The inhibition mechanism of the uptake of lamivudine via human organic anion transporter 1 by Stellera chamaejasme L. extracts.

Stellera chamaejasme L. is a traditional Chinese medicine with a long history to treat stubborn skin ulcer, and it also has antiviral and antitumor effects. Neochamaejasmine B (NCB), Neochamaejasmine A (NCA) and Chamaechromone (CMC) are the major components in dried roots of Stellera chamaejasme L.. Our studies suggested that NCB, NCA and CMC are inhibitors of Organic anion transporter 1 (OAT1). OAT1 is encoded by solute carrier family 22 member 6 gene (SLC22A6) in humans and plays a critical role in the organic anion drug uptake and excretion in the kidney. Lamivudine is the typical substrate of OAT1 and is frequently used in combination with other antiviral drugs in clinical antiviral treatments. The aim of this study is to investigate the interaction and its mechanism between these bi-flavone components in Stellera chamaejasme L. and lamivudine via OAT1 both in vitro and in vivo. In vitro, the uptake studies in Madin-Darby canine kidney (MDCK) cells overexpressing OAT1 suggested that NCB inhibited the uptake of 6-CFL and lamivudine.Similar results were obtained for NCA and CMC. NCB was a noncompetitive and competitive inhibitor interaction with OAT1. IC50 values of NCB, NCA and CMC for inhibiting OAT1-mediated lamivudine transport were 2.46, 8.35 and 0.61 µmol·L-1, respectively. In vivo, the pharmacokinetic results of lamivudine in rats showed that the mean area under the plasma concentration-time curve (AUC0-∞) and maximal plasma concentration (Cmax) of lamivudine after co-administration is increased 2.94-fold and 1.87-fold, respectively, compared to lamivudine administration alone. The results of interactions between lamivudine and these bi-flavone components in Stellera chamaejasme L. extracts via OAT1 in vivo are consistent with studies in vitro. The inhibition of OAT1-mediated uptake of lamivudine by NCB, NCA and CMC is the possible mechanism for Stellera chamaejasme L. extracts improving the oral bioavailability of lamivudine in rats.

The inhibition mechanism of the uptake of lamivudine via human organic anion transporter 1 by Stellera chamaejasme L. extracts / 中国天然药物

Stellera chamaejasme L. is a traditional Chinese medicine with a long history to treat stubborn skin ulcer, and it also has antiviral and antitumor effects. Neochamaejasmine B (NCB), Neochamaejasmine A (NCA) and Chamaechromone (CMC) are the major components in dried roots of Stellera chamaejasme L.. Our studies suggested that NCB, NCA and CMC are inhibitors of Organic anion transporter 1 (OAT1). OAT1 is encoded by solute carrier family 22 member 6 gene (SLC22A6) in humans and plays a critical role in the organic anion drug uptake and excretion in the kidney. Lamivudine is the typical substrate of OAT1 and is frequently used in combination with other antiviral drugs in clinical antiviral treatments. The aim of this study is to investigate the interaction and its mechanism between these bi-flavone components in Stellera chamaejasme L. and lamivudine via OAT1 both in vitro and in vivo. In vitro, the uptake studies in Madin-Darby canine kidney (MDCK) cells overexpressing OAT1 suggested that NCB inhibited the uptake of 6-CFL and lamivudine.Similar results were obtained for NCA and CMC. NCB was a noncompetitive and competitive inhibitor interaction with OAT1. IC values of NCB, NCA and CMC for inhibiting OAT1-mediated lamivudine transport were 2.46, 8.35 and 0.61 μmol·L, respectively. In vivo, the pharmacokinetic results of lamivudine in rats showed that the mean area under the plasma concentration-time curve (AUC) and maximal plasma concentration (C) of lamivudine after co-administration is increased 2.94-fold and 1.87-fold, respectively, compared to lamivudine administration alone. The results of interactions between lamivudine and these bi-flavone components in Stellera chamaejasme L. extracts via OAT1 in vivo are consistent with studies in vitro. The inhibition of OAT1-mediated uptake of lamivudine by NCB, NCA and CMC is the possible mechanism for Stellera chamaejasme L. extracts improving the oral bioavailability of lamivudine in rats.

High-performance liquid chromatography-diode array detector in determination of five flavonoids in Stellera chamaejasme L / 第二军医大学学报

Objective To develop a high-performance liquid chromatography (HPLC) method for simultaneous determination of five flavonoids (chamaechromone, neochamaejasmin A, chamaejasmine, isochamaejasmin and 7-methoxylneochaejasmin A) in the extraction of Stellera chamaejasme L.. Methods An HPLC-diode array detector (DAD) method was established. The HPLC-DAD condition was as follows: chromatographic column was Agilent Eclipse Plus C18 (3.0 mm×100 mm, 3.5 μm); mobile phase A was 0.1% formic acid-water solution; mobile phase B was acetonitrile; gradient of acetonitrile was 20% (0-6 min), 20%-35% (6-10 min) and 35% (10-50 min); flow rate was 0.4 mL/min; equilibration time was 10 min; acquisition time was 50 min; column temperature was 25 ℃; DAD detection wavelength was 290 nm; and sample injection volume was 5 μL. Results Chamaechromone, neochamaejasmin A, chamaejasmine, isochamaejasmin and 7-methoxylneochaejasmin A were successfully separated using this method, with good linear relationship between 4.680-468.0, 2.016-201.6, 3.784-378.4, 5.520-552.0 and 0.974-97.40 µg/mL, respectively. The precision, stability, repeatability and recovery of the five flavonoids were good with this method. Conclusion HPLC-DAD has good stability and repeatability, and can be used to determine the contents of five flavonoids (chamaechromone, neochamaejasmin A, chamaejasmine, isochamaejasmin and 7-methoxylneochaejasmin A) in Stellera chamaejasme L.

Neochamaejasmin B increases the bioavailability of chamaechromone coexisting in Stellera chamaejasme L. via inhibition of MRP2 and BCRP.

Chamaechromone and neochamaejasmin B (NCB) are the most abundant components in the dried roots of the toxic perennial herb Stellera chamaejasme L. and have pharmacological activities. The objective of this study was to investigate the transport mechanism of these two components in vivo and in vitro. The transport and cellular accumulation studies in Madin-Darby canine kidney (MDCK) cells overexpressing human multidrug resistance protein 2 (MRP2) or P-gp and LLC-PK1 cells overexpressing human breast cancer resistance protein (BCRP) were performed. The results showed that chamaechromone was a good substrate of MRP2 and BCRP but not a substrate of P-gp. NCB was found to be a MRP2 inhibitor in transfected cells and significantly enhanced the cellular accumulation of chamaechromone in MDCK cells overexpressing MRP2. Similar results were obtained in LLC-PK1-BCRP cells. In addition, the influence of NCB on the bioavailability of chamaechromone following their co-administration was also determined in rats. The results showed that the area under the plasma concentration-time curve and maximal plasma concentration of chamaechromone in rats were increased by 48.9% and 81.9%, respectively. The mechanism of improving the oral bioavailability of chamaechromone was attributable to the inhibition of the BCRP and MRP2-mediated efflux of chamaechromone by NCB.

Metabolites characterization of chamaechromone in vivo and in vitro by using ultra-performance liquid chromatography/Xevo G2 quadrupole time-of-flight tandem mass spectrometry.

ETHNOPHARMACOLOGICAL RELEVANCE: Stellera chamaejasme L. (Thymelaeaceae) was a toxic perennial herb and widely used as pesticide and dermatological agents in China. Chamaechromone was a major component in the dried roots of Stellera chamaejasme with anti-HBV and insecticidal activity. Analysis of metabolic profile in vivo and in vitro plays a pivotal role to unravel how TCM works. And the metabolites of chamaechromone might influence the effects and toxicity of Stellera chamaejasme. Moreover, the metabolic routes of chamaechromone provide an important basis for toxicological safety evaluation. Until now, little is known about the metabolism of chamaechromone. The current study was designed to characterize the whole metabolic pathways of chamaechromone in vitro and in vivo. MATERIALS AND METHODS: Twenty-four rats were randomly divided into four groups, including two oral administration groups (100mgkg(-1)), one intravenous injection group (5 mgkg(-1)), and one control group. The metabolites in rat urine and feces and bile were identified by UPLC/Q-TOF MS analysis and ß-glucuronidase hydrolysis. Moreover, the possible metabolic mechanism was further confirmed by Phase I and Phase II metabolism and catechol-O-methyltransferase methylation in rat liver S9 fraction and degradation in rat intestinal bacteria. RESULTS: A total of 24 metabolites from chamaechromone were detected and identified in vivo and in vitro, 20 of which were novel. And the major metabolic processes were hydroxylation, methylation, glucuronation, acetylation, dehydroxylation and degradation. CONCLUSIONS: The present study revealed the whole metabolic pathways of chamaechromone in rat through both in vitro and in vivo experiments for the first time. And chamaechromone could undergo extensive phase I and phase II metabolism in rat. These findings would provide an important basis for the further study and clinical application of chamaechromone. In addition, the results of this work have showed the feasibility of the UPLC/Q-TOF-MS approach for rapid and reliable characterization of metabolites.

Determination of chamaechromone in rat urine and feces by HPLC-MS and the excretion study / 中国药学杂志

OBJECTIVE: To develop a new method for the determination of chamaechromone in urine and feces of rats and investigate its excretion characteristics. METHODS: A liquid chromatography tandem mass spectrometry (LC-MS) method was developed. Chromatographic separation was performed on Xbridge™G18 column (2.1 mm×50 mm, 3.5 μm) with linear gradient elution u-sing water and methanol, both of which were acidified with 0.1% aqueous formic acid. The flow rate was 0.4 mL·min-1. Detection was performed on a triple-quadrupole tandem mass spectrometer using positive ion mode electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The monitored MS/MS ion transitions were m/z 543.3→198.9 and 481.9→258.3 for chamaechromone and rosuvastatin, respectively. RESULTS: Good linearity was observed over the concentration range of 8-6 400 ng·mL-1 in 0.1 mL of both rat urine and feces. The limit of detection (LOD) was 4 ng·mL: The intra-assay and inter-assay variabilities were less than 14% in both urine and feces. Cumulative urinary and fecal excretion ratios within 48 h at a dose of 100 mg·kg-1 after oral administration of chamaechromone accounted for (1.84±0.290)% and (41.5±6.917)% of the dose respectively. CONCLUSION: This method shows excellent sensitivity, precision, and accuracy, and is successfully applied to evaluate the excretion of chamaechromone in urine and feces of rats. The results indicate that feces are the major excretion route of chamaechromone after oral administration. And the most efficient excretion happens in 12-24 h after administration.