Simple and rapid method for 336 multiresidual pesticide analysis in saliva, determination of their chemical stabilities, and biomonitoring of farmers.

Simultaneous multiresidual pesticide analysis of saliva samples was performed using scaled-down QuEChERS extraction with LC-MS/MS and GC-MS/MS. The optimum extraction procedure using acidified acetonitrile was applicable to 336 pesticides (287 for LC-MS/MS and 49 for GC-MS/MS). To determine pesticide multiresidues in saliva, 100 µL of the sample was extracted with 200 µL of 0.1% formic acid in acetonitrile, and the initial extract was partitioned with 40 mg of MgSO4 and 10 mg of NaCl. The organic supernatants (120 µL) were then mixed with acetonitrile (30 µL) for matrix-matching (4:1, v/v), and the final extract solution was injected into the LC-MS/MS (4 µL) and GC-MS/MS (2 µL) systems. The established analytical method showed a good LOQs between 5 and 25 ng/mL with reliable accuracy/precision values and recovery results (50-140%) for the target pesticides. Under the two different storage conditions, most of the analytes did not undergo chemical changes in the saliva samples, whereas some pesticides were more stable in freeze-thaw processes than those left at room temperature. Biomonitoring of farmers (ten mixers and ten sprayers) was successfully applied using the validated method, and two carbamates (fenobucarb and propamocarb) were determined at trace concentrations (12.5-675.0 ng/mL from 11 positively detected samples).

Involvement of Organic Anion Transporters in the Pharmacokinetics and Drug Interaction of Rosmarinic Acid.

We investigated the involvement of drug transporters in the pharmacokinetics of rosmarinic acid in rats as well as the transporter-mediated drug interaction potential of rosmarinic acid in HEK293 cells overexpressing clinically important solute carrier transporters and also in rats. Intravenously injected rosmarinic acid showed bi-exponential decay and unchanged rosmarinic acid was mainly eliminated by urinary excretion, suggesting the involvement of transporters in its renal excretion. Rosmarinic acid showed organic anion transporter (OAT)1-mediated active transport with a Km of 26.5 µM and a Vmax of 69.0 pmol/min in HEK293 cells overexpressing OAT1, and the plasma concentrations of rosmarinic acid were increased by the co-injection of probenecid because of decreased renal excretion due to OAT1 inhibition. Rosmarinic acid inhibited the transport activities of OAT1, OAT3, organic anion transporting polypeptide (OATP)1B1, and OATP1B3 with IC50 values of 60.6 µM, 1.52 µM, 74.8 µM, and 91.3 µM, respectively, and the inhibitory effect of rosmarinic acid on OAT3 transport activity caused an in vivo pharmacokinetic interaction with furosemide by inhibiting its renal excretion and by increasing its plasma concentration. In conclusion, OAT1 and OAT3 are the major transporters that may regulate the pharmacokinetic properties of rosmarinic acid and may cause herb-drug interactions with rosmarinic acid, although their clinical relevance awaits further evaluation.

Epimagnolin targeting on an active pocket of mammalian target of rapamycin suppressed cell transformation and colony growth of lung cancer cells.

Mammalian target of rapamycin (mTOR) has a pivotal role in carcinogenesis and cancer cell proliferation in diverse human cancers. In this study, we observed that epimagnolin, a natural compound abundantly found in Shin-Yi, suppressed cell proliferation by inhibition of epidermal growth factor (EGF)-induced G1/S cell-cycle phase transition in JB6 Cl41 cells. Interestingly, epimagnolin suppressed EGF-induced Akt phosphorylation strongly at Ser473 and weakly at Thr308 without alteration of phosphorylation of MAPK/ERK kinases (MEKs), extracellular signal-regulated kinase (ERKs), and RSK1, resulting in abrogation of the phosphorylation of GSK3ß at Ser9 and p70S6K at Thr389. Moreover, we found that epimagnolin suppressed c-Jun phosphorylation at Ser63/73, resulting in the inhibition of activator protein 1 (AP-1) transactivation activity. Computational docking indicated that epimagnolin targeted an active pocket of the mTOR kinase domain by forming three hydrogen bonds and three hydrophobic interactions. The prediction was confirmed by using in vitro kinase and adenosine triphosphate-bead competition assays. The inhibition of mTOR kinase activity resulted in the suppression of anchorage-independent cell transformation. Importantly, epimagnolin efficiently suppressed cell proliferation and anchorage-independent colony growth of H1650 rather than H460 lung cancer cells with dependency of total and phosphorylated protein levels of mTOR and Akt. Inhibitory signaling of epimagnolin on cell proliferation of lung cancer cells was observed mainly in mTOR-Akt-p70S6K and mTOR-Akt-GSK3ß-AP-1, which was similar to that shown in JB6 Cl41 cells. Taken together, our results indicate that epimagnolin potentiates as chemopreventive or therapeutic agents by direct active pocket targeting of mTOR kinase, resulting in sensitizing cancer cells harboring enhanced phosphorylation of the mTORC2-Akt-p70S6k signaling pathway.

In Vitro Metabolism of 25B-NBF, 2-(4-Bromo-2,5-Dimethoxyphenyl)-N-(2-Fluorobenzyl)ethanamine, in Human Hepatocytes Using Liquid Chromatography⁻Mass Spectrometry.

25B-NBF, 2-(4-bromo-2,5-dimethoxyphenyl)-N-(2-fluorobenzyl)ethanamine, is a new psychoactive substance classified as a phenethylamine. It is a potent agonist of the 5-hydroxytryptamine receptor, but little is known about its metabolism and elimination properties since it was discovered. To aid 25B-NBF abuse screening, the metabolic characteristics of 25B-NBF were investigated in human hepatocytes and human cDNA-expressed cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes using liquid chromatography⁻high resolution mass spectrometry. At a hepatic extraction ratio of 0.80, 25B-NBF was extensively metabolized into 33 metabolites via hydroxylation, O-demethylation, bis-O-demethylation, N-debenzylation, glucuronidation, sulfation, and acetylation after incubation with pooled human hepatocytes. The metabolism of 25B-NBF was catalyzed by CYP1A1, CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, and UGT2B7 enzymes. Based on these results, it is necessary to develop a bioanalytical method for the determination of not only 25B-NBF but also its metabolites in biological samples for the screening of 25B-NBF abuse.

Licochalcone A attenuates acne symptoms mediated by suppression of NLRP3 inflammasome.

Activation of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome by Propionibacterium acnes (P. acnes) is critical for inducing inflammation and aggravating the development of acne lesions. We searched for available small-molecule inhibitors of the NLRP3 inflammasome that could be topically administered for the treatment of acne. We found that licochalcone A, a chalconoid isolated from the root of Glycyrrhiza inflate, was an effective inhibitor for P. acnes-induced NLRP3 inflammasome activation. Licochalcone A blocked P. acnes-induced production of caspase-1(p10) and IL-1ß in primary mouse macrophages and human SZ95 sebocytes, indicating the suppression of NLRP3 inflammasome. Licochalcone A suppressed P. acnes-induced ASC speck formation and mitochondrial reactive oxygen species. Topical application of licochalcone A to mouse ear skin attenuated P. acnes-induced skin inflammation as shown by histological assessment, ear thickness measurement, and inflammatory gene expression. Licochalcone A reduced caspase-1 activity and IL-1ß production in mouse ear injected with P. acnes. This study demonstrated that licochalcone A is effective in the control of P. acnes-induced skin inflammation as an efficient inhibitor for NLRP3 inflammasome. Our study provides a new paradigm for the development of anti-acne therapy via targeting NLRP3 inflammasome.

A Comprehensive In Vivo and In Vitro Assessment of the Drug Interaction Potential of Red Ginseng.

Purpose: Red ginseng is one of the world's most popular herbal medicines; it exhibits a wide range of pharmacologic activities and is often co-ingested with other herbal and conventional medicines. This open-label, randomized, 3-period study investigated the in vivo herb-drug interaction potential for red ginseng extract with cytochrome P-450 (CYP) enzymes and organic anion-transporting polypeptide (OATP) 1B1. METHODS: Fifteen healthy male volunteers (22-28 years; 57.1-80.8 kg) were administered a single dose of cocktail probe substrates (caffeine 100 mg, losartan 50 mg, omeprazole 20 mg, dextromethorphan 30 mg, midazolam 2 mg, and pitavastatin 2 mg) and single or multiple doses of red ginseng extract for 15 days. FINDINGS: The pharmacokinetic profiles of the probe substrates and metabolites after single- or multiple-dose administration of red ginseng extracts were comparable to the corresponding profiles of the control group. The geometric mean ratio of AUC0-t and 90% CIs for the probe substrate drugs between the control and multiple doses of red ginseng for 15 days were within 0.8 to 1.25 (CYP2C9, CYP3A4, and OATP1B1 probe substrates) or slightly higher (CYP1A2, CYP2C19, and CYP2D6 probe substrates). Additional assessments of the in vitro drug interaction potential of red ginseng extracts and the ginsenoside Rb1 on drug-metabolizing enzymes and transporters using human liver microsomes, cryopreserved human hepatocytes, and transporter-overexpressed cells were negative. IMPLICATIONS: Red ginseng poses minimal risks for clinically relevant CYP- or OATP-mediated drug interactions and is well tolerated. Clinical Research Information Service registry no.

Selective inhibition of CYP2C8 by fisetin and its methylated metabolite, geraldol, in human liver microsomes.

Fisetin is a flavonol compound commonly found in edible vegetables and fruits. It has anti-tumor, antioxidant, and anti-inflammatory effects. Geraldol, the O-methyl metabolite of fisetin in mice, is reported to suppress endothelial cell migration and proliferation. Although the in vivo and in vitro effects of fisetin and its metabolites are frequently reported, studies on herb-drug interactions have not yet been performed. This study was designed to investigate the inhibitory effect of fisetin and geraldol on eight isoforms of human cytochrome P450 (CYP) by using cocktail assay and LC-MS/MS analysis. The selective inhibition of CYP2C8-catalyzed paclitaxel hydroxylation by fisetin and geraldol were confirmed in pooled human liver microsomes (HLMs). In addition, an IC50 shift assay under different pre-incubation conditions confirmed that fisetin and geraldol shows a reversible concentration-dependent, but not mechanism-based, inhibition of CYP2C8. Moreover, Michaelis-Menten, Lineweaver-burk plots, Dixon and Eadie-Hofstee showed a non-competitive inhibition mode with an equilibrium dissociation constant of 4.1 µM for fisetin and 11.5 µM for geraldol, determined from secondary plot of the Lineweaver-Burk plot. In conclusion, our results indicate that fisetin showed selective reversible and non-competitive inhibition of CYP2C8 more than its main metabolite, geraldol, in HLMs.

Investigation of pharmacokinetic parameters of bakuchicin isolated from Psoralea corylifolia in mice.

Bakuchicin is a furanocoumarin isolated from the seeds of Psoralea corylifolia, which is used in oriental medicine. However, limited information on the pharmacokinetics of bakuchicin is available and in addition, no determined method has been devised to quantify bakuchicin levels in the plasma. In the present study, we developed and validated a quantification method using liquid chromatography (LC) coupled with tandem mass spectrometry (LC-MS/MS), which was applied to a pharmacokinetic investigation in mouse plasma. LC was performed using an ACE 5 C18 column, and a mixture of acetonitrile and water containing 0.1% formic acid was used as the mobile phase at a flow rate of 220µL/min. Bakuchicin transition ions in multiple reaction-monitoring modes using positive ionization were observed at m/z 187.0 to m/z 131.0. Bakuchicin and the internal standard (reserpine) had retention times of 4.5 and 4.3min, respectively. Acceptable linearity (r2=0.996) was observed over the concentration range of 20-1000ng/mL, with a lower quantification limit of 20ng/mL in mouse plasma. This method was successfully applied to determine the pharmacokinetic parameters of bakuchicin in mouse plasma and showed that the bioavailability of bakuchicin was 58.3% at 5mg/kg oral administration.

In Vitro Metabolic Pathways of the New Anti-Diabetic Drug Evogliptin in Human Liver Preparations.

Evogliptin ((R)-4-((R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(tert-butoxymethyl)-piperazin-2-one), is a new dipeptidyl peptidase IV inhibitor used for the treatment of type II diabetes mellitus. The in vitro metabolic pathways of evogliptin were identified in human hepatocytes, liver microsomes, and liver S9 fractions using liquid chromatography-Orbitrap mass spectrometry (LC-HRMS). Five metabolites of evogliptin-4-oxoevogliptin (M1), 4(S)-hydroxyevogliptin (M2), 4(R)-hydroxyevogliptin (M3), 4(S)-hydroxyevogliptin glucuronide (M4), and evogliptin N-sulfate (M5)-were identified in human liver preparations by comparison with authentic standards. We characterized the cytochrome P450 (CYP) enzymes responsible for evogliptin hydroxylation to 4(S)-hydroxyevogliptin (M2) and 4(R)-hydroxyevogliptin (M3) and the UGT enzymes responsible for glucuronidation of 4(S)-hydroxyevogliptin (M2) to 4(S)-hydroxy-evogliptin glucuronide (M4). CYP3A4/5 played the major role in the hydroxylation of evogliptin to 4(S)-hydroxyevogliptin (M2) and 4(R)-hydroxyevogliptin (M3). Glucuronidation of 4(S)-hydroxy-evogliptin (M2) to 4(S)-hydroxyevogliptin glucuronide (M4) was catalyzed by the enzymes UGT2B4 and UGT2B7. These results suggest that the interindividual variability in the metabolism of evogliptin in humans is a result of the genetic polymorphism of the CYP and UGT enzymes responsible for evogliptin metabolism.

Organic anion transporter 3- and organic anion transporting polypeptides 1B1- and 1B3-mediated transport of catalposide.

We investigated the in vitro transport characteristics of catalposide in HEK293 cells overexpressing organic anion transporter 1 (OAT1), OAT3, organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, organic cation transporter 1 (OCT1), OCT2, P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP). The transport mechanism of catalposide was investigated in HEK293 and LLC-PK1 cells overexpressing the relevant transporters. The uptake of catalposide was 319-, 13.6-, and 9.3-fold greater in HEK293 cells overexpressing OAT3, OATP1B1, and OATP1B3 transporters, respectively, than in HEK293 control cells. The increased uptake of catalposide via the OAT3, OATP1B1, and OATP1B3 transporters was decreased to basal levels in the presence of representative inhibitors such as probenecid, furosemide, and cimetidine (for OAT3) and cyclosporin A, gemfibrozil, and rifampin (for OATP1B1 and OATP1B3). The concentration-dependent OAT3-mediated uptake of catalposide revealed the following kinetic parameters: Michaelis constant (K m) =41.5 µM, maximum uptake rate (V max) =46.2 pmol/minute, and intrinsic clearance (CL int) =1.11 µL/minute. OATP1B1- and OATP1B3-mediated catalposide uptake also showed concentration dependency, with low CL int values of 0.035 and 0.034 µL/minute, respectively. However, the OCT1, OCT2, OAT1, P-gp, and BCRP transporters were apparently not involved in the uptake of catalposide into cells. In addition, catalposide inhibited the transport activities of OAT3, OATP1B1, and OATP1B3 with half-maximal inhibitory concentration values of 83, 200, and 235 µM, respectively. However, catalposide did not significantly inhibit the transport activities of OCT1, OCT2, OAT1, P-gp, or BCRP. In conclusion, OAT3, OATP1B1, and OATP1B3 are major transporters that may regulate the pharmacokinetic properties and may cause herb-drug interactions of catalposide, although their clinical relevance awaits further evaluation.

Metabolism-mediated drug interaction potential of HS-23, a new herbal drug for the treatment of sepsis in human hepatocytes and liver microsomes.

HS-23, an extract of the dried flower buds of Lonicera japonica, is a new botanical drug currently being evaluated in a phase I clinical study in Korea for the treatment of sepsis. The in vitro induction and inhibition potentials of HS-23 on the drug-metabolizing enzymes using human hepatocytes and liver microsomes were assessed to evaluate herb-drug interaction according to botanical drug guideline and drug interaction guidance of FDA. HS-23 slightly inhibited CYP2A6, CYP2B6, CYP2C9, CYP2C19, and CYP3A4 enzyme activities in human liver microsomes with IC50 values of 80.6, 160.7, 169.5, 85.4, and 76.6 µg/mL, respectively. HS-23 showed negligible inhibition of CYP1A2, CYP2C8, CYP2D6, UGT1A1, UGT1A4, UGT1A9, and UGT2B7 activities in human liver microsomes. Based on these results, HS-23 may not inhibit the metabolism of CYP2A6, CYP2B6, CYP2C9, CYP2C19, and CYP3A4-catalyzed drugs in humans. HS-23 did not affect the mRNA expression of CYP1A2, CYP2B6, and CYP3A4 after 48 h treatment at three concentrations (0.5, 5, and 50 µg/mL) in three independent human hepatocytes, indicating that HS-23 has no effect on herb-drug interactions that up- or down-regulate CYP1A2, CYP2B6, and CYP3A4. These results indicate that the administration of HS-23 in human may not cause clinically relevant inhibition and induction of these cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes and HS-23 may be promising therapeutic agent for treatment of sepsis.

Inhibitory effects of cedrol, ß-cedrene, and thujopsene on cytochrome P450 enzyme activities in human liver microsomes.

Cedrol, ß-cedrene, and thujopsene are bioactive sesquiterpenes found in cedar essential oil and exert antiseptic, anti-inflammatory, antispasmodic, tonic, astringent, diuretic, sedative, insecticidal, and antifungal activities. These compounds are used globally in traditional medicine and cosmetics. The aim of this study was to investigate the inhibitory effects of cedrol, ß-cedrene, and thujopsene on the activities of eight major human cytochrome P-450 (CYP) enzymes using human liver microsomes to assess potential ß-cedrene-, cedrol-, and thujopsene-drug interactions. Cedrol, ß-cedrene, and thujopsene were found to be potent competitive inhibitors of CYP2B6-mediated bupropion hydroxylase with inhibition constant (Ki) values of 0.9, 1.6, and 0.8 µM, respectively, comparable with that of a selective CYP2B6 inhibitor, thioTEPA (Ki, 2.9 µM). Cedrol also markedly inhibited CYP3A4-mediated midazolam hydroxylation with a Ki value of 3.4 µM, whereas ß-cedrene and thujopsene moderately blocked CYP3A4. Cedrol, ß-cedrene, and thujopsene at 100 µM negligibly inhibited CYP1A2, CYP2A6, and CYP2D6 activities. Only thujopsene was found to be a mechanism-based inhibitor of CYP2C8, CYP2C9, and CYP2C19. Cedrol and thujopsene weakly inhibited CYP2C8, CYP2C9, and CYP2C19 activities, but ß-cedrene did not. These in vitro results indicate that cedrol, ß-cedrene, and thujopsene need to be examined for potential pharmacokinetic drug interactions in vivo due to their potent inhibition of CYP2B6 and CYP3A4.

Evaluation of the transporter-mediated herb-drug interaction potential of DA-9801, a standardized dioscorea extract for diabetic neuropathy, in human in vitro and rat in vivo.

BACKGROUND: Drug transporters play important roles in the absorption, distribution, and elimination of drugs and thereby, modulate drug efficacy and toxicity. With a growing use of poly pharmacy, concurrent administration of herbal extracts that modulate transporter activities with drugs can cause serious adverse reactions. Therefore, prediction and evaluation of drug-drug interaction potential is important in the clinic and in the drug development process. DA-9801, comprising a mixed extract of Dioscoreae rhizoma and Dioscorea nipponica Makino, is a new standardized extract currently being evaluated for diabetic peripheral neuropathy in a phase II clinical study. METHOD: The inhibitory effects of DA-9801 on the transport functions of organic cation transporter (OCT)1, OCT2, organic anion transporter (OAT)1, OAT3, organic anion transporting polypeptide (OATP)1B1, OATP1B3, P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP) were investigated in HEK293 or LLC-PK1 cells. The effects of DA-9801 on the pharmacokinetics of relevant substrate drugs of these transporters were also examined in vivo in rats. RESULTS: DA-9801 inhibited the in vitro transport activities of OCT1, OCT2, OAT3, and OATP1B1, with IC50 values of 106, 174, 48.1, and 273 µg/mL, respectively, while the other transporters were not inhibited by 300 µg/mL DA-9801. To investigate whether this inhibitory effect of DA-9801 on OCT1, OCT2, and OAT3 could change the pharmacokinetics of their substrates in vivo, we measured the pharmacokinetics of cimetidine, a substrate for OCT1, OCT2, and OAT3, and of furosemide, a substrate for OAT1 and OAT3, by co-administration of DA-9801 at a single oral dose of 1,000 mg/kg. Pre-dose of DA-9801 5 min or 2 h prior to cimetidine administration decreased the Cmax of cimetidine in rats. However, DA-9801 did not affect the elimination parameters such as half-life, clearance, or amount excreted in the urine, suggesting that it did not inhibit elimination process of cimetidine, which is governed by OCT1, OCT2, and OAT3. Moreover, DA-9801 did not affect the pharmacokinetic characteristics of furosemide, as evidenced by its unchanged pharmacokinetic parameters. CONCLUSION: Inhibitory effects of DA-9801 on OCT1, OCT2, and OAT3 observed in vitro may not necessarily translate into in vivo herb-drug interactions in rats even at its maximum effective dose.

Low adherence to upfront and extended adjuvant letrozole therapy among early breast cancer patients in a clinical practice setting.

OBJECTIVE: The aim of this study was to investigate the prevalence and causes of early discontinuation and non-adherence to upfront and extended adjuvant letrozole therapy in breast cancer patients. METHODS: Adherence was assessed using medical charts and longitudinal pharmacy records of 609 patients who initiated adjuvant letrozole between January 2002 and April 2011. A Cox proportional hazards regression model was adopted to identify potential predictors of non-adherence. RESULTS: The overall adherence rate after 1 year of therapy was 79.5%, with cumulative rates declining to 63.7% after 3 years and 57.1% after 5 years. A significantly lower rate of adherence in the extended adjuvant group was observed compared with the upfront adjuvant group (49.0 vs. 72.5%, p < 0.001). Adverse events (50.4%) were the major cause of early discontinuation, with musculoskeletal pain (73.2%) being the single most cited reason. Additional factors correlating with non-adherence in the upfront adjuvant group included a delay in initiation of adjuvant hormone therapy, breast-conserving surgery, calcium supplements, bisphosphonate therapy and concomitant medication for co-morbidity. CONCLUSIONS: We observed that approximately 57% of patients fully adhered to letrozole therapy over a 5-year treatment period, and that the adherence to extended letrozole was meaningfully lower than the upfront adjuvant letrozole in a clinical practice setting.

Pharmacokinetics of chlorogenic acid and corydaline in DA-9701, a new botanical gastroprokinetic agent, in rats.

1.Few studies describing the pharmacokinetic properties of chlorogenic acid (CA) and corydaline (CRD) which are marker compounds of a new prokinetic botanical agent, DA-9701, have been reported. The aim of the present study is to evaluate the pharmacokinetic properties CA and CRD following intravenous and oral administration of pure CA (1-8 mg/kg) or CRD (1.1-4.5 mg/kg) and their equivalent dose of DA-9701 to rats. 2. Dose-proportional AUC and dose-independent clearance (10.3-12.1 ml/min/kg) of CA were observed following its administration. Oral administration of CA as DA-9701 did not influence the oral pharmacokinetic parameters of CA. Incomplete absorption of CA, its decomposition in the gastrointestinal tract, and/or pre-systemic metabolism resulted in extremely low oral bioavailability (F) of CA (0.478-0.899%). 3. CRD showed greater dose-normalized AUC in the higher dose group than that in lower dose group(s) after its administration due to saturation of its metabolism via decreased non-renal clearance (by 51.3%) and first-pass extraction. As a result, the F of CRD following 4.5 mg/kg oral CRD (21.1%) was considerably greater than those of the lower dose groups (9.10 and 13.8%). However, oral administration of CRD as DA-9701 showed linear pharmacokinetics as a result of increased AUC and F in lower-dose groups (by 182% and 78.5%, respectively) compared to those of pure CRD. The greater oral AUC of CRD for DA-9701 than for pure CRD could be due to decreased hepatic and/or GI first-pass extraction of CRD by other components in DA-9701.

Targeting of magnolin on ERKs inhibits Ras/ERKs/RSK2-signaling-mediated neoplastic cell transformation.

Mitogen-activated protein kinases play a key role in cell proliferation, cell cycle progression and cell transformation, and activated Ras/extracellular signal-regulated kinases (ERKs)/ribosomal S6 kinase 2 (RSK2) signaling pathways have been widely identified in many solid tumors. In this study, we found that magnolin, a compound found in the Magnolia species, directly targeted and inhibited ERK1 and ERK2 kinase activities with IC50 values of 87 and 16.5 nM by competing with adenosine triphosphate in an active pocket. Further, we demonstrated that magnolin inhibited epidermal growth factor (EGF)-induced p90RSKs phosphorylation at Thr359/Ser363, but not ERKs phosphorylation at Thr202/Tyr204, and this resulted in inhibition of cell proliferation by suppression of the G1/S cell cycle transition. Additionally, p38 kinases, Jun N-terminal kinases and Akts were not involved in the magnolin-mediated inhibitory signaling. Magnolin targeting of ERK1 and 2 activities suppressed the phosphorylation of RSK2 and downstream target proteins including ATF1 and c-Jun and AP-1, a dimer of Jun/Fos, and the transactivation activities of ATF1 and AP-1. Notably, ERKs inhibition by magnolin suppressed EGF-induced anchorage-independent cell transformation and colony growth of Ras(G12V)-harboring A549 human lung cancer cells and NIH3T3 cells stably expressing Ras(G12V) in soft agar. Taken together, these results demonstrated that magnolin might be a naturally occurring chemoprevention and therapeutic agent capable of inhibiting cell proliferation and transformation by targeting ERK1 and ERK2.

Effect of honokiol on cytochrome P450 and UDP-glucuronosyltransferase enzyme activities in human liver microsomes.

Honokiol is a bioactive component isolated from the medicinal herbs Magnolia officinalis and Magnolia grandiflora that has antioxidative, anti-inflammatory, antithrombotic, and antitumor activities. The inhibitory potentials of honokiol on eight major human cytochrome P450 (CYP) enzymes 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4, and four UDP-glucuronosyltransferases (UGTs) 1A1, 1A4, 1A9, and 2B7 in human liver microsomes were investigated using liquid chromatography-tandem mass spectrometry. Honokiol strongly inhibited CYP1A2-mediated phenacetin O-deethylation, CYP2C8-mediated amodiaquine N-deethylation, CYP2C9-mediated diclofenac 4-hydroxylation, CYP2C19-mediated [S]-mephenytoin 4-hydroxylation, and UGT1A9-mediated propofol glucuronidation with K(i) values of 1.2, 4.9, 0.54, 0.57, and 0.3 µM, respectively. Honokiol also moderately inhibited CYP2B6-mediated bupropion hydroxylation and CYP2D6-mediated bufuralol 1'-hydroxylation with K(i) values of 17.5 and 12.0 µM, respectively. These in vitro results indicate that honokiol has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP1A2, CYP2C8, CYP2C9, CYP2C19, and UGT1A9.

Pharmacokinetics of hederacoside C, an active ingredient in AG NPP709, in rats.

1. Hederacoside C (HDC) is one of the active ingredients in Hedera helix leaf extract (Ivy Ex.) and AG NPP709, a new botanical drug to treat acute respiratory infection and chronic inflammatory bronchitis. However, information regarding its pharmacokinetic properties remains limited. 2. Here, we report the pharmacokinetics of HDC in rats after intravenous administration of HDC (3, 12.5, and 25 mg/kg) and after oral administration of HDC, Ivy Ex., and AG NPP709 (equivalent to 12.5, 25, and 50 mg/kg HDC). 3. Linear pharmacokinetics of HDC were identified upon its intravenous administration at doses of 3-25 mg/kg. Intravenous administration of HDC results in relatively slow clearance (1.46-2.08 mL/min/kg) and a small volume of distribution at steady state (138-222 mL/kg), while oral administration results in a low absolute oral bioavailability (F) of 0.118-0.250%. The extremely low F of HDC may be due to poor absorption of HDC from the gastrointestinal (GI) tract and/or its decomposition therein. 4. The oral pharmacokinetics of HDC did not differ significantly among pure HDC, Ivy Ex., and AG NPP709.

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.

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.