Development of a UHPLC-MS/MS method for the quantification of ilaprazole enantiomers in rat plasma and its pharmacokinetic application / 药物分析学报

In Korea and China, ilaprazole is a widely used proton pump inhibitor in the treatment of gastric ulcers. In this study, a specific and sensitive LC-MS/MS method has been developed and validated for the quantification of ilaprazole enantiomers in the rat plasma, using R-lansoprazole as the internal standard. The enantioseparation was achieved on a CHIRALPAK AS-RH column (4.6 mm × 150 mm, i.d. 5μm), with a mobile phase composed of 10 mM ammonium acetate aqueous solution and acetonitrile (60:40, V/V), at a flow-rate of 0.5 mL/min. The method was validated over the concentration range of 0.5-300 ng/mL for both, R- and S -ilaprazole. The lower limit of quantification was 0.5 ng/mL for both enantiomers. The relative standard deviation (RSD) of intra- and inter-day precision of R-ilaprazole and S-ilaprazole was less than 10.9％, and the relative error accuracy (RE) ranged from -0.5％-2.0％. Finally, the method was successfully evaluated in rats in a stereoselective pharmacokinetic study of the ilaprazole racemate.

Upregulation of miR-489-3p and miR-630 inhibits oxaliplatin uptake in renal cell carcinoma by targeting OCT2

Renal cell carcinoma (RCC) is one of the most common malignant tumors affecting the urogenital system, accounting for 90% of renal malignancies. Traditional chemotherapy options are often the front-line choice of regimen in the treatment of patients with RCC, but responses may be modest or limited due to resistance of the tumor to anticarcinogen. Downregulated expression of organic cation transporter OCT2 is a possible mechanism underlying oxaliplatin resistance in RCC treatment. In this study, we observed that miR-489-3p and miR-630 suppress OCT2 expression by directly binding to the OCT2 3'-UTR. Meanwhile, 786-O-OCT2-miRNAs stable expression cell models, we found that miRNAs could repress the classic substrate 1-methyl-4-phenylpyridinium (MPP), fluorogenic substrate ,-dimethyl-4-(2-pyridin-4-ylethenyl) aniline (ASP), and oxaliplatin uptake by OCT2 both and in xenografts. In 33 clinical samples, miR-489-3p and miR-630 were significantly upregulated in RCC, negatively correlating with the OCT2 expression level compared to that in adjacent normal tissues, using tissue microarray analysis and qPCR validation. The increased binding of c-Myc to the promoter of pri-miR-630, responsible for the upregulation of miR-630 in RCC, was further evidenced by chromatin immunoprecipitation and dual-luciferase reporter assay. Overall, this study indicated that miR-489-3p and miR-630 function as oncotherapy-obstructing microRNAs by directly targeting OCT2 in RCC.

Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

RNA interference (RNAi) is useful for selective gene silencing. Cytochrome P450 3A4 (CYP3A4), which metabolizes approximately 50% of drugs in clinical use, plays an important role in drug metabolism. In this study, we aimed to develop a short hairpin RNA (shRNA) to modulate CYP3A4 expression. Three new shRNAs (S1, S2 and S3) were designed to target the coding sequence (CDS) of CYP3A4, cloned into a shRNA expression vector, and tested in different cells. The mixture of three shRNAs produced optimal reduction (55%) in CYP3A4 CDS-luciferase activity in both CHL and HEK293 cells. Endogenous CYP3A4 expression in HepG2 cells was decreased about 50% at both mRNA and protein level after transfection of the mixture of three shRNAs. In contrast, CYP3A5 gene expression was not altered by the shRNAs, supporting the selectivity of CYP3A4 shRNAs. In addition, HepG2 cells transfected with CYP3A4 shRNAs were less sensitive to Ginkgolic acids, whose toxic metabolites are produced by CYP3A4. These results demonstrate that vector-based shRNAs could modulate CYP3A4 expression in cells through their actions on CYP3A4 CDS, and CYP3A4 shRNAs may be utilized to define the role of CYP3A4 in drug metabolism and toxicity.

Establishment of in vitro evaluation model for CYP2B6 induction and its application to screen inducers among TCMs / 药学学报

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.

Analysis of chiral non-steroidal anti-inflammatory drugs flurbiprofen, ketoprofen and etodolac binding with HSA / 药物分析学报

The protein binding of non-steroidal anti-inflammatory drugs flurbiprofen, ketoprofen and etodolac with human serum albumin （HSA） was investigated using indirect chiral high performance liquid chromatography （HPLC） and ultrafiltration techniques. S-（-）-1-（1-naphthyl）- ethylamine （S-NEA） was utilized as chiral derivatization reagent and pre-column derivatization RP-HPLC method was established for the separation and assay of the three pairs of enantiomer. The method had good linear relationship over the investigated concentration range without interference. The average extraction efficiency was higher than 85% in different systems, and the intra-day and inter-day precisions were less than 15%. In serum albumin, the protein binding of etodolac enantiomers showed significant stereoselectivity that the affinity of S-enantiomer was stronger than R-enantiomer, and the stereoselectivity ratio reached 6.06; Flurbiprofen had only weak stereoselectivity in HSA, and ketoprofen had no stereoselectivity at all. Scatchard curves showed that all the three chiral drugs had two types of binding sites in HSA.

CYP2D6*1, CYP2D6*10 co-expressed with CYPOR in Bac-to-Bac expression system and activity determination / 药学学报

CYP2D6 is an important drug-metabolizing enzyme. The polymorphism of CYP2D6 leads to metabolism difference and the different reactions of drugs in the individuals and different races are normal phenomenon in clinical medication. CYP2D6*10 is an important subtype in Asian people and 51.3% Chinese are classified with this subtype. To obtain recombinant active CYP2D6*1/CYP2D6*10 in baculovirus system by optimizing coexpression with CYPOR, and detect their activity to catalyze dextromethorphan, three recombinants pFastBac-CYP2D6*1, pFastBac-CYP2D6*10 and pFastBac-CYPOR were constructed and transformed into DH10Bac cell to obtain the recombinant Bacmid-CYPOR, Bacmid-CYP2D6*1 and Bacmid-CYP2D6*10. And then the recombinant CYP2D6*1 and CYP2D6*10 virus were obtained by transfecting Sf9. Then homogenate protein activity was determined with dextromethorphan as substrate. The multiple of infection (MOI) and its ratio of recombinant CYP2D6 virus to CYPOR virus were adjusted by detecting the activity of the homogenate protein. The Km and Vmax are 26.67 +/- 2.71 micromol x L(-1) (n=3) and 666.7 +/- 56.78 pmol x nmol(-1) (CYP2D6) x min(-1) (n=3) for CYP2D6*1 to catalyze dextromethaphan. The Km and Vmax are 111.36 +/- 10.89 micromol x L(-1) (n=3) and 222.2 +/- 20.12 pmol x nmol(-1) (CYP2D6) x min(-1) (n=3) for CYP2D6*10 to catalyze dextromethorphan. There is significant difference between CYP2D6*1 and CYP2D6*10 for Vmax and Km (P < 0.01). The clearance ratio of CYP2D6*1 is 25.0 and the clearance ratio of CYP2D6*10 is 2.0. The expressed CYP2D6*1 and CYP2D6*10 are useful tools to screen the metabolism profile of many xenobiotics and endobiotics in vitro, which are benefit to understand individual metabolism difference.

The role of ADME evaluation in translation research of innovative drug / 药学学报

New Chemical Entities (NCEs) development is a systematic long-term project that involves multiple disciplines. The translation research will help to build an advanced R&D system from the basic laboratory research, preclinical studies and clinical evaluation to clinical application of drug, for the purpose of shortening the R&D cycle and accelerate the launch of new drugs. In new drug R&D and its clinical application, drug disposition (absorption, distribution, metabolism, excretion, ADME) properties are important criteria for assessing drug-likeness of candidates. ADME evaluation of NCEs plays an important role in the translation research throughout innovative drug R&D process. Therefore, ADME evaluation at the early stage of drug design and development will be helpful to improve the success rate and reduce costs, and further access to safe, effective drugs.

Analysis of chiral non-steroidal anti-inflammatory drugs flurbiprofen, ketoprofen and etodolac binding with HSA / 药物分析学报

The protein binding of non-steroidal anti-inflammatory drugs flurbiprofen,ketoprofen and etodolac with human serum albumin (HSA) was investigated using indirect chiral high performance liquid chromatography (HPLC) and ultrafiltration techniques.S-(-)-2-(1-naphthyl)-ethylamine (S-NEA) was utilized as chiral derivatization reagent and pre-column derivatization RP-HPLC method was established for the separation and assay of the three pairs of enantiomer.The method had good linear relationship over the investigated concentration range without interference.The average extraction efficiency was higher than 85％ in different systems,and the intra-day and inter-day precisions were less than 15％.In serum albumin,the protein binding of etodolac enantiomers showed significant stereoselectivity that the affinity of S-enantiomer was stronger than R-enantiomer,and the stereoselectivity ratio reached 6.06; Flurbiprofen had only weak stereoselectivity in HSA,and ketoprofen had no stereoselectivity at all.Scatchard curves showed that all the three chiral drugs had two types of binding sites in HSA.