Quantitation of TNP-470 and its metabolites in human plasma: sample handling, assay performance and stability.

A selective and sensitive assay for the determination of TNP-470 and two of its metabolites, AGM-1883 and M-II, in human plasma was developed. The assay involved liquid-liquid extraction followed by analysis using high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry. Because TNP-470 is most stable in a pH of 4-5, an acidification procedure was utilized to prevent degradation of TNP-470 during sample collection which involved acidifying the whole blood sample collected with 5 mg of citric acid per ml of blood. Liquid-liquid extraction using an organic solvent mixture was chosen over solid-phase extraction to minimize the degradation of TNP-470 during solvent evaporation.

Ex vivo inhibition of beta-thromboglobulin release following administration to man of ABT-299, a novel prodrug of a potent platelet activating factor antagonist.

OBJECTIVE AND DESIGN: ABT-299 is a prodrug that is converted by serum esterase to a potent platelet activating factor (PAF) antagonist (A-85783). In order to evaluate the pharmacological activity of this antagonist in man the effect of ABT-299 given to healthy volunteers on ex vivo PAF-induced beta-thromboglobulin (beta-TG) release in blood was assessed. SUBJECTS: 37 healthy male volunteers, age 18 to 40 (mean age of 23.6 years) and free of medication, participated in the study. TREATMENT: Subjects were administered intravenously 0.8 mg, 2 mg, or 70 mg doses of ABT-299 (6-7 subjects per group) or placebo (9 subjects, pooled). METHODS: Peripheral blood taken over 12 h after dosing was used for ex vivo beta-TG release and, in the case of the 70 mg dose, measurement of plasma drug concentration. Data were compared by Student's t-test. RESULTS: All three doses produced highly significant inhibition (p < 0.005 compared to predose values) of PAF-induced beta-TG release (units/ml plasma +/- SEM) 12 h after drug administration (54 +/- 14 vs. 405 +/- 51, n = 8; 79 +/- 23 vs. 480 +/- 127, n = 7; 21 +/- 10 vs. 327 +/- 72, n = 6, respectively) whereas there was no significant difference in beta-TG release in the placebo group (449 +/- 90 vs. 307 +/- 49, n = 9). Inhibition was associated with the rapid appearance in plasma of A-85783 and the pyridine N-oxide metabolite of A-85783. Within 2 h, the plasma concentration of the metabolite exceeded that of the parent drug. Both the parent drug and the metabolite exhibited potent in vitro inhibition of PAF-induced beta-TG release (A2 values of 4 and 1 nM respectively). CONCLUSIONS: These studies are the first to illustrate the utility of the beta-TG release assay for assessing ex vivo activity of PAF antagonists. These studies also demonstrate that the administration of ABT-299 to man results in potent, long lasting inhibition of PAF-mediated platelet activation, due in part to the pyridine-N-oxide metabolite, and support the potential therapeutic utility of this prodrug in treating PAF-mediated diseases.

Analytical method development for the simultaneous quantitation of dexmedetomidine and three potential metabolites in plasma.

Dexmedetomidine, a novel alpha 2-adrenergic receptor agonist, is being developed as an anesthetic adjunct for perioperative use. An assay method has been developed for the sensitive quantitation of dexmedetomidine and three metabolites in plasma: MPV-1305, MPV-1306 and MPV-1709. The method involves solid-phase extraction (C18 cartridge) of dexmedetomidine and metabolites followed by a two-step derivatization. The first step utilized BF3-MeOH to simultaneously mask a primary alcohol in MPV-1305 and a carboxylic acid in MPV-1306. The second step applied PFB-Cl to derivatize the imidazole ring for sensitive detection of these compounds by GC-negative chemical ionization MS at pg/ml concentrations. MPV-1709 was not derivatized in the process and was detected by GC-positive chemical ionization MS. Optimization of extraction and derivatization is discussed. The method is suitable for quantitation of dexmedetomidine, MPV-1305, MPV-1306 and MPV-1709 over concentration ranges of 0.1, 40, 0.5-100, 0.5-100 and 1.0-500 ng/ml, respectively. The method showed excellent specificity, linearity and sensitivity and is useful for profiling the pharmacokinetic disposition of these compounds.

Pharmacokinetics of sertindole and dehydrosertindole in volunteers with normal or impaired renal function.

OBJECTIVE: To study the effect of renal impairment on the pharmacokinetics of sertindole. METHODS: A single 4 mg oral dose of sertindole was given to normal subjects (n = 6) and subjects with various degrees of impaired renal function (n = 18) classified into mild, moderate, and severe/hemodialysis based on their creatinine clearance). The relationships between the pharmacokinetic parameters and the degree of renal impairment were investigated using regression analysis with creatinine clearance as an explanatory variable along with body weight. Subjects were also genotyped for CYP2D6-A or 2D6-B mutations. RESULTS: The mean CL/f and t1/2 values of sertindole ranged from 14 to 31 1.h-1 and from 73 to 93 h, respectively, and were not significantly related to creatinine clearances. There was no indication of any influence of creatinine clearance on the fraction of sertindole (0.994-0.995) binding to plasma proteins. The total fraction of the sertindole dose removed by dialysis was less than 0.1% Subjects with B/B genotype (n = 2) for CYP2D6 were associated with a distinctly lower clearance of sertindole (6.3 vs 25.3 1.h-1) than subjects with wt/wt genotype for CYP2D6. CONCLUSIONS: Since the pharmacokinetics of sertindole are unchanged by renal impairment, dosage adjustment does not appear to be necessary for subjects with various degrees of renal insufficiency or subjects with renal failure requiring hemodialysis.

Liquid chromatography combined with tandem mass spectrometry for the confirmation of sarafloxacin in catfish tissue.

A specific chromatographic LC/MS/MS assay is described for the confirmatory identification of residues of sarafloxacin, an arylfluoroquinolone antibacterial agent, in catfish tissue. This confirmatory method takes advantage of the specificity provided by sample preparation, liquid chromatography, and tandem mass spectrometry. This kind of multidimensional analysis is commonly used in environmental, pharmacokinetic, residue, and other studies. However, we demonstrate the addition of a previously unreported criterion, the use of ion ratio ranges from the tandem mass spectrometry (MS/MS) experiment as an aid in confirmation. Using the described method, we were able to achieve MS/MS product ion ratios with <7% variation during 1 day of analysis for over 25 injections. We believe the addition of this criterion will increase the scientific certainty of the confirmatory method.

In vivo calibration of microdialysis probes for exogenous compounds.

Several approaches for calibrating microdialysis probes for exogenous compounds in vivo are described which avoid the error introduced by in vitro calibration. These methods are based on establishing a steady state of the exogenous compound by a continuous (zero-order) iv infusion. The steady-state concentration is estimated by three methods that directly determine the in vivo concentration. The methods are (a) extrapolation of dialysate concentrations at various flow rates to the concentration at zero flow, (b) dialysis with concentrations of analyte added to the perfusion medium above and below the expected concentration to determine the concentration at no net flux across the membrane, and (c) dialysis at a very slow perfusion rate (57 nL/min) where the recovery is expected to be better than 90%. Using these approaches, the recovery for cocaine in the brain was found to be (8.9 +/- 0.68)%, as compared to an in vitro recovery of (5.1 +/- 0.18)% at 24 degrees C and (7.4 +/- 0.18)% at 37 degrees C, at a perfusion rate of 1.2 microL/min through a 0.3- X 2-mm microdialysis probe. The in vivo concentration of cocaine in the rat brain for an intravenous dose of 0.3 mg/kg per min was found to be 17.1 +/- 1.3 microM.

Anomalous effect of mazindol on dopamine uptake as measured by in vivo voltammetry and microdialysis.

The effect of mazindol on the dopamine (DA) uptake system in the rat striatum was studied by in vivo voltammetry and microdialysis. An increase in the maximal uptake rate was observed by voltammetry 30 min after i.p. administration of 1, 5, 10, 15, and 20 mg/kg doses but not after a dose of 25 mg/kg, while stimulated release was enhanced at all doses. The increased maximal uptake is in contrast to increased levels of extracellular DA observed with microdialysis following mazindol administration. This divergence of action is anomalous in the context of current understanding of the DA uptake system.

Analysis of 2-DG autoradiograms using image-averaging and image-differencing procedures for systems-level description of neurobehavioral function.

Computer assisted 2-deoxyglucose (2-DG) autoradiography has been used to provide functional maps of areas of altered neural activity related to changes in an animal's behavior or state. The standard procedure for comparison of autoradiograms between different treatment groups has been to take measurement samples from predefined neuroanatomical regions and to average these across brains to attain statistical sensitivity for detecting treatment effects. Unfortunately, when sampling is restricted to predefined areas, important topographic information is lost along with the ability to reveal an unexpected change in neural activity. To preserve the rich topographical detail of metabolic information and to enhance the capacity to uncover novel areas of altered metabolic activity, we have developed a system for averaging entire images from 2-DG autoradiograms and for comparing the average images from two experimental groups by creating an image of differences. This procedure does not rely on sampling only preselected regions, but still allows statistical comparisons between experimental groups. The procedures we describe can be readily and inexpensively adapted for use in individual laboratories and are based on modifications of preexisting image analysis software. We show that, when average and difference images are created using standardized protocols for sectioning brain tissue and editing section images, they are impressively resolved and realistic and can serve as effective topographic descriptions of group differences in neural activity of functional and behavioral relevance.

Differences in the pharmacokinetics of cocaine in naive and cocaine-experienced rats.

Enhanced cocaine concentrations in brain and blood observed after an intraperitoneal challenge dose in rats exposed to cocaine for 10 days by subcutaneous administration are traced to a change in the absorption process from the site of an intraperitoneal injection to general circulation. This conclusion is reached by three sets of corroborating results: (a) Adipose tissue of rats treated for 10 days with repeat subcutaneous injections of cocaine did not reveal a buildup of cocaine in sufficient concentrations to account for the twofold increase in brain and blood concentrations seen during intraperitoneal administration; (b) administration of the drug by an intravenous route after 10-day cocaine treatment did not show a significant difference between treatment and control groups; (c) nonlinear regression on the intravenous and intraperitoneal data sets using a two-compartment open model indicated a difference in the absorption process but not in the metabolic and blood-brain transfer processes.

In vivo microdialysis and thermospray tandem mass spectrometry of the dopamine uptake blocker 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)-piper azine (GBR-12909).

Microdialysis in conjunction with thermospray tandem mass spectrometry was employed in following the time course of the experimental drug GBR-12909 in vivo. GBR-12909 is 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-3(phenylpropyl)-piperazine. An important feature of microdialysis exploited in the method is the elimination of sample cleanup procedures. The detection limit was determined to be 100 pg and the relative standard deviation of estimates for standard solution in the range of 50 nmol/L to 1 mumol/L concentrations was found to be 17%. Important factors in obtaining high sensitivity and reproducibility were carrier phase composition and operation in the flow injection mode. The maximum concentration of GBR-12909 in the brain for a dose of 100 mg/kg i.p. was determined to be 250 nmol/L with the maximal concentration occurring approximately 2 h postinjection. This represents a 40-fold lower concentration of GBR-12909 in the brain as compared to cocaine concentration obtained at a dose of 30 mg/kg, which was estimated earlier under similar experimental conditions. This observation could explain the discrepancy between relative in vivo and in vitro potencies of the two drugs.