concentrations of efavirenz, 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz were measured by LC-MS/MS. Compared to placebo treatment, rifampin increased the oral clearance (by ∼2.5-fold) and decreased maximum plasma concentration (Cmax) and area under the plasma concentration-time curve (AUC0-∞) of efavirenz (by ∼1.6- and ∼2.5-fold respectively) (p < 0.001). Rifampin treatment substantially increased the Cmax and AUC0-12h of 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz, metabolic ratio (AUC0-72h of metabolites to AUC0-72h efavirenz) and the amount of metabolites excreted in urine (Ae0-12hr) (all, p < 0.01). Female subjects had longer elimination half-life (1.6-2.2-fold) and larger weight-adjusted distribution volume (1.6-1.9-fold) of efavirenz than male subjects (p < 0.05) in placebo and rifampin treated groups respectively. In conclusion, rifampin enhances CYP2B6-mediated efavirenz 8-hydroxylation in vivo. The metabolism of a single oral dose of efavirenz may be a suitable in vivo marker of CYP2B6 activity to evaluate induction drug interactions involving this enzyme.

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.

Simple and accurate quantitative analysis of ten antiepileptic drugs in human plasma by liquid chromatography/tandem mass spectrometry.

A simple, accurate, and sensitive liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method has been developed for the simultaneous quantification of 10 antiepileptic drugs (AEDs; gabapentin (GBP), levetiracetam (LEV), valproic acid (VPA), lamotrigine (LTG), carbamazepine-10,11-epoxide (CBZ-epoxide), zonisamide (ZNS), oxcarbazepine (OXC), topiramate (TPM), carbamazepine (CBZ), phenytoin (PHT)) in human plasma as a tool for drug monitoring. d(10)-Phenytoin (d(10)-PHT) and d(6-)valproic acid (d(6)-VPA) were used as internal standards for the positive- and negative-ionization modes, respectively. Plasma samples were precipitated by the addition of acetonitrile, and supernatants were analyzed on a C18 reverse-phase column using an isocratic elution. Detection was carried out in selected reaction monitoring (SRM) mode. The calibration curves were linear over a 50-fold concentration range, with correlation coefficients (r(2)) greater than 0.997 for all AEDs. The intra- and inter-day precision was less than 12%, and the accuracy was between 85.9 and 114.5%. This method was successfully used in the identification and quantitation of AEDs in patients undergoing mono- or polytherapy for epilepsy.

Simultaneous quantification of rosiglitazone and its two major metabolites, N-desmethyl and p-hydroxy rosiglitazone in human plasma by liquid chromatography/tandem mass spectrometry: application to a pharmacokinetic study.

We present a simple, rapid, and sensitive liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method for the simultaneous quantification of rosiglitazone and its two major metabolites via CYP2C8/9, N-desmethyl and p-hydroxy rosiglitazone, in human plasma. The procedure was developed and validated using rosiglitazone-d(3) as the internal standard. Plasma samples (0.1 ml) were prepared using a simple deproteinization procedure with 0.2 ml of acetonitrile containing 40 ng/ml of rosiglitazone-d(3). Chromatographic separation was carried out on a Luna C18 column (100 mm x 2.0 mm, 3-microm particle size) using an isocratic mobile phase consisting of a 60:40 (v/v) mixture of acetonitrile and 0.1% formic acid((aq)). Each sample was run at 0.2 ml/min for a total run time of 2.5 min per sample. Detection and quantification were performed using a mass spectrometer in selected reaction-monitoring mode with positive electrospray ionization at m/z 358.1-->135.1 for rosiglitazone, m/z 344.2-->121.1 for N-desmethyl rosiglitazone, m/z 374.1-->151.1 for p-hydroxy rosiglitazone, and m/z 361.1-->138.1 for rosiglitazone-d(3). The linear ranges of concentration for rosiglitazone, N-desmethyl rosiglitazone, and p-hydroxy rosiglitazone were 1-500, 1-150, and 1-25 ng/ml, respectively, with a lower limit of quantification of 1 ng/ml for all analytes. The coefficient of variation for assay precision was less than 14.4%, and the accuracy was 93.3-112.3%. No relevant cross-talk and matrix effect were observed. This method was successfully applied to a pharmacokinetic study after oral administration of a 4-mg rosiglitazone tablet to healthy male Korean volunteers.

Determination of two HMG-CoA reductase inhibitors, pravastatin and pitavastatin, in plasma samples using liquid chromatography-tandem mass spectrometry for pharmaceutical study.

We developed a method for determining pravastatin or pitavastatin, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, in plasma using liquid chromatography and tandem mass spectrometry (LC-MS/MS). Pravastatin, pitavastatin and the internal standard fluvastatin were extracted from plasma with solid-phase extraction columns and eluted with methanol. After drying the organic layer, the residue was reconstituted in mobile phase (acetonitrile:water, 90:10, v/v) and injected onto a reversed-phase C(18) column. The isocratic mobile phase was eluted at 0.2 mL/min. The ion transitions recorded in multiple reaction monitoring mode were m/z 423 --> 101, 420 --> 290 and 410 --> 348 for pravastatin, pitavastatin and fluvastatin, respectively. The coefficient of variation of the assay precision was less than 12.4%, the accuracy exceeded 89%. The limit of detection was 1 ng/mL for all analytes. This method was used to measure the plasma concentration of pitavastatin or pravastatin from healthy subjects after a single 4 mg oral dose of pitavastatin or 40 mg oral dose of pravastatin. This is a very simple, sensitive and accurate analytic method to determine the pharmacokinetic profiles of pitavastatin or pravastatiny.

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 (-)-alpha and (+)-alpha 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 (CL(int)) of the formation of N-desbenzylbenidipine and dehydrobenidipine metabolites from (-)-alpha isomer was similar to those from the (+)-alpha isomer (1.9 +/- 0.1 versus 2.3 +/- 2.3 microl/min/pmol P450 and 0.5 +/- 0.2 versus 0.6 +/- 0.6 microl/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 CL(int) values of CYP3A4-mediated metabolite formation from (-)-alpha isomer were similar to those from (+)-alpha isomer (17.7 versus 14.4 microl/min/pmol P450, respectively). The total CL(int) values of CYP3A5-mediated metabolite formation from (-)-alpha isomer were also similar to those from (+)-alpha isomer (8.3 versus 11.0 microl/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.