Biological Products ; analysis ; pharmacology ; Chromatography, Liquid ; methods ; Drug Evaluation, Preclinical ; methods ; Gas Chromatography-Mass Spectrometry ; methods ; Magnetic Resonance Spectroscopy ; Plants, Medicinal ; chemistry

Human tissue kallikrein-binding protein (Kallistatin, KAL), a secretory protein that participates in the regulation of multiple signaling pathways by binding to the extracellular receptor, however, at present has not been reported about the intracellular activity, and whether it has the similar biological activity with extracellular activity. Here we constructed no signal peptide KAL (NSK) into the adeno-associated virus vector to explore the intracellular activity of KAL. Both the endothelial cell and lung cancer cells could express KAL, but not secreted after rAAV2-NSK transfection. The proliferation and migration of human umbilical vein endothelial cells (HUVECs) were inhibited, but the apoptosis rate was not affected. The proliferation rates, mobility and tubule formation of all the three tested lung cancer cells, such as NCI-H446, NCI-H460 and A549, were inhibited to different extents. This cellular study not only confirmed the intracellular activity, but also suggested it may serve as a kind of "balance factor" in multi-targeted controlling, which may provide a new train of thoughts to explain the regulatory contradiction in PI3K-Akt signaling pathways by KAL.
Apoptosis ; Cell Proliferation ; Dependovirus ; Genetic Vectors ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Lung Neoplasms ; metabolism ; Serpins ; metabolism ; Signal Transduction ; Transfection

A rapid, sensitive and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of clevidipine butyrate and its primary metabolite clevidipine acid in dog blood. After one-step protein precipitation with methanol, the chromatographic separation was carried out on an Ecosil C18 column (150 mm x 4.6 mm, 5 µm) with a gradient mobile phase consisting of methanol and 5 mmol · L(-1) ammonium formate. A chromatographic total run time of 13.0 min was achieved. The quantitation analysis was performed using multiple reaction monitoring (MRM) at the specific ion transitions of m/z 454.1 [M-H]- --> m/z 234.1 for clevidipine butyrate, m/z 354.0 [M-H]- --> m/z 208.0 for clevidipine acid and m/z 256.1 [M-H]- --> m/z 227.1 for elofesalamide (internal standard, IS) in the negative ion mode with electrospray ionization (ESI) source. The linear calibration curves for clevidipine butyrate and clevidipine acid were obtained in the concentration ranges of 0.5-100 ng · mL and 1-200 ng · mL(-1), separately. The lower limit of quantification of clevidipine butyrate and clevidipine acid were 0.5 ng · mL(-1) and 1 ng · mL(-1). The intra and inter-assay precisions were all below 12.9%, the accuracies were all in standard ranges. Stability testing indicated that clevidipine butyrate and clevidipine acid in dog blood with the addition of denaturant methanol was stable under various processing and/or handling conditions. The validated method has been successfully applied to a pharmacokinetic study of clevidipine butyrate injection to 8 healthy Beagle dogs following intravenous infusion at a flow rate of 5 mg · h(-1) for 0.5 h.
Animals ; Butyrates ; blood ; pharmacokinetics ; Calibration ; Chromatography, Liquid ; Dogs ; Infusions, Intravenous ; Pyridines ; blood ; pharmacokinetics ; Tandem Mass Spectrometry

The paper is to report the development of a high-performance liquid chromatographic/tandem mass spectrometry (HPLC-MS/MS) method for the determination of icaritin (ICT) in rat plasma. After precipitated with acetonitrile from the plasma, ICT was isolated chromatographically on a Dikma C18 column. The mobile phase consisted of acetonitrile-water-acetic acid (72 : 28 : 1.5, v/v/v). Electrospray ionization (ESI) source was applied and operated in the positive ion mode. Multiple reaction monitoring (MRM) mode with the transitions of m/z 387 --> m/z 313 and m/z 331 --> m/z 315 were used to quantify ICT and the internal standard, respectively. The linear calibration curve was obtained in the concentration range of 2.5-1,000 ng x mL(-1). The lower limit of quantification was 2.5 ng x mL(-1). The inter- and intra-day precision (RSD) were less than 9.63%, and the accuracy (relative error) was within +/-7.42%. The method was proved to be suitable for the pharmacokinetics of ICT, which offers advantages of high sensitivity and selectivity.
Administration, Oral ; Animals ; Chromatography, High Pressure Liquid ; methods ; Epimedium ; chemistry ; Female ; Flavonoids ; administration & dosage ; blood ; isolation & purification ; pharmacokinetics ; Male ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar ; Reproducibility of Results ; Sensitivity and Specificity ; Spectrometry, Mass, Electrospray Ionization ; methods ; Tandem Mass Spectrometry ; methods

To develop and validate a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of tamsulosin in dog plasma after oral administration of controlled-release tablet of tamsulosin hydrochloride, the samples and the internal standard, diphenhydramine, were extracted from dog plasma by n-hexane-dichloromethane (2 : 1), and separated on a Bonchrom XBP-C18 column using a mobile phase consisted of methanol-acetonitrile-ammonium formate (10 mmol x L(-1)) (30 : 40 : 30, v/v/v), at a flow rate of 0.4 mL x min(-1). Mass spectrometric detection was operated on a triple quadrupole tandem mass spectrometer equipped with atmospheric pressure chemical ionization (APCI) source in positive mode. Quantification was performed using selected reaction monitoring (SRM) of the transitions m/z 409 --> 228 for tamsulosin and m/z 256 --> 167 for the internal standard, respectively. The linear concentration ranges of the calibration curves for tamsulosin were 0.02 - 50 ng x mL(-1). The lower limit of quantification was 0.02 ng x mL(-1). The accuracy ranged from -2.61% to 8.82% in terms of relative error (RE). The intra- and inter-day relative standard deviation (RSD) across three-run validations were lower than 9.72%. The method was proved to be highly sensitive, selective, and had been successfully applied to the pharmacokinetic study after an oral administration of 0.4 mg tamsulosin hydrochloride controlled release preparations to dogs.
Administration, Oral ; Animals ; Area Under Curve ; Chromatography, Liquid ; methods ; Delayed-Action Preparations ; Dogs ; Male ; Sensitivity and Specificity ; Sulfonamides ; administration & dosage ; blood ; pharmacokinetics ; Tablets ; Tandem Mass Spectrometry ; methods

A sensitive and simple high-performance liquid chromatographic tandem mass spectrometric (LC-MS/MS) method for the determination of verapamil and norverapamil in human plasma was established and utilized in a pharmacokinetic study in healthy patients. Protein was precipitated by methanol in plasma samples, and the analytes and internal standard were separated on an Agilent Zorbax Eclipse C₁₈ column (50 mm×4.6 mm, 5 μm) with a gradient procedure using methanol-acetonitrile (50∶50) as the organic phase and 0.1% formic acid - 5% acetonitrile - 10 mmol·L^-1 ammonium formate solution as the mobile phase at flow rate of 0.5 mL·min^-1. Electrospray ionization (ESI) and multiple reaction monitoring (MRM) detection modes were used for quantitative detection of verapamil, norverapamil and verapamil-d₆ (IS). In the mode of multiple reaction monitoring of positive-ions, the monitoring ion pairs of verapamil, norverapamil and the verapamil-d₆ were m/z 445.0→165.2, m/z 441.0→165.2 and m/z 461.1→165.2, respectively. The quantitative lower limit (LLOQ) for the determination of verapamil and norverapamil concentrations in human plasma can reach 0.1 ng·mL^-1 in this assay. The calibration curve concentration ranged from 0.1 to 50 ng·mL^-1 with high linearity (r² > 0.997). The matrix effect of verapamil and norverapamil was 99.2%-100% and 101%-102%, respectively. The recovery of verapamil and norverapamil was 86.8%-95.9% and 87.4%-94.8%, respectively. This method has good specificity and high sensitivity. The determination of the verapamil and norverapamil was not subject to the matrix effect and stable extraction recovery was achieved in this assay. This method could be used to determine the concentration of verapamil and norverapamil in human plasma and suitable for human pharmacokinetic studies after approved by ethics committee.

A significant number of organic carboxylic acids have been shown to influence the absorption and distribution of drugs mediated by organic anion transporters (OATs). In this study, uptake experiments were performed to assess the inhibitory effects of cinnamic acid, ferulic acid, oleanolic acid, deoxycholic acid, and cynarin on hOAT1, hOAT3, hOATP1B1, and hOATP2B1. After a drug-drug interaction (DDI) investigation, cinnamic acid, ferulic acid, deoxycholic acid, and cynarin were found and validated to inhibit hOAT1 in a competitive manner, and deoxycholic acid was found to be an inhibitor of all four transporters. The apparent 50% inhibitory concentrations of cinnamic acid, ferulic acid, deoxycholic acid, and cynarin were estimated to be 133.87, 3.69, 90.03 and 6.03 μmol·L(-1) for hOAT1, respectively. The apparent 50% inhibitory concentrations of deoxycholic acid were estimated to be 9.57 μmol·L(-1) for hOAT3, 70.54 μmol·L(-1) for hOATP1B1, and 168.27 μmol·L(-1) for hOATP2B1. Because cinnamic acid, ferulic acid, and cynarin are ingredients of food or food additives, the present study suggests there are new food-drug interactions to be disclosed. In addition, deoxycholic acid may be used as a probe for studying the correlation of OATs and OATPs.
Carboxylic Acids ; pharmacology ; Cinnamates ; pharmacology ; Coumaric Acids ; pharmacology ; Deoxycholic Acid ; pharmacology ; Diet ; Drug Interactions ; HEK293 Cells ; Humans ; Organic Anion Transport Protein 1 ; antagonists & inhibitors ; Organic Anion Transporters ; antagonists & inhibitors ; Plant Extracts ; pharmacology ; Plants, Medicinal ; chemistry

A simple and sensitive method was developed for quantitation of obeticholic acid in rat plasma with liquid chromatography-tandem mass spectrometry (LC-MS/MS). After liquid-liquid extraction by methyl tert-butyl ether, the chromatographic separation was carried out on an ACE Excel 2 Super C18 column (50 mm×2.1 mm ID, 1.7 μm) with a gradient mobile phase consisting of acetonitrile and 2 mmol·L^-1 ammonium formate at a flow rate of 0.2 mL·min^-1. The quantitation analysis was performed using multiple reaction monitoring (MRM) at the specific ion transitions of m/z 418.9[M-H]^-→401.2 for obeticholic acid and m/z 469.0[M-H]^-→ 425.2 for glycyrrhetinic acid (internal standard) in the negative ion mode with electrospray ionization (ESI) source. This validated LC-MS/MS method yielded a good linearity over the range of 5 -5 000 ng·mL^-1 with the lower limit of quantitation (LLOQ) of 5 ng·mL^-1. The intra and inter-assay precisions (RSD) were all less than 9.82% and the accuracy (RE) was within ±6.90%. The extraction recovery of obeticholic acid was from 85.4% to 88.5%, and the matrix effect of obeticholic acid ranged from 78.9% to 82.5%. Stability test suggest that obeticholic acid in rat plasma was stable for 24 h on workbench, up to 1 month at -70℃, and after three cycles of freeze-thaw. Extracted samples were stable for more than 24 h in an auto-sampler at 6℃. The precision was less than 7.25%, and the accuracy was within ±11.2%, after being diluted 10 times by blank rat plasma. The method has been successfully applied to a pharmacokinetic study of obeticholic acid in rats following oral administration at the dose of 2.5 mg·kg^-1.

A LC-MS/MS method for quantification of norfloxacin in human plasma had been developed. This method was applied to the pharmacokinetics study of norfloxacin in the human. The plasma sample was precipitated by methanol and ciprofloxacin was used as the internal standard (IS). Chromatographic separation was performed on a Symmetry® C₁₈ column(100 mm×4.6 mm, 5 μm). Mobile phase contains 0.3% formic acid and 5% methanol in deionized water at a flow rate of 0.45 mL·min^-1. Norfloxacin and ciprofloxacin (IS) were ionized with an ESI source and operated in positive ion mode. The detected ions were m/z 320.3→302.1 (norfloxacin), m/z 332.3→314.1 (ciprofloxacin). This LC-MS/MS method yielded a linearity over the range of 10-1 000 ng·mL^-1 with the lower limit of quantitation (LLOQ) of 10 ng·mL^-1. The intra and inter-assay precisions (RSD%) were within the range of 2.64%-7.23% and the accuracy (RE%) was less than ±5.00%. The pharmacokinetic parameters t_max, C_max, AUC_0-t, and t_1/2 were 1.28±0.364 h, 627±171 ng·mL^-1, 2 938±850 h·ng·mL^-1, and 6.01±1.36 h, respectively. This LC-MS /MS method was proven simple, sensitive, rapid and suitable for pharmacokinetics study of norfloxacin in the human and Approved by the Medical Ethics Committee of Liuzhou Workers' Hospital.

Bentysrepinine (Y101), a derivative of phenylalanine dipeptide, is a novel drug candidate for the treatment of hepatitis B virus (HBV) infection. Our previous preclinical pharmacokinetic study showed that its in vivo absorption and distribution characteristics were probably related to transmembrane transport after Y101 was administered intragastically in rats. In this study, Caco-2 and MDCK-MDR1 cell models were used to investigate interactions between Y101 and P-gp through the apparent permeation coefficient (P_app) and efflux ratio (R_E); the results showed that Y101 was a substrate of P-gp. In addition, gene-transfected cell models, HEK293-hOATP1B1, HEK293-hOATP2B1 and CHO-PEPT1 were used to evaluate the affinity to OATP1B1,