Combined translational pharmacometrics approach to support the design and conduct of the first-in-human study of DWP16001.

AIMS: The objective of this study was to characterize the pharmacokinetics (PK)/pharmacodynamics (PD) of DWP16001, a novel sodium-glucose cotransporter 2 inhibitor, and predict efficacious doses for the first-in-human study using various translational approaches. METHODS: A mechanistic PK/PD model was developed for DWP16001 using nonlinear mixed-effect modelling to describe animal PK/PD properties. Using allometry and in silico physiologically based equations, human PK parameters were predicted. Human PD parameters were scaled by applying interspecies difference and in vitro drug-specific factors. Human parameters were refined using early clinical data. Model-predicted PK and PD outcomes were compared to observations before and after parameter refinement. RESULTS: The PK/PD model of DWP16001 was developed using a 2-compartment model with first-order absorption and indirect response. Efficacious doses of 0.3 and 2 mg of DWP16001 were predicted using human half-maximal inhibitory concentration values translated from Zucker Diabetic Fatty rats and normal rats, respectively. After parameter refinement, doses of 0.2 and 1 mg were predicted to be efficacious for each disease model, which improved the prediction results to within a 1.2-fold difference between the model prediction and observation. CONCLUSIONS: This study predicted efficacious human doses of DWP16001 using population PK/PD modelling and a combined translational pharmacometrics approach. Early clinical data allowed the methods used to translate in vitro and in vivo findings to clinical PK/PD values for DWP16001 to be optimized. This study has shown that a refinement step can be readily applied to improve model prediction and further support the study design and conduct of a first-in-human study.

SGLT2 inhibition ameliorates nano plastics-induced premature endothelial senescence and dysfunction.

Nano plastics (NPs) have been a significant threat to human health and are known to cause premature endothelial senescence. Endothelial senescence is considered one of the primary risk factors contributing to numerous cardiovascular disorders. Recent studies have suggested that inhibition of sodium glucose co-transporter (SGLT2) ameliorates endothelial senescence and dysfunction. Therefore, our study intends to explore the role of SGLT2 in NPs-induced endothelial senescence and dysfunction. Porcine coronary artery and its endothelial cells were treated with NPs in the presence or absence of Enavogliflozin (ENA), a SGLT2 inhibitor and then SGLTs expression, senescence markers and vascular function were evaluated. NPs significantly up-regulated SGLT2 and ENA significantly decreased NPs-induced senescence-associated-ß-gal activity, cell-cycle arrest, and senescence markers p53 and p21 suggesting that inhibition of SGLT2 prevents NPs-induced endothelial senescence. In addition, ENA decreased the formation of reactive oxygen species with the downregulation of Nox2, and p22phox. Furthermore, SGLT2 inhibition also up regulated the endothelial nitric oxide synthase expression along with improving vascular function. In conclusion, premature endothelial senescence by NPs is, at least in part, associated with SGLT2 and it could be a potential therapeutic target for preventing and/or treating environmental pollutants-induced cardiovascular disorders mediated by endothelial senescence and dysfunction.

Physiologically Based Pharmacokinetic Modelling to Predict Pharmacokinetics of Enavogliflozin, a Sodium-Dependent Glucose Transporter 2 Inhibitor, in Humans.

Enavogliflozin is a sodium-dependent glucose cotransporter 2 (SGLT2) inhibitor approved for clinical use in South Korea. As SGLT2 inhibitors are a treatment option for patients with diabetes, enavogliflozin is expected to be prescribed in various populations. Physiologically based pharmacokinetic (PBPK) modelling can rationally predict the concentration-time profiles under altered physiological conditions. In previous studies, one of the metabolites (M1) appeared to have a metabolic ratio between 0.20 and 0.25. In this study, PBPK models for enavogliflozin and M1 were developed using published clinical trial data. The PBPK model for enavogliflozin incorporated a non-linear urinary excretion in a mechanistically arranged kidney model and a non-linear formation of M1 in the liver. The PBPK model was evaluated, and the simulated pharmacokinetic characteristics were in a two-fold range from those of the observations. The pharmacokinetic parameters of enavogliflozin were predicted using the PBPK model under pathophysiological conditions. PBPK models for enavogliflozin and M1 were developed and validated, and they seemed useful for logical prediction.

Pharmacokinetics and Tissue Distribution of Enavogliflozin in Mice and Rats.

This study investigated the pharmacokinetics and tissue distribution of enavogliflozin, a novel sodium-glucose cotransporter 2 inhibitor that is currently in phase three clinical trials. Enavogliflozin showed dose-proportional pharmacokinetics following intravenous and oral administration (doses of 0.3, 1, and 3 mg/kg) in both mice and rats. Oral bioavailability was 84.5-97.2% for mice and 56.3-62.1% for rats. Recovery of enavogliflozin as parent form from feces and urine was 39.3 ± 3.5% and 6.6 ± 0.7%, respectively, 72 h after its intravenous injection (1 mg/kg), suggesting higher biliary than urinary excretion in mice. Major biliary excretion was also suggested for rats, with 15.9 ± 5.9% in fecal recovery and 0.7 ± 0.2% in urinary recovery for 72 h, following intravenous injection (1 mg/kg). Enavogliflozin was highly distributed to the kidney, which was evidenced by the AUC ratio of kidney to plasma (i.e., 41.9 ± 7.7 in mice following its oral administration of 1 mg/kg) and showed slow elimination from the kidney (i.e., T1/2 of 29 h). It was also substantially distributed to the liver, stomach, and small and large intestine. In addition, the tissue distribution of enavogliflozin after single oral administration was not significantly altered by repeated oral administration for 7 days or 14 days. Overall, enavogliflozin displayed linear pharmacokinetics following intravenous and oral administration, significant kidney distribution, and favorable biliary excretion, but it was not accumulated in the plasma and major distributed tissues, following repeated oral administration for 2 weeks. These features may be beneficial for drug efficacy. However, species differences between rats and mice in metabolism and oral bioavailability should be considered as drug development continues.

In Vitro Metabolism of DWP16001, a Novel Sodium-Glucose Cotransporter 2 Inhibitor, in Human and Animal Hepatocytes.

DWP16001 is currently in a phase 2 clinical trial as a novel anti-diabetes drug for the treatment of type 2 diabetes by selective inhibition of sodium-glucose cotransporter 2. This in vitro study was performed to compare the metabolism of DWP16001 in human, dog, monkey, mouse, and rat hepatocytes, and the drug-metabolizing enzymes responsible for the metabolism of DWP16001 were characterized using recombinant human cytochrome 450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes expressed from cDNAs. The hepatic extraction ratio of DWP16001 in five species ranged from 0.15 to 0.56, suggesting that DWP16001 may be subject to species-dependent and weak-to-moderate hepatic metabolism. Five phase I metabolites (M1-M5) produced by oxidation as well as three DWP16001 glucuronides (U1-U3) and two hydroxy-DWP16001 (M1) glucuronides (U4, U5), were identified from hepatocytes incubated with DWP16001 by liquid chromatography-high resolution mass spectrometry. In human hepatocytes, M1, M2, M3, U1, and U2 were identified. Formation of M1 and M2 from DWP16001 was catalyzed by CYP3A4 and CYP2C19. M3 was produced by hydroxylation of M1, while M4 was produced by hydroxylation of M2; both hydroxylation reactions were catalyzed by CYP3A4. The formation of U1 was catalyzed by UGT2B7, but UGT1A4, UGT1A9, and UGT2B7 contributed to the formation of U2. In conclusion, DWP16001 is a substrate for CYP3A4, CYP2C19, UGT1A4, UGT1A9, and UGT2B7 enzymes. Overall, DWP16001 is weakly metabolized in human hepatocytes, but there is a potential for the pharmacokinetic modulation and drug-drug interactions, involved in the responsible metabolizing enzymes of DWP16001 in humans.

Comparative Pharmacokinetics and Pharmacodynamics of a Novel Sodium-Glucose Cotransporter 2 Inhibitor, DWP16001, with Dapagliflozin and Ipragliflozin.

Since sodium-glucose cotransporter 2 (SGLT2) inhibitors reduced blood glucose level by inhibiting renal tubular glucose reabsorption mediated by SGLT2, we aimed to investigate the pharmacokinetics and kidney distribution of DWP16001, a novel SGLT2 inhibitor, and to compare these properties with those of dapagliflozin and ipragliflozin, representative SGLT2 inhibitors. The plasma exposure of DWP16001 was comparable with that of ipragliflozin but higher than that of dapagliflozin. DWP16001 showed the highest kidney distribution among three SGLT2 inhibitors when expressed as an area under curve (AUC) ratio of kidney to plasma (85.0 ± 16.1 for DWP16001, 64.6 ± 31.8 for dapagliflozin and 38.4 ± 5.3 for ipragliflozin). The organic anion transporter-mediated kidney uptake of DWP16001 could be partly attributed to the highest kidney uptake. Additionally, DWP16001 had the lowest half-maximal inhibitory concentration (IC50) to SGLT2, a target transporter (0.8 ± 0.3 nM for DWP16001, 1.6 ± 0.3 nM for dapagliflozin, and 8.9 ± 1.7 nM for ipragliflozin). The inhibition mode of DWP16001 on SGLT2 was reversible and competitive, but the recovery of the SGLT2 inhibition after the removal of SGLT2 inhibitors in CHO cells overexpressing SGLT2 was retained with DWP16001, which is not the case with dapagliflozin and ipragliflozin. In conclusion, selective and competitive SGLT2 inhibition of DWP16001 could potentiate the efficacy of DWP16001 in coordination with the higher kidney distribution and retained SGLT2 inhibition of DWP16001 relative to dapagliflozin and ipragliflozin.

Evaluation of DA-9801, a new herbal drug for diabetic neuropathy, on metabolism-mediated interaction.

DA-9801, the mixture extract of Dioscoreae rhizoma and Dioscorea nipponica Makino, is a new herbal drug currently being evaluated in a phase II clinical study for the treatment of diabetic peripheral neuropathy in Korea. The inhibitory potentials of DA-9801, D. rhizoma extract, D. nipponica Makino extract, and dioscin, an active component of DA-9801, on eight human cytochrome P450 (CYP) enzymes and four UDP-glucuronosyltransferase (UGT) enzymes were investigated in human liver microsomes using liquid chromatography-tandem mass spectrometry. DA-9801 showed slight inhibition of CYP1A2, CYP2C8, UGT1A1, and UGT1A9 enzyme activities with IC(50) values of 396.4, 449.9, 226.0, and 408.8 µg/mL, respectively. D. rhizoma extract showed negligible inhibition of CYP and UGT activities, but D. nipponica extract slightly inhibited CYP1A2, CYP2C8, CYP2C9, UGT1A1, and UGT1A9 activities with IC(50) values of 264.2, 237.1, 206.8, 302.4, and 383.1 µg/mL, respectively. DA-9801 showed volume per dose index values of 0.44-0.88 L for a 200-mg dose, suggesting that they may not cause the inhibition of the metabolism of CYP1A2, CYP2C8, UGT1A1, and UGT1A9-catalyzed drugs in humans. These results suggest that the administration of DA-9801 in human may not cause clinically relevant inhibition of these enzymes.

In vitro and in vivo metabolism of verproside in rats.

Verproside, a catalpol derivative iridoid glycoside isolated from Pseudolysimachion rotundum var. subintegrum, is a biologically active compound with anti-inflammatory, antinociceptic, antioxidant, and anti-asthmatic properties. Twenty-one metabolites were identified in bile and urine samples obtained after intravenous administration of verproside in rats using liquid chromatography-quadrupole Orbitrap mass spectrometry. Verproside was metabolized by O-methylation, glucuronidation, sulfation, and hydrolysis to verproside glucuronides (M1 and M2), verproside sulfates (M3 and M4), picroside II (M5), M5 glucuronide (M7), M5 sulfate (M9), isovanilloylcatalpol (M6), M6 glucuronide (M8), M6 sulfate (M10), 3,4-dihydroxybenzoic acid (M11), M11 glucuronide (M12), M11 sulfates (M13 and M14), 3-methyoxy-4-hydroxybenzoic acid (M15), M15 glucuronides (M17 and M18), M15 sulfate (M20), 3-hydroxy-4-methoxybenzoic acid (M16), M16 glucuronide (M19), and M16 sulfate (M21). Incubation of verproside with rat hepatocytes resulted in thirteen metabolites (M1-M11, M13, and M14). Verproside sulfate, M4 was a major metabolite in rat hepatocytes. After intravenous administration of verproside, the drug was recovered in bile (0.77% of dose) and urine (4.48% of dose), and O-methylation of verproside to picroside II (M5) and isovanilloylcatalpol (M6) followed by glucuronidation and sulfation was identified as major metabolic pathways compared to glucuronidation and sulfation of verproside in rats.

In vitro metabolism of corydaline in human liver microsomes and hepatocytes using liquid chromatography-ion trap mass spectrometry.

Corydaline is a pharmacologically active isoquinoline alkaloid isolated from Corydalis tubers. It exhibits the antiacetylcholinesterase, antiallergic, antinociceptive, and gastric emptying activities. The purposes of this study were to establish in vitro metabolic pathways of corydaline in human liver microsomes and hepatocytes by identification of their metabolites using liquid chromatography-ion trap mass spectrometry. Human liver microsomal incubation of corydaline in the presence of an NADPH-generating system resulted in the formation of nine metabolites, namely, four O-desmethylcorydaline [M1 (yuanhunine), M2 (9-O-desmethylcorydaline), M3 (isocorybulbine), and M4 (corybulbine)], three di-O-desmethylcorydaline [M5 (9,10-di-O-desmethylcorydaline), M6 (2,10-di-O-desmethylcorydaline), and M7 (3,10-di-O-desmethylcorydaline)], M8 (hydroxyyuanhunine), and M9 (hydroxycorydaline). Incubation of corydaline in human hepatocytes produced four metabolites including M1, M5, M6, and M9. O-Demethylation and hydroxylation were the major metabolic pathways for the metabolism of corydaline in human liver microsomes and hepatocytes.

Anticancer activity of undecapeptide analogues derived from antimicrobial peptide, brevinin-1EMa.

In spite of great advances in cancer therapy, cancer remains the major cause of death throughout the world. The increasing resistance of cancer cells towards current anticancer drugs requires development of anticancer agents with a new mode of action. Some antimicrobial peptides have become therapeutic candidates as new anticancer agents. As part of an effort to develop new antimicrobial and/or anticancer agents from natural peptides with low molecular weights, we have investigated the shortest bioactive analogues, which were derived from a 24-residue antimicrobial peptide, Brevinin-1EMa. Recently, we found four bioactive undecapeptides derived from a cationic, amphipathic α-helical, 11-residue peptide (named herein GA-W2: FLGWLFKWASK-NH(2)) (Won et al., 2011). In order to identify the potential of these peptides as anticancer agents, we investigated the anticancer activity of four undecapeptides against seven tumor cell lines such as A498 (kidney), A549 (lung), HCT116 (colon), MKN45 (stomach), PC-3 (prostate), SK-MEL-2 (skin) and SK-OV-3 (ovary). GA-K4 (FLKWLFKWAKK-NH(2)), which had the most potent antimicrobial activity of the four undecapeptides, also exhibited the most potent anticancer activity and synergistic effect in combination with doxorubicin. Therefore, GA-K4 peptide may be a potentially useful candidate as an anticancer peptide agent.

Effect of a New Prokinetic Agent DA-9701 Formulated with Corydalis Tuber and Pharbitidis Semen on Cytochrome P450 and UDP-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes.

DA-9701 is a new botanical drug composed of the extracts of Corydalis tuber and Pharbitidis semen, and it is used as an oral therapy for the treatment of functional dyspepsia in Korea. The inhibitory potentials of DA-9701 and its component herbs, Corydalis tuber and Pharbitidis semen, on the activities of seven major human cytochrome P450 (CYP) enzymes and four UDP-glucuronosyltransferase (UGT) enzymes in human liver microsomes were investigated using liquid chromatography-tandem mass spectrometry. DA-9701 and Corydalis tuber extract slightly inhibited UGT1A1-mediated etoposide glucuronidation, with 50% inhibitory concentration (IC(50)) values of 188 and 290 µg/mL, respectively. DA-9701 inhibited CYP2D6-catalyzed bufuralol 1'-hydroxylation with an inhibition constant (K(i)) value of 6.3 µg/mL in a noncompetitive manner. Corydalis tuber extract competitively inhibited CYP2D6-mediated bufuralol 1'-hydroxylation, with a K(i) value of 3.7 µg/mL, whereas Pharbitidis semen extract showed no inhibition. The volume in which the dose could be diluted to generate an IC(50) equivalent concentration (volume per dose index) value of DA-9701 for inhibition of CYP2D6 activity was 1.16 L/dose, indicating that DA-9701 may not be a potent CYP2D6 inhibitor. Further clinical studies are warranted to evaluate the in vivo extent of the observed in vitro interactions.

Effect of efavirenz on UDP-glucuronosyltransferase 1A1, 1A4, 1A6, and 1A9 activities in human liver microsomes.

Efavirenz is a non-nucleoside reverse transcriptase inhibitor used for the treatment of human immunodeficiency virus type 1 infections. Drug interactions of efavirenz have been reported due to in vitro inhibition of CYP2C9, CYP2C19, CYP3A4, and UDP-glucuronosyltransferase 2B7 (UGT2B7) and in vivo CYP3A4 induction. The inhibitory potentials of efavirenz on the enzyme activities of four major UDP-glucuronosyltransferases (UGTs), 1A1, 1A4, 1A6, and 1A9, in human liver microsomes were investigated using liquid chromatography-tandem mass spectrometry. Efavirenz potently inhibited UGT1A4-mediated trifluoperazine N-glucuronidation and UGT1A9-mediated propofol glucuronidation, with K(i) values of 2.0 and 9.4 µM, respectively. [I]/K(i) ratios of efavirenz for trifluoperazine N-glucuronidation and propofol glucuronidation were 6.5 and 1.37, respectively. Efavirenz also moderately inhibited UGT1A1-mediated 17ß-estradiol 3-glucuronidation, with a K(i) value of 40.3 µM, but did not inhibit UGT1A6-mediated 1-naphthol glucuronidation. Those in vitro results suggest that efavirenz should be examined for potential pharmacokinetic drug interactions in vivo due to strong inhibition of UGT1A4 and UGT1A9.

Hydrophilic interaction chromatography-electrospray ionization tandem mass spectrometric analysis of anastrozole in human plasma and its application to a pharmacokinetic study.

A hydrophilic interaction chromatography-electrospray ionization tandem mass spectrometric method was developed for determination of anastrozole in human plasma. Anastrozole and irbesartan (internal standard) were extracted from human plasma with a mixture of dichloromethane and methyl tert-butyl ether (30:70, v/v). Analysis of the analytes was performed on a Luna HILIC column with the mobile phase of acetonitrile-10 m m ammonium formate (95:5, v/v) and detected by electrospray ionization tandem mass spectrometry in the selected reaction monitoring mode. The standard curve was linear (r(2) = 0.9992) over the concentration range of 0.10-50.0 ng/mL using 200 µL of plasma sample. The coefficient of variation and relative error for intra- and inter-assay at four QC levels were 1.2-10.0% and -7.2-3.2%, respectively. The present method was applied successfully to the pharmacokinetic study of anastrozole after oral administration of 1 mg anastrozole tablet to healthy male volunteers.

Evaluation of metabolism-mediated herb-drug interactions.

As the use of herbal medicines increases, the public health consequences of drug-herb interactions are becoming more significant. Herbal medicines share the same drug metabolizing enzymes and drug transporters, including cytochrome P450 enzymes (CYPs), glucuronosyltransferases (UGTs), and P-glycoprotein, with several clinically important drugs. Interactions of several commonly used herbal medicines, such as Ginko biloba, milk thistle, and St. John's wort, with therapeutic drugs including warfarin, midazolam, alprazolam, indinavir, saquinavir, digoxin, nifedipine, cyclosporine, tacrolimus, irinotecan, and imatinib in humans have been reported. Many of these drugs have very narrow therapeutic indices. As the herb-drug interactions can significantly alter pharmacokinetic and pharmacodynamic properties of administered drugs, the drugs interacting with herbal medicines should be identified by appropriate in vitro and in vivo methods. A good understanding of the mechanisms of herb-drug interactions is also essential for assessing and minimizing clinical risks. In vitro methods are useful for providing mechanistic information and evaluating multiple components in herbal medicines. This review describes major factors affecting the metabolism of herbal medicines, mechanisms of herb-drug interactions mediated by CYPs and UGTs, and several in vitro methods to assess the herb-drug interactions. Finally, drug interactions of Ginkgo biloba and St. John's wort, as representative herbal medicines, are described.

Corydaline inhibits multiple cytochrome P450 and UDP-glucuronosyltransferase enzyme activities in human liver microsomes.

Corydaline is a bioactive alkaloid with various antiacetylcholinesterase, antiallergic, and antinociceptive activities found in the medicinal herb Corydalis Tubers. The inhibitory potential of corydaline on the activities of seven major human cytochrome P450 and four UDP-glucuronosyltransferase enzymes in human liver microsomes was investigated using LC-tandem MS. Corydaline was found to inhibit CYP2C19-catalyzed S-mephenytoin-4'-hydroxylatoin and CYP2C9-catalyzed diclofenac 4-hydroxylation, with K(i) values of 1.7 and 7.0 mM, respectively. Corydaline also demonstrated moderate inhibition of UGT1A1-mediated 17b-estradiol 3-glucuronidation and UGT1A9-mediated propofol glucuronidation with K(i) values of 57.6 and 37.3 mM, respectively. In the presence of corydaline, CYP3A-mediated midazolam hydroxylation showed a decrease with increasing preincubation time in a dose-dependent manner with K(i) values of 30.0 mM. These in vitro results suggest that corydaline should be evaluated for potential pharmacokinetic drug interactions in vivo due to potent inhibition of CYP2C19 and CYP2C9.

In vitro metabolism of magnolin and characterization of cytochrome P450 enzymes responsible for its metabolism in human liver microsomes.

Magnolin is a major bioactive component found in Shin-i, the dried flower buds of Magnolia fargesii; it has anti-inflammatory and anti-histaminic activities. Incubation of magnolin in human liver microsomes with an nicotinamide adenine dinucleotide phosphate-generating system resulted in the formation of five metabolites, namely, O-desmethyl magnolin (M1 and M2), didesmethylmagnolin (M3), and hydroxymagnolin (M4 and M5). In this study, we characterized the human liver cytochrome P450 (CYP) enzymes responsible for the biotransformation of three major metabolites--M1, M2, and M4--of magnolin. CYP2C8, CYP2C9, CYP2C19, and CYP3A4 were identified as the major enzymes responsible for the formation of the two O-desmethyl magnolins (M1 and M2), on the basis of a combination of correlation analysis and experiments, including immunoinhibition of magnolin in human liver microsomes and metabolism of magnolin by human cDNA-expressed CYP enzymes. CYP2C8 played a predominant role in the formation of hydroxymagnolin (M4). These results suggest that the pharmacokinetics of magnolin may not be affected by CYP2C8, CYP2C9, CYP2C19, and CYP3A4 responsible for the metabolism of magnolin or by the co-administration of appropriate CYP2C8, CYP2C9, CYP2C19, and CYP3A4 inhibitors or inducers due to the involvement of multiple CYP enzymes in the metabolism of magnolin.

Liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry for the simultaneous determination of dimethoxyaschantin, dimethylliroresinol, dimethylpinoresinol, epimagnolin A, fargesin and magnolin in rat plasma.

A rapid, selective, and sensitive liquid chromatography-atmospheric pressure chemical ionization (APCI) tandem mass spectrometry method was developed for the simultaneous determination of dimethoxyaschantin, dimethylliroresinol, dimethylpinoresinol, epimagnolin A, fargesin and magnolin, the pharmacologically active ingredients of Magnolia fargesii in rat plasma. These tetrahydrofurofuranoid lignans were extracted from rat plasma using tert-butyl methyl ether at pH 7.4. The analytes were separated on a Pinnacle DB biphenyl column with 65% methanol in 10 mm ammonium formate (pH 3.0) and detected by APCI tandem mass spectrometry in the selective reaction monitoring mode. The calibration curves were linear (r(2) ≥ 0.996) over the concentration range of 20.0-1000 ng/mL for six tetrahydrofurofuranoid lignans. The lower limit of quantification for these lignans was 20.0 ng/mL with 50 µL of plasma sample. The intra- and inter-assay coefficient of variation and relative error for the six tetrahydrofurofuranoid lignans at four quality control concentrations were 0.2-9.9% and -8.5-8.2%, respectively. There was no matrix effect for the six tetrahydrofurofuranoid lignans and tolterodine (internal standard). The pharmacokinetics of dimethylliroresinol, dimethylpinoresinol, epimagnolin A, fargesin and magnolin were evaluated after oral administration of a purified extract isolated from dried flower buds of Magnolia fargesii at doses of 5.5, 11.0 and 22.0 mg/kg in male rats.

In vitro metabolism of jaceosidin and characterization of cytochrome P450 and UDP-glucuronosyltransferase enzymes in human liver microsomes.

Jaceosidin is an active component in Artemisia species as well as Eupatorium species and it exhibits antiallergic, anticancer, antioxidant, anti-inflammatory, and antimutagenic activities. Jaceosidin was metabolized to jaceosidin glucuronide, 6-O-desmethyljaceosidin, hydroxyjaceosidin, 6-O-desmethyljaceosidin glucuronide, and hydroxyjaceosidin glucuronide in human liver microsomes. This study characterized the human liver cytochrome P450 (CYP) and UDPglucuronosyltransferase (UGT) enzymes responsible for the metabolism of jaceosidin. CYP1A2 was identified as the major enzyme responsible for the formation of 6-O-desmethyljaceosidin and hydroxyjaceosidin from jaceosidin on the basis of a combination of correlation analysis and experiments including immuno-inhibition, chemical inhibition in human liver microsomes, and metabolism by human cDNA-expressed CYP enzymes. Jaceosidin glucuronidation was catalyzed by UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, and UGT1A10. These results suggest that the pharmacokinetics of jaceosidin may be dramatically affected by polymorphic CYP1A2, UGT1A1, and UGT1A7 responsible for the metabolism of jaceosidin or by the coadministration of relevant CYP1A2 or UGT inhibitors or inducers.

Quantification of a novel PPARγ partial agonist (S)-2-ethoxy-3-(4-{3-methyl-5-[4-(3-methyl-isoxazol-5-yl)-phenyl]thiophen-2-ylmethoxy}-phenyl)-propionic acid (PAM-1616) in rat plasma using liquid chromatography-tandem mass spectrometry.

(S)-2-Ethoxy-3-(4-{3-methyl-5-[4-(3-methyl-isoxazol-5-yl)-phenyl]thiophen-2-ylmethoxy}-phenyl)-propionic acid (PAM-1616) is a novel peroxisome proliferators-activated receptor γ (PPARγ) partial agonist with excellent antihyperglycemic activity. It is a promising new drug candidate for the treatment of type-2 diabetes with reduced possibility of edema in vitro/in vivo. In order to evaluate the pharmacokinetics of PAM-1616, a reliable, selective and sensitive highperformance liquid chromatography/electrospray ionization tandem mass spectrometry was developed for the quantification of PAM-1616 in rat plasma. The analytes were extracted from rat plasma with ethyl acetate, separated on an Atlantis dC(18) column with a mobile phase of 75% acetonitrile in 10 mM ammonium formate (pH 4.5), and detected by tandem mass spectrometry in the selective reaction monitoring mode. The calibration curve was linear (r (2) = 0.999) over the concentration range of 0.05-20.0 µg/mL and the lower limit of quantification was 0.05 µg/mL. The coefficient of variation and relative error at four QC levels were 1.8% to 14.3% and -10.0% to 6.5%, respectively. The present method was successfully applied to the pharmacokinetic study of PAM-1616 after intravenous administration of PAM-1616 potassium at a dose of 1 mg/kg in rats.

Effects of eupatilin and jaceosidin on cytochrome p450 enzyme activities in human liver microsomes.

Eupatilin and jaceosidin are bioactive flavones found in the medicinal herbs of the genus Artemisia. These bioactive flavones exhibit various antioxidant, antiinflammatory, antiallergic, and antitumor activities. The inhibitory potentials of eupatilin and jaceosidin on the activities of seven major human cytochrome P450 enzymes in human liver microsomes were investigated using a cocktail probe assay. Eupatilin and jaceosidin potently inhibited CYP1A2-catalyzed phenacetin O-deethylation with 50% inhibitory concentration (IC(50)) values of 9.4 microM and 5.3 microM, respectively, and CYP2C9-catalyzed diclofenac 4-hydroxylation with IC(50) values of 4.1 microM and 10.2 microM, respectively. Eupatilin and jaceosidin were also found to moderately inhibit CYP2C19-catalyzed [S]-mephenytoin 4'-hydroxylation, CYP2D6-catalyzed bufuralol 1'-hydroxylation, and CYP2C8-catalyzed amodiaquine N-deethylation. Kinetic analysis of human liver microsomes showed that eupatilin is a competitive inhibitor of CYP1A2 with a K(i) value of 2.3 microM and a mixed-type inhibitor of CYP2C9 with a K(i) value of 1.6 microM. Jaceosidin was shown to be a competitive inhibitor of CYP1A2 with a K(i) value of 3.8 microM and a mixed-type inhibitor of CYP2C9 with K(i) value of 6.4 microM in human liver microsomes. These in vitro results suggest that eupatilin and jaceosidin should be further examined for potential pharmacokinetic drug interactions in vivo due to inhibition of CYP1A2 and CYP2C9.