Subject(s)
Arthritis, Juvenile , Coronavirus Infections , Gastrointestinal Microbiome , Pandemics , Pneumonia, Viral/epidemiology , Betacoronavirus , COVID-19 , Child , China , Humans , SARS-CoV-2ABSTRACT
Ginkgolic acids (GAs) in natural product Ginkgobiloba L. are the pharmacological active but also toxic components. Two compounds, GA (C15:1) and GA (C17:1) are the most abundant GAs. In this study, several in vitro and in vivo models were applied to investigate transport mechanism of GAs. A rapid and sensitive LC-MS/MS method for the simultaneous determination of GA (C15:1) and GA (C17:1) was applied to analyze the biological specimens. The Papp(APâBL) values of GA (C15:1) and GA (C17:1) were 1.66-2.13×10(-)(6)cm/s and 1.34-1.85×10(-)(6)cm/s determined using MDCK and MDCK-MDR1 cell monolayers, respectively. The Papp(BLâAP) were remarkably greater in the MDCK-MDR1 cell line, which were 6.77-11.2×10(-)(6)cm/s for GA (C15:1) and 4.73-5.15×10(-)(6)cm/s for GA (C17:1). Similar results were obtained in LLC-PK1 and LLC-PK1-BCRP cell monolayers. The net efflux ratio of GA (C15:1) and GA (C17:1) in both cell models was greater than 2 and markedly reduced by the presence of Cyclosporin A (CsA) or GF120918, inhibitors of P-gp and BCRP, suggesting that GAs are P-gp and BCRP substrates. The results from a rat bioavailability study also showed that co-administrating CsA intravenously (20mg/kg) could significantly increase GA (C15:1) and GA (C17:1) AUC0-t by 1.46-fold and 1.53-fold and brain concentration levels of 1.43-fold and 1.51-fold, respectively, due to the inhibition of P-gp and BCRP efflux transporters by CsA.
Subject(s)
ATP-Binding Cassette Transporters/antagonists & inhibitors , Cyclosporine/pharmacology , Neoplasm Proteins/antagonists & inhibitors , Salicylates/pharmacokinetics , ATP Binding Cassette Transporter, Subfamily B/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Animals , Biological Availability , Biological Transport , Brain/drug effects , Brain/metabolism , Cell Culture Techniques , Cell Survival/drug effects , Dogs , LLC-PK1 Cells , Madin Darby Canine Kidney Cells , Male , Neoplasm Proteins/genetics , Rats, Sprague-Dawley , Salicylates/blood , Salicylates/toxicity , Substrate Specificity , Swine , Tissue Distribution , TransfectionABSTRACT
This paper is to report the development of a high-throughput in vitro system to screen hPXR/CAR mediated CYP2B6 drug inducers, and the application of it into the quick determination of induction activity toward CYP2B6 by various commonly used traditional Chinese medicines (TCMs) extract. Dual reporter gene assays were performed. The hPXR/CAR expression vectors and the reporter vector pGL3-CYP2B6-Luc involved in the distal and proximal promoters of CYP2B6 were co-transfected into HepG2 cells. Relative luciferase activities in cell lysate were analyzed after 48 h treatment of blank vehicle or drugs to determine the induction activity toward CYP2B6 by various commonly used TCMs extract. The positive hPXR/hCAR activators rifampicin and CITCO were applied to make sure that the reporter gene model was successfully established. Then 5 kinds of commonly used TCM extracts and 1 herbal compound were successfully investigated, some were found to activate hPXR or hCAR and therefore have the potential to induce CYP2B6 enzyme. This is the first domestic article to report the hCAR3-mediated CYP2B6 induction model and the establishment of a reporter gene system for hPXR/CAR-mediated CYP2B6 induction can be an effective and systemic in vitro method to investigate the drug inducers of CYP2B6 and to explain the mechanism involved.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Drugs, Chinese Herbal/pharmacology , Plants, Medicinal/chemistry , Receptors, Cytoplasmic and Nuclear , Receptors, Steroid , Aryl Hydrocarbon Hydroxylases/genetics , Aryl Hydrocarbon Hydroxylases/metabolism , Constitutive Androstane Receptor , Cytochrome P-450 CYP2B6 , Drugs, Chinese Herbal/isolation & purification , Genes, Reporter , Genetic Vectors , Hep G2 Cells , High-Throughput Screening Assays , Humans , Luciferases/genetics , Luciferases/metabolism , Oximes/pharmacology , Plasmids , Pregnane X Receptor , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Steroid/genetics , Receptors, Steroid/metabolism , Rifampin/pharmacology , Thiazoles/pharmacology , TransfectionABSTRACT
AIM: To investigate the stereoselective binding of mexiletine or ketoprofen enantiomers with different recombinant domains of human serum albumin (HSA). METHODS: Three domains (HSA DOM I, II and III) were expressed in Pichia pastoris GS115 cells. Blue Sepharose 6 Fast Flow was employed to purify the recombinant HSA domains. The binding properties of the standard ligands, digitoxin, phenylbutazone and diazepam, and the chiral drugs to HSA domains were investigated using ultrafiltration. The concentrations of the standard ligands, ketoprofen and mexiletine were analyzed with HPLC. RESULTS: The recombinant HSA domains were highly purified as shown by SDS-PAGE and Western blotting analyses. The standard HSA ligands digitoxin, phenylbutazone and diazepam selectively binds to DOM I, DOM II and DOM III, respectively. For the chiral drugs, R-ketoprofen showed a higher binding affinity toward DOM III than S-ketoprofen, whereas S-mexiletine bound to DOM II with a greater affinity than R-mexiletine. CONCLUSION: The results demonstrate that HSA DOM III possesses the chiral recognition ability for the ketoprofen enantiomers, whereas HSA DOM II possesses that for the mexiletine enantiomers.