Breast cancer resistance protein-mediated efflux of luteolin glucuronides in HeLa cells overexpressing UDP-glucuronosyltransferase 1A9.

PURPOSE: UDP-glucuronosyltransferases (UGTs) are responsible for the formation of glucuronides of polyphenolic flavonoids. This study investigated the UGT1A9-mediated glucuronidation of luteolin and the kinetics of luteolin glucuronide efflux. METHOD: HeLa cells overexpressing UGT1A9 (HeLa-UGT1A9) were used to determine the kinetics of breast cancer resistance protein (BCRP)-mediated transport of luteolin glucuronides. Human UGT isoforms were used to determine glucuronidation rates. RESULTS: UGT1A9 was found to catalyze the production of four luteolin glucuronides, including three known monoglucuronides and a novel 3', 4'-diglucuronide. Ko143, a potent specific inhibitor of BCRP, significantly inhibited efflux of luteolin monoglucuronides from HeLa1A9 cells and increased their intracellular levels in a dose-dependent manner. The formation of luteolin diglucuronide was observed when intracellular concentration of total monoglucuronides went above 0.07 nM. CONCLUSIONS: Intracellular accumulation of diglucuronide was detected at high monoglucuronide concentrations (>0.07 nM). Diglucuronide production is speculated to be a compensatory pathway for luteolin disposition.

Pharmacokinetic characterization of oxymatrine and matrine in rats after oral administration of radix Sophorae tonkinensis extract and oxymatrine by sensitive and robust UPLC-MS/MS method.

The purpose of this study is to systematically investigate the pharmacokinetic (PK) behaviors of radix Sophorae tonkinensis (S. tonkinensis) using oxymatrine (OMT) and matrine (MT) as the target markers (2 mg/kg OMT and 1.3 mg/kg MT, oral administration). The PK characteristics in radix S. tonkinensis extracts were also compared with those of pure OMT. A fast ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed. OMT absorption was very fast, and no significant differences were observed (p>0.05) in tmax, CL, and t1/2 for both pure OMT and extracts. Cmax and AUC0→∞ of pure OMT were significantly higher than those of S. tonkinensis extracts (Cmax, 61.64±6.65 vs. 43.24±10.14 ng/mL; AUC, 9894.48±2234.99 vs. 4730.30±3503.8 min ng/mL) (p<0.05). However, the absolute OMT bioavailability of pure OMT was higher than that of the compound in radix S. tonkinensis extracts (6.79±2.52% vs. 1.87±2.66%). By contrast, the bioavailability of total alkaloids (OMT+MT) after pure OMT administration was 81.14±8.83%, similar to that of radix S. tonkinensis extracts (69.36±17.37%) (p>0.05). It was presumed that OMT absorption has no effect on the bioavailability of the two alkaloids. Other constituents in radix S. tonkinensis extracts can influence the transformation of OMT to MT, which directly leads to variations in the PK behavior of OMT. In addition, the protein binding of OMT and MT in plasma was very low (4.80%-8.95% for OMT, 5.10-10.55% for MT). In conclusion, OMT in radix S. tonkinensis extracts exhibits different PK behaviors with pure OMT through the transformation of OMT to MT due to other complex ingredients.

Monoester-Diterpene Aconitum Alkaloid Metabolism in Human Liver Microsomes: Predominant Role of CYP3A4 and CYP3A5.

Aconitum, widely used to treat rheumatoid arthritis for thousands of years, is a toxic herb that can frequently cause fatal cardiac poisoning. Aconitum toxicity could be decreased by properly hydrolyzing diester-diterpene alkaloids into monoester-diterpene alkaloids. Monoester-diterpene alkaloids, including benzoylaconine (BAC), benzoylmesaconine (BMA), and benzoylhypaconine (BHA), are the primary active and toxic constituents of processed Aconitum. Cytochrome P450 (CYP) enzymes protect the human body by functioning as the defense line that limits the invasion of toxicants. Our purposes were to identify the CYP metabolites of BAC, BMA, and BHA in human liver microsomes and to distinguish which isozymes are responsible for their metabolism through the use of chemical inhibitors, monoclonal antibodies, and cDNA-expressed CYP enzyme. High-resolution mass spectrometry was used to characterize the metabolites. A total of 7, 8, and 9 metabolites were detected for BAC, BMA, and BHA, respectively. The main metabolic pathways were demethylation, dehydrogenation, demethylation-dehydrogenation, hydroxylation and didemethylation, which produced less toxic metabolites by decomposing the group responsible for the toxicity of the parent compound. Taken together, the results of the chemical inhibitors, monoclonal antibodies, and cDNA-expressed CYP enzymes experiments demonstrated that CYP3A4 and CYP3A5 have essential functions in the metabolism of BAC, BMA, and BHA.