Jian Jiang

Intestinal absorption mechanisms of berberine, palmatine, jateorhizine, and coptisine: involvement of P-glycoprotein

The absorption and transport mechanisms of berberine, palmatine, jateorhizine, and coptisine were studied using a Caco-2 cells uptake and transport model, with the addition of cyclosporin A and verapamil as P-glycoprotein (P-gp) inhibitors and MK-571 as a multidrug resistance-associated protein 2 (MRP2) inhibitor. In the uptake experiment, berberine, palmatine, jateorhizine, and coptisine were all taken into Caco-2 cells, and their uptakes were increased in the presence of cyclosporin A or verapamil. In the transport experiment, Papp (AP-BL) was between 0.1 and 1.0 × 106 cm/sec for berberine, palmatine, jateorhizine, and coptisine and was lower than Papp (BL-AB). ER values were all >2. Cyclosporin A and verapamil both increased Papp (AP-BL) but decreased Papp (BL-AB) for berberine, palmatine, jateorhizine, and coptisine; ER values were decreased by >50%. MK-571 had no influence on the transmembrane transport of berberine, palmatine, jateorhizine, and coptisine. At a concentration of 1–100 μM, berberine, palmatine, jateorhizine, and coptisine had no significant effects on the bidirection transport of Rho123. Berberine, palmatine, jateorhizine, and coptisine were all P-gp substrates; and at the range of 1–100 μM, berberine, palmatine, jateorhizine, and coptisine had no inhibitory effects on P-gp.

Effect of danshen extract on the activity of CYP3A4 in healthy volunteers.

AIMS To assess the effect of danshen extract on CYP3A4 activity using midazolam as an in vivo probe. METHODS A sequential, open-label, two-period pharmacokinetic interaction study design was used to compare midazolam pharmacokinetic parameters before and after 14 days of administration of danshen tablets. Twelve healthy volunteers received a single oral dose (15 mg) of midazolam followed by danshen tablets (four tablets orally, three times a day) for 14 days. On the last day of the study they received four danshen tablets with a 15 mg midazolam tablet and plasma concentrations of midazolam and its corresponding metabolite 1-hydroxylmidazolam were measured prior to and after the administration of danshen tablets periodically for 12 h. RESULTS The 90% confidence intervals of C(max,)t(1/2), CL/F and AUC(0,infinity) of midazolam before and after administration of danshen tablets were (0.559, 0.849), (0.908, 1.142), (1.086, 1.688) and (0.592, 0.921), respectively; and those of C(max), t(1/2) and AUC(0,infinity) of 1-hydroxylmidazolam after vs. before administration of danshen tablets were (0.633, 0.923), (0.801, 1.210) and (0.573, 0.980), respectively. Ratios of geometric LS means of C(max(1OHMid)) : C(max(Mid)) and AUC(max(1OHMid)) : AUC(max(Mid)) (after vs. before 14-day danshen) were 1.072 and 1.035, respectively. CONCLUSIONS Our findings suggest that multiple dose administration of danshen tablets may induce CYP3A4 in the gut. Accordingly, caution should be taken when danshen products are used in combination with therapeutic drugs metabolized by CYP3A.

Opposite Effects of Single-Dose and Multidose Administration of the Ethanol Extract of Danshen on CYP3A in Healthy Volunteers

The aim of this study was to investigate the effect of single- and multidose administration of the ethanol extract of danshen on in vivo CYP3A activity in healthy volunteers. A sequential, open-label, and three-period pharmacokinetic interaction study design was used based on 12 healthy male individuals. The plasma concentrations of midazolam and its metabolite 1-hydroxymidazolam were measured. Treatment with single dose of the extract caused the mean C max of midazolam to increase by 87% compared with control. After 10 days of the danshen extract intake, the mean AUC0–12, C max, and t 1/2 of midazolam were decreased by 79.9%, 66.6%, and 43.8%, respectively. The mean clearance of midazolam was increased by 501.6% compared with control. The in vitro study showed that dihydrotanshinone I in the extract could inhibit CYP3A, while tanshinone IIA and cryptotanshinone could induce CYP3A. In conclusion, a single-dose administration of the danshen extract can inhibit intestinal CYP3A, but multidose administration can induce intestinal and hepatic CYP3A.

Inhibitory effects of gypenosides on seven human cytochrome P450 enzymes in vitro.

Among the various possible causes for drug interactions, pharmacokinetic factors such as inhibition of drug-metabolizing enzymes, especially cytochrome P450 (CYP) enzymes, are regarded as the most frequent and clinically important. Gypenosides is widely used as functional food and over-the-counter drug in East Asia. In this study, the in vitro inhibitory effects of gypenosides on the major human CYP enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) activities in human liver microsomes were examined using liquid chromatography-tandem mass spectrometry. Gypenosides showed the strongest inhibition of CYP2D6, followed by CYP2C8, CYP3A4 and CYP2C9. The IC50 values were 1.61 μg/mL, 20.06 μg/mL, 34.76 μg/mL (CYP3A4/midazolam), 46.73 μg/mL (CYP3A4/testosterone), and 54.52 μg/mL, respectively. Gypenosides exhibited competitive inhibition of CYP2D6 (Ki=1.18). In conclusion, Gypenosides might cause herb-drug interactions via inhibition of CYP2D6. An in vivo study is needed to examine this further.

Effects of danshen ethanol extract on the pharmacokinetics of fexofenadine in healthy volunteers.

This study investigated the effect of multidose administration of danshen ethanol extract on fexofenadine pharmacokinetics in healthy volunteers. A sequential, open-label, two-period pharmacokinetic interaction design was used. 12 healthy male volunteers received a single oral dose of fexofenadine (60 mg) followed by danshen ethanol extract (1 g orally, three times a day) for 10 days, after which they received 1 g of the danshen extract with fexofenadine (60 mg) on the last day. The plasma concentrations of fexofenadine was measured by LC-MS/MS. After 10 days of the danshen extract administration, the mean AUC and C max⁡ of the fexofenadine was decreased by 37.2% and 27.4% compared with the control, respectively. The mean clearance of fexofenadine was increased by 104.9%. The in vitro study showed that tanshinone IIA and cryptotanshinone could induce MDR1 mRNA. This study showed that multidose administration of danshen ethanol extract could increase oral clearance of fexofenadine. The increased oral clearance of fexofenadine is attributable to induction of intestinal P-glycoprotein.

Metabolic Interaction of the Active Constituents of Coptis chinensis in Human Liver Microsomes

Coptis chinensis is commonly used in traditional Chinese medicine. The study investigated metabolic interaction of the active constituents (berberine, coptisine, palmatine, and jatrorrhizine) of Coptis chinensis in human liver microsomes. After incubation of the four constituents of Coptis chinensis in HLMs, the metabolism of the four constituents was observed by HPLC. The in vitro inhibition experiment between the active constituents was conducted, and IC50 value was estimated. Coptisine exhibited inhibitions against the formation of the two metabolites of berberine with IC50 values of 6.5 and 8.3 μM, respectively. Palmatine and jatrorrhizine showed the weaker inhibitory effect on the formation of the metabolites of berberine. Berberine showed a weak inhibitory effect on the production of coptisine metabolite with an IC50 value of 115 μM, and palmatine and jatrorrhizine had little inhibitory effect on the formation of coptisine metabolite. Berberine, coptisine, and jatrorrhizine showed no inhibitory effect on the generation of palmatine metabolite (IC50 > 200 μM). The findings suggested that there are different degrees of metabolic interaction between the four components. Coptisine showed the strongest inhibition toward berberine metabolism.

Simultaneous determination of ezetimibe and its glucuronide metabolite in human plasma by solid phase extraction and liquid chromatography–tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed to quantify ezetimibe (EZM) and its major glucuronide (ezetimibe glucuronide, EZM-G) in human plasma simultaneously. The analytes were purified by solid phase extraction (SPE) without hydrolysis. Separation of the analytes was achieved using acetonitrile-water (0.08% formic acid) (70:30, v/v) as the mobile phase at a flow rate of 0.8 mL/min on an Agilent Extend C18 column. The analytes were detected by LC-MS/MS using negative ionization in multiple reaction monitoring (MRM) mode. The mass transition pairs of m/z 408.4→271.0 and m/z 584.5→271.0 were used to detect EZM and EZM-G, respectively. The analytical method was linear over the concentration range of 0.1-20 ng/mL for EZM and 0.5-200 ng/mL for EZM-G. Within- and between-run precision for EZM was no more than 8.6% and 12.8%; and for EZM-G was no more than 9.0% and 8.7%, respectively. This method was reproducible and reliable, and was successfully used to analyze human plasma samples for application in a bioequivalence study.

Activation of CYP3A-mediated testosterone 6β-hydroxylation by tanshinone IIA and midazolam 1-hydroxylation by cryptotanshinone in human liver microsomes

This study evaluated the in vitro activation of CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation by tanshinone I, tanshinone IIA, and cryptotanshinone. The abilities of tanshinones to activate CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation in human liver microsomes (HLMs) were tested. Substrate- and effector-dependent activation of CYP3A by tanshinones were both observed. Cryptotanshinone was shown to activate CYP3A-mediated midazolam 1-hydroxylation in a concentration-dependent manner. In contrast, tanshinone IIA and tanshinone I did not activate this hydroxylation reaction. In addition, tanshinone IIA activated CYP3A-mediated testosterone 6β-hydroxylation, whereas cryptotanshinone and tanshinone I did not. The results from our study enhance the understanding of CYP3A activation by tanshinone IIA and cryptotanshinone in HLMs. Additionally, these data allow for an accurate prediction of the magnitude and likelihood of Danshen-drug interactions.

Metabolic characteristics of Tanshinone I in human liver microsomes and S9 subcellular fractions

Abstract Tanshinone I (TSI) is a lipophilic diterpene in Salvia miltiorrhiza with versatile pharmacological activities. However, metabolic pathway of TSI in human is unknown. In this study, we determined major metabolites of TSI using a preparation of human liver microsomes (HLMs) by HPLC-UV and Q-Trap mass spectrometer. A total of 6 metabolites were detected, which indicated the presence of hydroxylation, reduction as well as glucuronidation. Selective chemical inhibition and purified cytochrome P450 (CYP450) isoform screening experiments revealed that CYP2A6 was primarily responsible for TSI Phase I metabolism. Part of generated hydroxylated TSI was glucuronidated via several glucuronosyltransferase (UGT) isoforms including UGT1A1, UGT1A3, UGT1A7, UGT1A9, as well as extrahepatic expressed isoforms UGT1A8 and UGT1A10. TSI could be reduced to a relatively unstable hydroquinone intermediate by NAD(P)H: quinone oxidoreductase 1 (NQO1), and then immediately conjugated with glucuronic acid by a panel of UGTs, especially UGT1A9, UGT1A1 and UGT1A8. Additionally, NQO1 could also reduce hydroxylated TSI to a hydroquinone intermediate, which was immediately glucuronidated by UGT1A1. The study demonstrated that hydroxylation, reduction as well as glucuronidation were the major pathways for TSI biotransformation, and six metabolites generated by CYPs, NQO1 and UGTs were found in HLMs and S9 subcellular fractions.

Charactering the metabolism of cryptotanshinone by human P450 enzymes and uridine diphosphate glucuronosyltransferases in vitro

Cryptotanshinone (CT) is the main active component in the root of Salvia miltiorrhiza Bunge (SMB) that displays antibacterial, anti-inflammatory and anticancer activities. In this study, we characterized phase I and phase II metabolism of CT in human liver microsomes in vitro and identified the metabolic enzymes (CYPs and UGTs) involved. The metabolites of CT generated by CYPs were detected using LC-MS/MS and the CYP subtypes involved in the metabolic reactions were identified using chemical inhibitors of CYP enzymes and recombinant human CYP enzymes (CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Glucuronidation of CT was also examined, and the UGT subtypes involved in the metabolic reactions were identified using recombinant human UGT enzymes (1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17). After adding NADPH to the human liver microsomes incubation system, CT was transformed into 6 main dehydrogenation and hydroxylation metabolites. CYP2A6, CYP3A4 and CYP2C19 were the major contributors to the transformation of its hydroxylation metabolites. CYP2C19, CYP1A2 and CYP3A4 were the major contributors to the transformation of its hydrogenation metabolites in human liver microsomes. This study showed that the metabolites at m/z of 473 were mediated by UGT1A9 and that the metabolites at m/z of 489 were mediated by UGT2B7 and UGT2B4. CT was extensively metabolized by UGTs following metabolism by CYPs in the liver.