Simultaneous quantification of seven active metabolites of roxifiban in human plasma by LC/MS/MS in the presence of an interfering displacer at millimolar concentrations.

Roxifiban (DMP 754) is a glycoprotein (GP) IIb/IIIa antagonist. Following oral administration to humans, roxifiban is metabolized to its primary active zwitterionic form, XV459, and several minor, active, hydrolyzed and hydroxylated metabolites, namely, M1a (DPC-AD3508), M1b (DPC-AD6128), M2 (SW156), M3 (DPC-AG2185), M8a (DPC-AF5814), and M8b (DPC-AF5818). Quantification of these metabolites in humans was not workable with a previous analytical method due to ion suppression of at least four of the analytes by a competitive displacer, DMP 728. This compound, which is another GP IIb/IIIa antagonist with very high affinity for the platelet receptor, was added to harvested blood samples in millimolar quantity to liberate XV459 from the GP IIb/IIIa receptor. An automated ion exchange solid phase extraction (IX-SPE) procedure was developed to selectively extract the seven metabolites of roxifiban and its deuterated internal standard while specifically excluding DMP 728. Among the six hydroxylation metabolites, there were two pairs of epimeric diastereomers (M1a/M1b and M8a/M8b) and one pair of geometric isomers (M2/M3), corresponding to three critical chromatographic pairs that needed to be base-line resolved because of the lack of specificity of MS/MS detection for these isomers. A new LC/MS/MS assay was developed to simultaneously quantify the seven metabolites in human plasma. The assay method was validated under GLP conditions over the concentration range of 0.5 to 80 nM for each of the analytes and successfully applied to assaying approximately 500 plasma samples from clinical trials.

Salivary histatin 5 induces non-lytic release of ATP from Candida albicans leading to cell death.

Salivary histatins are potent in vitro antifungal proteins and have promise as therapeutic agents against oral candidiasis. We performed pharmacological studies directed at understanding the biochemical basis of Hst 5 candidacidal activity. Three inhibitors of mitochondrial metabolism: carbonyl cyanide p-chlorophenylhydrazone, dinitrophenol, and azide inhibited Hst 5 killing of Candida albicans, while not inhibiting cellular ATP production. In contrast, Hst 5 caused a drastic reduction of C. albicans intracellular ATP content, which was a result of an efflux of ATP. Carbonyl cyanide p-chlorophenylhydrazone, dinitrophenol, and azide inhibited Hst 5-induced ATP efflux, thus establishing a correlation between ATP release and cell killing. Furthermore, C. albicans cells were respiring and had polarized membranes at least 80 min after ATP release, thus implying a non-lytic exit of cellular ATP in response to Hst 5. Based on evidence that transmembrane ATP efflux can occur in the absence of cytolysis through a channel-like pathway and that released ATP can act as a cytotoxic mediator by binding to membrane purinergic receptors, we evaluated whether extracellular ATP released by Hst 5 may have further functional role in cell killing. Consistent with this hypothesis, purinergic agonists BzATP and adenosine 5'O-(thiotriphosphate) induced loss of C. albicans cell viability and purinergic antagonists prevented Hst 5 killing.

Role of E box and initiator region in the expression of the rat follicle-stimulating hormone receptor.

The promoter for the rat follicle-stimulating hormone receptor (FSHR) gene contains a conserved consensus E box sequence and an initiator-like region (InR) sequence. Deletion analysis and transient transfections showed that a 114-base pair region (-143 to -30) that encompasses the E box and the InR was sufficient for greater than 75% of promoter function. DNase I footprint analysis showed that the E box and InR were protected by nuclear proteins from rat Sertoli cells, and the E box region was shown by electrophoretic mobility shift assays (EMSA) to be a site of Sertoli protein interactions. Mutations in the E box disrupted these interactions and reduced FSHR promoter activity. Co-transfection of the inhibitor of DNA binding (Id) with an FSHR/luciferase construct into mouse Sertoli 1 cells reduced FSHR promoter activity. Using EMSA, the upstream stimulatory factor was shown to be a component of the complexes that interacted with the E box in the FSHR promoter. Binding of proteins from rat Sertoli cells to the InR was demonstrated using EMSA. Also, an oligonucleotide that represented the sequence of the terminal deoxynucleotidyltransferase InR displaced the complexes at the FSHR InR. Mutations in the InR resulted in a significant reduction of FSHR promoter activity.

Determination of alosetron in human plasma or serum by high-performance liquid chromatography with robotic sample preparation.

A method of analysis for the determination of alosetron in human plasma or serum has been developed. The method was fully automated using a laboratory robot in order to improve analytical precision, efficiency and safety. The assay involved solid-phase extraction with reversed-phase HPLC separation and fluorescence detection. A validation exercise over the concentration range of 0.1 to 20 ng/ml demonstrated the selectivity, linearity, sensitivity, accuracy, precision, extraction efficiency, ruggedness and stability of the method. The method has been applied in support of numerous human pharmacokinetic/biopharmaceutic studies over the last five years.

Flow cytometric assay of modulation of P-glycoprotein function in whole blood by the multidrug resistance inhibitor GG918.

We sought to develop an assay for measuring the inhibition of P-glycoprotein (Pgp) function in whole blood as an indicator of in vivo drug activity. Since the CD56(+) subset of peripheral blood lymphocytes (PBLs) has been shown to express functional Pgp, the changes in rhodamine 123 (R123) uptake by CD56(+) PBLs from GG918-treated and untreated whole blood were used as the basis for these studies. In an ex vivo study, heparin-treated whole blood was obtained from normal volunteers, and GG918 and R123 were added at various concentrations for time course analyses of dye loading. GG918 concentrations from 2.5 to 800 nm were tested in incubations ranging from 15 min to 3 h prior to R123 addition. R123 loading times ranged from 0 to 80 min. Flow cytometric analyses of the CD56(+) PBLs indicated that the resolution of Pgp inhibition was dependent on inhibitor concentration and time of R123 loading and independent of the R123 concentrations tested. In this ex vivo assay model, a dose-dependent response was seen for GG918 with a 2-fold increase in cellular R123 intensity being produced at a drug concentration of 80 nm. When this assay method was applied to blood samples from volunteers dosed p.o. with GG918, similar shifts in R123 fluorescence of the CD56(+) PBLs were observed with significant increases in R123 intensity occurring at serum concentrations as low as 40 nm. In contrast to assays in which target cell populations are enriched prior to testing, the addition of the substrate (R123) directly to the blood sample combined with the segregation of the target cells by specific immunofluorescence provides the investigator an indication of in situ activity of circulating drug. Thus, CD56(+) PBLs may prove useful as a surrogate target for monitoring multidrug resistance inhibitor activity in situ.

Automated high-performance liquid chromatographic analysis of (-)-2'-deoxy-3'-thiacytidine in biological fluids using the automated sequential trace enrichment of dialysate systems.

A fully automated HPLC procedure was developed for the analysis of a small volume of perfusion solutions from an isolated perfused rat kidney study. The method involved separation of (-)-2'-deoxy-3'-thiacytidine (3TC) from the matrix by dialysis with 10 mM potassium phosphate buffer pH 3.0. 3TC was subsequently separated from the dialysate as it flowed through a SCX cation-exchange cartridge. The trapped 3TC was then eluted with a mobile phase of 50 mM ammonium acetate buffer (pH 5.5)-methanol (90.5:9.5, v/v) at a flow-rate of 1.0 ml/min for 2 min. The eluent was directed to the HPLC system and chromatographed with a BDS C18 analytical column at a temperature of 45 degrees C. Detection of 3TC was carried out by UV absorption at 274 nm. The procedure was validated from 25 to 10,000 ng/ml. Coefficients of variance (C.V.) of 3TC quality control samples were less than 9%. C.V.s of the standard curve samples were also less than 10% except for the 25 ng/ml samples (11.5%). The mean interpolated concentrations were within 8% of the nominal concentrations for all samples. No interference from concurrent drugs was observed. Preliminary results suggested that this procedure may also be used for human serum and urine samples.

Comparison of the pharmacokinetics of an ondansetron solution (8 mg) when administered intravenously, orally, to the colon, and to the rectum.

Ondansetron, an antagonist of the serotonin type 3 (5-HT3) receptor, is indicated for the treatment of chemotherapy-induced emesis. This study compares the pharmacokinetics, especially the bioavailability, of an ondansetron 8-mg solution when administered intravenously, orally, to the colon via nasogastric intubation, and to the rectum using a retention enema. Six healthy, male volunteers received ondansetron infused into the colon during the first treatment period. These subjects then received the remaining three treatments in random order, with a minimum 1-week washout period between treatments. Serial plasma samples were obtained for up to 24 hr after dosing in each treatment period. Absolute bioavailability after the oral dosing, colonic infusion, and rectal administration averaged 71 +/- 14, 74 +/- 26, and 58 +/- 18%, respectively. These values were not significantly different (P > 0.05). Values of Tmax and Cmax were also not significantly different among the nonparenteral routes. Mean absorption half-lives were 0.66, 1.1, and 0.75 hr after the oral, colonic, and rectal administrations, respectively. These results indicate that ondansetron is well absorbed in the intestinal segments studied including the upper small intestine, the colon, and the rectum and that sustained-release and suppository formulations of ondansetron are feasible.

Robotic solid phase extraction and high performance liquid chromatographic analysis of ranitidine in serum or plasma.

A fully automated assay for the analysis of ranitidine in serum and plasma, with and without an internal standard, was validated. It utilizes robotic solid phase extraction with on-line high performance liquid chromatographic (HPLC) analysis. The ruggedness of the assay was demonstrated over a three-year period. A Zymark Py Technology II robotic system was used for serial processing from initial aspiration of samples from original collection containers, to final direct injection onto the on-line HPLC system. Automated serial processing with on-line analysis provided uniform sample history and increased productivity by freeing the chemist to analyse data and perform other tasks. The solid phase extraction efficiency was 94% throughout the assay range of 10-250 ng/mL. The coefficients of variation for within- and between-day quality control samples ranged from 1 to 6% and 1 to 5%, respectively. Mean accuracy for between-day standards and quality control results ranged from 97 to 102% of the respective theoretical concentrations.

Pharmacokinetics of cefuroxime axetil and cefaclor: relationship of concentrations in serum to MICs for common respiratory pathogens.

The pharmacokinetics of single doses of cefaclor at 250 and 375 mg and cefuroxime axetil at 250 mg administered under optimal conditions (i.e., cefuroxime axetil after food and cefaclor in the fasted state) were studied in 24 healthy male volunteers. Drug concentrations in serum were related to MICs for common respiratory tract pathogens by using data generated from a recently completed national survey. The time the concentrations in serum exceeded the MICs for Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella (formerly Branhamella) catarrhalis were significantly greater (P less than 0.05) for cefuroxime axetil at 250 mg than for cefaclor at 250 or 375 mg. With the recommended dosing regimens (cefuroxime axetil at 250 mg and cefaclor at 375 mg twice daily or cefaclor at 250 mg three times daily), cefuroxime concentrations exceed the MIC for 90% of the strains tested for a greater time period than cefaclor concentrations with either regimen. The reasons for this difference are (i) the greater potency and slower clearance of cefuroxime compared with those of cefaclor and (ii) the greater sensitivity of these pathogens to cefuroxime.