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Objective To establish a LC-MS/MS assay for simultaneous determination of 25 psychotropic drugs in human blood and apply the method to analyze the relationship between(driving under the inference of drugs, DUID)and traffic violations. Methods Acetonitrile was added to the sample for precipitating the protein, the mixture was centrifuged for 10min at 4℃ , 14000r/min, the supernatant was filtered, and then the filtrate was analyzed with LC-MS/MS. The separation was performed on C18 chromatography column (50mm×3.0mm, 2.6μm), the mobile phase was consisted of 0.1% formic acid (phase A), acetonitrile:methanol=1:1 (phase B) with gradient elution. Results The calibration curves for 25 psychotropic drugs were linear over the ranges of 0.05~20ng/mL, R=0.9944~0.9996, the assay recoveries were 83.0%~99.7%, the method recoveries were 80.2%~97.4%, the intraday and interday precious were 1.6%~14% and 3.1%~14% respectively. The method was applied to analyze 3140 human samples stored by Center of Forensic Sciences of Hangzhou. Conclusion The method is sensitive and accurate for fast detection of 25 psychotropic drugs in human blood.
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To establish single- and double-transfected transgenic cells stably expressing hMATE1, hMATE1 cDNA was cloned by RT-PCR from human cryopreserved kidney tissue, and subcloned into pcDNA3.1(+) plasmid by virtue of both HindIII and Kpn I restriction enzyme sites. Subsequently, the recombined pcDNA3.1(+)- hMATE1 plasmid was transfected into MDCK, MDCK-hOCT1 or MDCK-hOCT2 cells using Lipofectamine 2000 Reagent. After a 14-day-cultivation with hygromycin B at the concentration of 400 µg · mL(-1), all clones were screened with DAPI and MPP+ as substrates to identify the best candidate. The mRNA content of hMATE1, the cellular accumulation of metformin with or without cimetidine as inhibitor, or transportation of cimetidine was further valuated. The results showed that all of the three cell models over expressed hMATE1 mRNA. The cellular accumulation of metformin in MDCK-hMATE1 was 17.6 folds of the control cell, which was significantly inhibited by 100 µmol · L(-1) cimetidine. The transcellular transport parameter net efflux ratios of cimetidine across MDCK-hOCT1/hMATE1 and MDCK-hOCT2/hMATE1 monolayer were 17.5 and 3.65, respectively. In conclusion, cell models with good hMATE1 function have been established successfully, which can be applied to study the drug transport or drug-drug interaction involving hMATE1 alone or together with hOCT1/2 in vitro.
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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.
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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.
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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.
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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.
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Objective To study and compare the metabolism of Chrysanthemum morifolium extracts(CME) in vitro intestinal flora from human,rat,Beagle dog,and rabbit.Methods Intestinal flora of human,rat,Beagle dog,and rabbit and CME were anaerobically cultured at 37 ℃ in vitro. After being extracted by acetic ether,the main metabolites of CME were separated and quantified by HPLC.Results In vitro,CME was easily metabolized by the intestinal flora and the main metabolites in incubation medium were determined in high concentration after 0.5 h.By LC-MS,luteolin and apigenin were identified,respectively;both metabolites were degraded with prolongation of incubation time and the concentrations of luteolin and apigenin were low after 24 h;moreover,CME was quickly metabolized by human,rat,Beagle dog intestinal flora,and gently by rabbit intesinal flora.Conclusion In vitro,CME is easily metabolized by intestinal flora human,rat,Beagle dog,and rabbit and the main metabolites are luteolin and apigenin.
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extraction hours. No significant difference of flavonoids content was observed among the different collecting time of C. morifolium. Conclusion The optimum extraction conditions were 75% ethanol, refluence for 1 5 h at 100 ℃ water bath for three times. The flavonoids would not be bound up with the harvest time.
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AIM: To investigate the effect of Dendranthema morifolium (Ramat) Tzvel (DM) on isolated rat heart and ventricular myocytes during ischemia/anoxia and reperfusion/reoxygenation.METHODS: The Langendorff perfused rat hearts were used to measure intraventricular pressure and coronary flow. The cell contraction and intracellular calcium transient in enzymatically isolated ventricular myocytes were determined. RESULTS: (1) DM (0.5 g/L) significantly attenuated the inhibitory effects induced by ischemia/reperfusion on left ventricular developed pressure (LVDP), ?dp/dt max, coronary flow and LVDP?HR, meanwhile increased the content of SOD and decreased the content of MDA in the myocardium; (2) DM (0.5 g/L) attenuated the inhibitory effects of anoxia and reoxygenation on [Ca 2+]i transient and cell contraction in isolated ventricular myocytes. CONCLUSION: DM attenuated the effects on contractility and intracellular calcium induced by ischemia/anoxia and reperfusion/reoxygenation in the isolated rat heart and the ventricular myocytes. The mechanism might be related to increase in SOD activity and maintaining [Ca 2+]i homeostasis.
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AIM: To study the effect of salvia miltiorrhiza (SM) on cell contraction and intracellular calcium of enzymatically isolated rat ventricular myocytes during normoxia and anoxia/reoxygenation. METHODS: Contraction and intracellular calcium were determined with video tracking system and spectrofluorometric method, and the chemical anoxic method was employed. RESULTS: The ?d L /d t max , dL of cell contraction and the amplitude of [Ca 2+ ] i in the cardiomyocytes following SM treatment were decreased in a dose-dependent manner. During anoxia, the ?d L /d t max , dL and amplitude of [Ca 2+ ] i were decreased, while the diastolic Ca 2+ level was elevated compared with control group. All the contractile parameters and the diastolic Ca 2+ level were back toward pretreatment values during reoxygenation, but could not return to control level. After the treatment with SM (3 g/L), ?d L /d t max and dL of cell contraction and the amplitude of [Ca 2+ ] i were higher and the diastolic Ca 2+ level was lower than those in anoxia/reoxygenation group. CONCLUSION: SM antagonized effects of anoxia and reoxygenation on cell contraction and intracellular calcium in isolated ventricular myocytes.
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AIM: To investigate the vasorelaxant effect and mechanism of EtOAc extract from Chrysanthemum morifolium Ramat (CME). METHODS: The effects of CME on the contraction of rat thoracic a orta were examined. RESULTS: CME caused concentration-dependent relaxation of aorta rings precontricted with phenylephrine and K+. The effect in endothelium-intac t aorta was more effective than that in endothelium-deduced aorta. NG-nitro-L- arginine methylester, methylene blue and glibenclamide attenuated the effect of C ME significantly. However, indomethacin, propranolol, tetraethylammonium, BaCl 2, 4-aminopyridine and 5-hydroxydecanoate did not affect CME effect. The effect of SKF-525A combined with L-NAME had no obvious difference with that of L-NAME o n CME-induced relaxation. NOS activity in aorta was increased markedly by CME in vitro. CME did not reduced the contraction elicited by PE in Ca 2+-f ree medium, but reduced the contraction induced by PE in K+-free solution or C a 2+ free following input Ca 2+. CONCLUSION: CME induces both endothelium-dependent and independe nt relaxation. NO and cGMP are likely involved in the endothelium-dependent rela xation, inhibition of voltage-dependent or receptor-operate Ca 2+ channel a nd activation of ATP-sensitive K+ channel contribute in part to the endotheliu m-independent relaxation by CME.