Development and validation of an LC-MS/MS method for the determination of a novel thienoquinolin urea transporter inhibitor PU-48 in rat plasma and its application to a pharmacokinetic study.

A specific, sensitive and stable high-performance liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitative determination of methyl 3-amino-6-methoxythieno [2,3-b]quinoline-2-carboxylate (PU-48), a novel diuretic thienoquinolin urea transporter inhibitor in rat plasma. In this method, the chromatographic separation of PU-48 was achieved with a reversed-phase C18 column (100 × 2.1 mm, 3 µm) at 35°C. The mobile phase consisted of acetonitrile and water with 0.05% formic acid added with a gradient elution at flow rate of 0.3 mL/min. Samples were detected with the triple-quadrupole tandem mass spectrometer with multiple reaction monitoring mode via electrospray ionization source in positive mode. The retention time were 6.2 min for PU-48 and 7.2 min for megestrol acetate (internal standard, IS). The monitored ion transitions were mass-to-charge ratio (m/z) 289.1 → 229.2 for PU-48 and m/z 385.3 → 267.1 for the internal standard. The calibration curve for PU-48 was linear over the concentration range of 0.1-1000 ng/mL (r2 > 0.99), and the lower limit of quantitation was 0.1 ng/mL. The precision, accuracy and stability of the method were validated adequately. The developed and validated method was successfully applied to the pharmacokinetic study of PU-48 in rats.

[Two types of MWNTs with different surface modifications induce differential expression of proteins in RAW264.7 cells].

OBJECTIVE: To compare the different expression of protein in RAW264.7 macrophage cells induced by two types of MWNTs (multi-walled carbon nanotubes) with different surface modifications (acid-treated MWNTs and tau-MWNTs modified by taurine). METHODS: Treating cells with both types of MWNTs in 20 mg/L and 24 h, with a blank-control group set. Cells are lysed by using urea and by ultrasonicating in ice bath, then total proteins of cells are extracted. Using two-dimensional gel electrophoresis to separate total proteins of cells, searching for the differential expressed protein spots on the images of the gels with silver staining. Identifying the differentially expressed proteins via mass spectrometry, and studying the mechanism of effects on cells imposed by two types of MWNTs at protein level. RESULTS: There are 13 spots of protein with notably differential expression among three treated groups (including blank controls). Their functions involve apoptosis-related, calcium-binding, cell-cycle related, DNA synthesis, folding of proteins, and energy metabolism, etc. The results are consistent with our previous studies about the cytotoxicity of Both types of MWNTs including induction of apoptosis and mitochodira damage. CONCLUSION: Both two types of MWNTs could induce alteration of protein expression in RAW264.7 cells. With different surface modifications, they imposed different effects. High throughout proteomics could be applied in toxicity assessment and mechanism investigation about carbon nanotubes.

Pharmacokinetics, Tissue Distribution and Excretion of a Novel Diuretic (PU-48) in Rats.

Methyl 3-amino-6-methoxythieno [2,3-b] quinoline-2-carboxylate (PU-48) is a novel diuretic urea transporter inhibitor. The aim of this study is to investigate the profile of plasma pharmacokinetics, tissue distribution, and excretion by oral dosing of PU-48 in rats. Concentrations of PU-48 within biological samples are determined using a validated high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. After oral administration of PU-48 (3, 6, and 12 mg/kg, respectively) in self-nanomicroemulsifying drug delivery system (SNEDDS) formulation, the peak plasma concentrations (Cmax), and the area under the curve (AUC0⁻∞) were increased by the dose-dependent and linear manner, but the marked different of plasma half-life (t1/2) were not observed. This suggests that the pharmacokinetic profile of PU-48 prototype was first-order elimination kinetic characteristics within the oral three doses range in rat plasma. Moreover, the prototype of PU-48 was rapidly and extensively distributed into thirteen tissues, especially higher concentrations were detected in stomach, intestine, liver, kidney, and bladder. The total accumulative excretion of PU-48 in the urine, feces, and bile was less than 2%. This research is the first report on disposition via oral administration of PU-48 in rats, and it provides important information for further development of PU-48 as a diuretic drug candidate.

[Chemotactic and mitogenic effect of rat chemokine-like factor 1 on aortic smooth muscle cells].

OBJECTIVE: To study the effects of rat chemokine-like factor 1 (rCklf1) on chemotaxis and proliferation of rat aortic smooth muscle cells(ASMC). METHODS: The recombinant eukaryotic expression vector pcDNA3.1/rCklf1 was transiently transfected into 293T cells. The supernatants were harvested for chemotactic and proliferation assays. pcDNA3.1/rCklf1 was also transfected into ASMC and the proliferation of transfected cells was detected by MTT assays. RESULTS: The 10 fold diluted supernatants of pcDNA3.1/rCklf1 transfected 293T cells had chemotactic effects on rat ASMC, which could be inhibited by pertussis toxin at the concentration of 10 microg/L. Furthermore, the 10 fold diluted supernatants of rCklf1 transfected 293T cells promoted the proliferation of ASMC (1.3 fold compared with the control) and transient overexpression of rCklf1 in ASMC had promoting activities on the proliferation of rat ASMC (1.5 fold compared with the control at 72 h). CONCLUSION: rCklf1 has mitogenic and GiPCR-dependent chemotactic effects on rat ASMC,indicating that rCklf1 may be involved in the pathological process of atherosclerosis.

[Difference analysis on proteome of Helicobacter pylori in patients with peptic ulcer, gastritis, and gastric cancer].

OBJECTIVE: To identify the different proteins of Helicobacter pylori (H. pylori) in gastric cancer, peptic ulcer, and gastritis initially. METHODS: H. pylori in the endoscopic biopsy specimens of gastric mucosa of patients with gastric cancer, peptic ulcer, or gastritis, 3 specimens for each disease, were separated and cultured. The whole-cell protein of the H. pylori was extracted by lysis buffer and sonication. The protein concentration of the bacteria cell lysates was measured by the Bradford method. The protein maps of H. pylori were obtained by two-dimensional gel electrophoresis (2-DE) and the different proteins in gastric cancer, peptic ulcer and gastritis were analyzed by Image Master v 5.0. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) were performed to identify the different proteins. The differential proteins were searched by the Mascot database at www.matrixscience.com. RESULTS: Four protein spots of H. pylori were over-expressed in the protein maps from gastric cancer in comparison with those from peptic ulcer and gastritis. Mass identification showed that the 4 proteins were thioredoxin, adenylate kinase, single-stranded DNA-binding protein, and ribosomal protein 50S L7/L12, with the Mowse scores of 94, 286, 139 and 132, and with the sequence coverage rates of 77%, 33%, 33%, and 28% respectively. CONCLUSION: Anti-oxidant and inhibiting apoptosis, thioredoxin may be related to gastric carcinogenesis induced by H. pylori. Proteomics technology has a widespread perspective in the field of relationship between pathogenic bacteria and gastric cancer.