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Mouse Plasma (mouse + plasma)
Selected AbstractsMouse recombinant protein C variants with enhanced membrane affinity and hyper-anticoagulant activity in mouse plasmaFEBS JOURNAL, Issue 22 2009Michael J. Krisinger Mouse anticoagulant protein C (461 residues) shares 69% sequence identity with its human ortholog. Interspecies experiments suggest that there is an incompatibility between mouse and human protein C, such that human protein C does not function efficiently in mouse plasma, nor does mouse protein C function efficiently in human plasma. Previously, we described a series of human activated protein C (APC) Gla domain mutants (e.g. QGNSEDY-APC), with enhanced membrane affinity that also served as superior anticoagulants. To characterize these Gla mutants further in mouse models of diseases, the analogous mutations were now made in mouse protein C. In total, seven mutants (mutated at one or more of positions P10S12D23Q32N33) and wild-type protein C were expressed and purified to homogeneity. In a surface plasmon resonance-based membrane-binding assay, several high affinity protein C mutants were identified. In Ca2+ titration experiments, the high affinity variants had a significantly reduced (four-fold) Ca2+ requirement for half-maximum binding. In a tissue factor-initiated thrombin generation assay using mouse plasma, all mouse APC variants, including wild-type, could completely inhibit thrombin generation; however, one of the variants denoted mutant III (P10Q/S12N/D23S/Q32E/N33D) was found to be a 30- to 50-fold better anticoagulant compared to the wild-type protein. This mouse APC variant will be attractive to use in mouse models aiming to elucidate the in vivo effects of APC variants with enhanced anticoagulant activity. [source] High sensitivity determination of valproic acid in mouse plasma using semi-automated sample preparation and liquid chromatography with tandem mass spectrometric detectionRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 24 2005Vincenzo Pucci A high-throughput liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) assay using automated sample preparation has been developed for the determination of valproic acid (VPA) in mouse plasma. A liquid-handling system was programmed to prepare calibration standard solutions in plasma, as well as quality controls and clinical samples. Plasma protein precipitation was performed on a 96-well plate, and the collected supernatant was directly injected into a reversed-phase LC/ESI-MS/MS system in the negative ionization mode. The calibration curve for VPA was linear over a dynamic range of 0.15,100,µg/mL. The limit of detection was 75,ng/mL and the lower limit of quantitation was 150,ng/mL. Intra- and inter-day validation assays of the semi-automated plasma analysis showed satisfactory accuracy and precision. Copyright © 2005 John Wiley & Sons, Ltd. [source] High-performance liquid chromatography/tandem mass spectrometry for the quantitative analysis of a novel taxane derivative (BAY59-8862) in biological samples and characterisation of its metabolic profile in rat bile samplesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 19 2001Cristina Sottani A sensitive, specific, accurate and reproducible high-performance liquid chromatography (HPLC) analytical method was developed and validated for the quantification of the novel oral taxane analogue BAY59-8862 in mouse plasma and tissue samples. A fully automated solid-phase extraction procedure was applied to the plasma after internal standard (IS) addition, with only 0.2,mL volume of the sample loaded on a CN-Sep-pak cartridge. In the case of the tissues a very simple acetonitrile extraction was used to recover BAY59-8862 and its internal standard from liver. The procedure for the quantification of BAY59-8862 and its IS (IDN5127) is based on high-performance liquid chromatography/ion spray-tandem mass spectrometry, operating in selected ion monitoring mode. The retention times of BAY and IS were 7.21 and 10.36,min, respectively. In both plasma and tissue specimens the assay was linear in the range 50,5000,ng/mL (ng/g). The overall precision and accuracy were assessed on three different days. The results for plasma were within 6.1% (precision) and between 99 and 112% (accuracy), and for the liver samples within 7.3% and between 104 and 118%, respectively. The LOD was 5,ng/mL and 20,ng/g in the plasma and liver, respectively. In addition, the biliary excretion of the compound in rats was studied. The study showed that an oxidative chemical reaction was the preferred metabolic pathway for biliary excretion, and two sets of mono- and dihydroxylated metabolites were detected by LC/ISP-MS/MS experiments. With this method, BAY59-8862 pharmacokinetic was determined in mice. The combined results demonstrate that the methodology can be considered a valid approach to conduct pharmacokinetic and metabolic studies during preclinical and clinical investigations. Copyright © 2001 John7 Wiley & Sons, Ltd. [source] High-throughput analysis in drug discovery: application of liquid chromatography/ion-trap mass spectrometry for simultaneous cassette analysis of ,-1a antagonists and their metabolites in mouse plasmaRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 8 2001Zongwei Cai The application of liquid chromatography/ion-trap mass spectrometry for simultaneous quantification of multiple drugs and detection of their metabolites for in vitro experiments was reported recently. In the current study, the use of these techniques was extended to in vivo pharmacokinetic (PK) studies of ,-1a antagonists. In combination with limited time-point PK, greatly increased throughput was demonstrated for the in vivo screening and investigation of in vivo,in vitro correlation. In addition to quantitative analyses, the technique allowed simultaneous detection of major in vivo metabolites without having to reanalyze the plasma samples. The drugs were individually dosed in mice intravenously via tail vein injection and the blood samples were collected 5,min and 2,h after dosing. After the plasma samples for the different drugs had been prepared separately, they were pooled for cassette analysis. The concentrations of five test compounds in the plasma samples at 2,h ranged from 36,1062,ng/mL, whereas their 5-min plasma levels were similar. From the same cassette analysis, major metabolites in the samples were also detected simultaneously through the interpretation of full-scan mass spectra. The metabolite identification confirmed the results from a previous report that the major sites of metabolism are hydroxylation of the phenyl ring not bearing the alkylsulfonamide substitutent, piperidine N-dealkylation, and N-demethylation of the alkylsulfonamide group. Copyright © 2001 John Wiley & Sons, Ltd. [source] Pharmacokinetics, biodistribution, and antitumor efficacy of a human glandular kallikrein 2 (hK2)-activated thapsigargin prodrugTHE PROSTATE, Issue 4 2006Samuel Janssen Abstract BACKGROUND Prostate cancer cells secrete unique proteases such as prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) that represent targets for the activation of prodrugs as systemic treatment of metastatic prostate cancer. Previously, a combinatorial peptide library was screened to identify a highly active peptide substrate for hK2. The peptide was coupled to an analog of the potent cytotoxin thapsigargin, L12ADT, to generate an hK2-activated prodrug that was efficiently hydrolyzed by purified hK2, stable to hydrolysis in human and mouse plasma in vitro and selectively toxic to hK2 producing prostate cancer cells in vitro. METHODS In the current study, toxicology, pharmacokinetics, prodrug biodistribution, and antitumor efficacy studies were performed to evaluate the hK2-activated prodrug in vivo. RESULTS The single intravenous maximally tolerated dose of prodrug was 6 mg/kg (i.e., 3.67 µmole/kg) which produced peak serum concentration of ,36 µM and had a half-life of ,40 min. In addition, over a 24 hr period <0.5% of free L12ADT analog was observed in plasma. The prodrug demonstrated significant antitumor effect in vivo while it was being administered, but prolonged intravenous administration was not possible due to local toxicity to tail veins. Subcutaneous administration of equimolar doses produced lower plasma AUC compared to intravenous dosing but equivalent intratumoral levels of prodrug following multiple doses. CONCLUSIONS The hK2-activated prodrug was stable in vivo. The prodrug, however, was rapidly cleared and difficult to administer over prolonged dosing interval. Additional studies are underway to assess antitumor efficacy with prolonged administration of higher subcutaneous doses of prodrug. Second-generation hK2-activated thapsigargin prodrugs with increased half-lives and improved formulations are also under development. © 2005 Wiley-Liss, Inc. [source] Determination of 3,,4,,5,,5,7-pentamethoxyflavone in the plasma and intestinal mucosa of mice by HPLC with UV detectionBIOMEDICAL CHROMATOGRAPHY, Issue 4 2009Hong Cai Abstract In a preliminary experiment 3,,4,,5,,5,7-pentamethoxyflavone (PMF) inhibited adenoma development in ApcMin mice, a model of the human heritable condition familial adenomatous polyposis. An HPLC method for tricin was modified and validated to permit measurement of PMF in mouse plasma and intestinal mucosa. HPLC analysis was carried out on a Hypersil-BDS C18 column with detection at 324 nm and tricin as internal standard. The assay was linear in the range of 100,2000 ng/mL plasma and 1.0,40 µg/mL mucosa. PMF in plasma was efficiently extracted using solid-phase columns. In the case of mucosa organic solvent protein precipitation displayed satisfactory accuracy and precision. The assay recovery at low, medium and high concentrations was between 85 and 103% for both biomatrices, with a relative standard deviation of <15%. The lower limits of quantitation for plasma and mucosa were 100 ng/mL and 1.0 µg/mL, respectively. This method allowed measurement of PMF steady-state median concentrations in plasma (1.08 nmol/mL, n = 11; 10th and 90th percentiles: 0.633 and 2.385 nmol/mL) and mucosa (108.5 nmol/g, n = 9; 10th and 90th percentiles: 38.9 and 164.4 nmol/g) in mice which had received PMF (0.2%, w/w) with their diet. Copyright © 2008 John Wiley & Sons, Ltd. [source] Pharmacokinetic study of p -coumaric acid in mouse after oral administration of extract of Ananas comosus L. leavesBIOMEDICAL CHROMATOGRAPHY, Issue 9 2006Zhen Meng Abstract Quantification of p- coumaric acid in mouse plasma following oral administration of Ananas comosus L. leaves was achieved by reversed-phase high-performance liquid chromatography using a mobile phase of water,acetonitrile (82:18, v/v) and UV detection at 310 nm. The method was linear (determination coefficient, r2 = 0.9997) within the tested range (0.04,1.28 µg/mL). Intra- and inter-day precision coefficients of variation and accuracy bias were acceptable (maximal CV value was 4.06% for intra-day and 4.19% for inter-day) over the entire range. The recoveries were 90.63, 97.98 and 100.01% for concentrations of 0.04, 0.32 and 1.28 µg/mL, respectively. This is a very rapid, sensitive and economical way to determine p- coumaric acid concentration in mouse plasma after oral administration of A. comosus leaves. The concentration,time curve was fitted to the one-compartment model. This is the first time that p- coumaric acid extracted from A. comosus leaves was detected by HPLC-UV method and its pharmacokinetic characteristic was comprehensively studied. Copyright © 2006 John Wiley & Sons, Ltd. [source] Quanti,cation of raf antisense oligonucleotide (rafAON) in biological matrices by LC-MS/MS to support pharmacokinetics of a liposome-entrapped rafAON formulationBIOMEDICAL CHROMATOGRAPHY, Issue 4 2005Jenifer L. Johnson Abstract An LC-MS/MS method was developed to quantify an antisense oligonucleotide against Raf-1 expression (rafAON) in monkey and mouse plasma and in mouse tissue homogenates from animals dosed with a liposome-entrapped rafAON easy-to-use formulation (LErafAON-ETU) intended for use as an antineoplastic agent. RafAON was extracted from mouse and monkey plasma using solid-phase extraction. Tissues were homogenized and sample cleanup was achieved by protein precipitation. RafAON and the internal standard (IS) were separated on a Hamilton PRP-1 column and quanti,ed by tandem mass spectrometry using an electrospray source in negative ion mode. The total run time was 4.0 min. The peak areas of two rafAON transitions were summed and plotted against the peak area of an IS transition to generate the standard curve. In monkey plasma the linear range was 50,10,000 ng/mL, and in mouse plasma it was 25,5000 ng/mL. The lower limit of quanti,cation was 500 ng/mL (10 µg/g tissue) in heart, kidney, liver, lung and spleen homogenates, and the standard curve was linear up to 10,000 ng/mL. Accuracy, precision and stability were evaluated and found to be acceptable in all three matrices. The assay was used to support pharmacokinetics and tissue distribution studies of LErafAON-ETU in mice and monkeys. Copyright © 2004 John Wiley & Sons, Ltd. [source] | ||||||||||