Pharmacokinetics, pharmacodynamics and food effect of LB30870, a novel direct thrombin inhibitor, after single oral doses in healthy men.

1. The safety, tolerability, pharmacokinetics, pharmacodynamics, and food effect of LB30870, a new selective thrombin inhibitor, were studied in 16 healthy men. 2. A double-blind, placebo-controlled single ascending dose study was done at oral doses of 5, 15, 30, 60, 120, and 240 mg under fasting conditions. An open, randomized, balanced cross-over food effect study was done at 60 mg dose. Plasma and urinary concentrations were measured up to 48 h post-dose. Coagulation and thrombin activity markers were measured at selected time points. 3. Cmax of LB30870 was at 1.3-3.0 h post-dose with a mean apparent terminal half-life (t1/2) of 2.8-4.1 h. AUC after doses above 15 mg appeared greater than dose-proportional. In fed state, AUC showed 80% reduction relative to fasting condition. 4. At doses 60 and 120 mg, peak activated partial thromboplastin time (aPTT) increased by 1.5- and 2-fold, respectively, from baseline. The aPTT and international normalized ratio (INR) were concentration-dependent, with less within-individual variability than ecarin clotting time (ECT), prothrombin time (PT), or thrombin time (TT). 5. Single oral doses of LB30870 up to 240 mg were well tolerated. The food effect must be overcome if LB30870 is to be used as an oral anti-coagulant.

Use of an intravenous microdose of 14C-labeled drug and accelerator mass spectrometry to measure absolute oral bioavailability in dogs; cross-comparison of assay methods by accelerator mass spectrometry and liquid chromatography-tandem mass spectrometry.

A technique utilizing simultaneous intravenous microdosing of (14)C-labeled drug with oral dosing of non-labeled drug for measurement of absolute bioavailability was evaluated using R-142086 in male dogs. Plasma concentrations of R-142086 were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and those of (14)C-R-142086 were measured by accelerator mass spectrometry (AMS). The absence of metabolites in the plasma and urine was confirmed by a single radioactive peak of the parent compound in the chromatogram after intravenous microdosing of (14)C-R-142086 (1.5 microg/kg). Although plasma concentrations of R-142086 determined by LC-MS/MS were approximately 20% higher than those of (14)C-R-142086 as determined by AMS, there was excellent correlation (r=0.994) between both concentrations after intravenous dosing of (14)C-R-142086 (0.3 mg/kg). The oral bioavailability of R-142086 at 1 mg/kg obtained by simultaneous intravenous microdosing of (14)C-R-142086 was 16.1%, this being slightly higher than the value (12.5%) obtained by separate intravenous dosing of R-142086 (0.3 mg/kg). In conclusion, on utilizing simultaneous intravenous microdosing of (14)C-labeled drug in conjunction with AMS analysis, absolute bioavailability could be approximately measured in dogs, but without total accuracy. Bioavailability in humans may possibly be approximately measured at an earlier stage and at a lower cost.

Development of a method for the detection and confirmation of the alpha-2 agonist amitraz and its major metabolite in horse urine.

Amitraz (N'-(2,4-dimethylphenyl)-N-[[(2,4-dimethylphenyl)imino]methyl]-N-methyl-methanimidamide) is an alpha-2 adrenergic agonist used in veterinary medicine primarily as a scabicide- or acaricide-type insecticide. As an alpha-2 adrenergic agonist, it also has sedative/tranquilizing properties and is, therefore, listed as an Association of Racing Commissioners International Class 3 Foreign Substance, indicating its potential to influence the outcome of horse races. We identified the principal equine metabolite of amitraz as N-2,4-dimethylphenyl-N'-methylformamidine by electrospray ionization(+)-mass spectrometry and developed a gas chromatographic-mass spectrometric (GC-MS) method for its detection, quantitation, and confirmation in performance horse regulation. The GC-MS method involves derivatization with t-butyldimethylsilyl groups; selected ion monitoring (SIM) of m/z 205 (quantifier ion), 278, 261, and 219 (qualifier ions); and elaboration of a calibration curve based on ion area ratios involving simultaneous SIM acquisition of an internal standard m/z 208 quantifier ion based on an in-house synthesized d(6) deuterated metabolite. The limit of detection of the method is approximately 5 ng/mL in urine and is sufficiently sensitive to detect the peak urinary metabolite at 1 h post dose, following administration of amitraz at a 75-mg/horse intravenous dose.

Comparison of heterogeneous and homogeneous radioactivity flow detectors for simultaneous profiling and LC-MS/MS characterization of metabolites.

Methods for simultaneous liquid chromatography-radioactivity monitor (LC-RAM) metabolite profiling and LC-tandem mass spectrometry (MS/MS) characterization of metabolites are described. Profiling and characterization of metabolites from three drug candidates from different therapeutic areas were compared using in-line heterogeneous LC-RAM-MS/MS and homogeneous LC-RAM-MS/MS methods. Although comparison shows that simultaneous metabolite profiling and characterization can be achieved using either heterogeneous or homogeneous-LC-RAM-MS/MS systems, a homogeneous system has the advantage in the following aspects, (1) sensitivity; (2) ease of method transfer; (3) less peak broadening problems due to the drug or metabolites adhering to the RAM cell; (4) accuracy in quantitation of the metabolites; and (5) the ability to load larger volumes of unprocessed biological fluids. Furthermore, the study shows that some of the possible metabolites that do not ionize well with electrospray ionization (ESI) and eluded detection by heterogeneous-LC-RAM detection could be very easily detected and characterized using a homogeneous-LC-RAM-MS/MS system.

Mass spectrometric and NMR characterization of metabolites of roxifiban, a potent and selective antagonist of the platelet glycoprotein IIb/IIIa receptor.

1. The methyl ester prodrug roxifiban is an orally active, potent and selective antagonist of the platelet glycoprotein GPIIb/IIIa receptor and is being developed for the prevention and treatment of arterial thrombosis. 2. Roxifiban was rapidly hydrolyzed to the zwitterion XV459 in vivo and by liver slices from the rat, mouse and human and by intestinal cores from dog. XV459 was metabolized to only a small extent in vitro and in vivo. 3. Studies with rat and dog given radiolabelled roxifiban showed limited oral absorption with the majority of the radiolabel being excreted in faeces. After i.v. doses of 14C-roxifiban, most of the radioactivity was recovered in the urine of rat whereas the dog excreted significant amounts of radioactivity in bile and urine. 4. XV459 could be metabolized extrahepatically by dog gut flora to produce an isoxazoline ring-opened metabolite. In vitro hepatic metabolism of XV459 was mainly by hydroxylation at the prochiral and chiral centres of the isoxazoline ring. These hydroxylated metabolites were not detected in the urine and plasma of human volunteers administered roxifiban. 5. Initial LC/MS identification of metabolites was achieved by dosing the rat with an equimolar mixture of d0:d4 roxifiban and detecting isotopic clusters of pseudomolecular ions. Unequivocal characterization of these metabolites was achieved by LC/MS, LC/NMR and high-field NMR techniques using synthetic standards of the metabolites. 6. The synthesis of one hydroxylated metabolite enabled the assignment of the correct stereochemistry of the substituted hydroxyl group on the isoxazoline ring.

Pharmacokinetics and pharmacodynamics of sibrafiban, an orally administered IIb/IIIa antagonist, in patients with acute coronary syndrome.

Sibrafiban is a double prodrug that is converted to the inactive single prodrug and to the active IIb/IIIa antagonist following oral administration. Pharmacokinetics (PK) and pharmacodynamics (PD) of oral sibrafiban and its metabolites were evaluated in patients postacute coronary syndrome receiving once- or twice-daily sibrafiban for up to 28 days at several dose levels. Mean peak concentrations of sibrafiban were < 5 ng/mL. Peak single prodrug concentrations occurred 1.7 +/- 1.0 (mean +/- SD) hours after sibrafiban dosing. Total apparent plasma clearance of the single prodrug was 40 +/- 15 L/h, and the elimination half-life was 2.3 +/- 0.8 hours. Mean values of the steady-state pharmacokinetics for total concentrations of the active drug over all doses were: time to peak plasma concentration, 5.0 +/- 1.7 hours; apparent clearance, 13.9 +/- 3.9 L/h; and half-life, 11.0 +/- 2.8 hours. Once-daily dosing resulted in high peak-trough excursions in active drug concentrations: trough concentrations were 21% +/- 6% of peak. Twice-daily dosing resulted in an AUC for the active drug on Day 28 that was 168% +/- 36% of that on Day 1, and steady-state trough concentrations were 54% +/- 10% of peak with sustained inhibition of platelet aggregation. Dose-adjusted steady-state active drug concentrations increased with increasing age and with decreasing renal function and body weight.

Pharmacokinetics and pharmacodynamics of Ro 44-3888 after single ascending oral doses of sibrafiban, an oral platelet aggregation inhibitor, in healthy male volunteers.

AIMS: This study constituted the first administration of the oral platelet inhibitor, sibrafiban, to humans. The aim was to investigate the pharmacokinetics and pharmacodynamics of Ro 44-3888, the active principle of sibrafiban, after single ascending oral doses of sibrafiban. Particular emphasis was placed on intersubject variability of the pharmacokinetic and pharmacodynamic parameters of Ro 44-3888. METHODS: The study consisted of three parts. Part I was an open ascending-dose study to determine target effect ranges of sibrafiban. Part II, a double-blind, placebo-controlled, parallel-group study, addressed the intersubject variability of pharmacokinetic and pharmacodynamic parameters of the active principle at a sibrafiban dose achieving an intermediate effect. Part III was a double-blind, placebo-controlled, ascending-dose design covering the complete plasma concentration vs pharmacodynamic response curve of sibrafiban. RESULTS: At sibrafiban doses between 5 mg and 12 mg, the pharmacokinetics of free Ro 44-3888 in plasma were linear whereas those of total Ro 44-3888 were non-linear because of the saturable binding to the glycoprotein IIb-IIIa receptor. Saturation of the GP IIb-IIIa receptor was reached at plasma concentrations of 15.9 ng ml-1. At sibrafiban doses up to 2 mg, ADP-induced platelet aggregation was inhibited by 50%, whereas the inhibition of TRAP-induced platelet aggregation was about 20-30%. At the higher doses, ADP-induced platelet aggregation was almost completely inhibited while a clear dose-response could be observed with TRAP-induced inhibition of platelet aggregation at sibrafiban doses of 5 to 12 mg. Ivy bleeding time increased very steeply with dose with a significant prolongation observed at doses of 5 to 7 mg of sibrafiban (5-7 min, >30 min in one case). At a sibrafiban dose of 12 mg, the stopping criterion for dose escalation (prolongation of the Ivy bleeding time >30 min in three out of four subjects per dose group) was reached. The interindividual coefficients of variation of the integrated pharmacokinetic and pharmacodynamic parameters (AUC and AUE) were below 20%, thus lying well within the pre-set level of acceptance. CONCLUSIONS: With a low intersubject variability of its pharmacokinetic and pharmacodynamic parameters, linear pharmacokinetics and pharmacodynamic effects closely related to its plasma concentrations, Ro 44-3888 has good pharmacological prerequisites for a well controllable therapy of secondary prevention of arterial thrombosis in patients with acute coronary syndrome.