Rapid and sensitive determination of formamidines and metabolites with HPLC-MS/MS using core-shell columns.

A number of poisoning and suicide cases involving formamidine pesticides have been reported, thus developing a rapid and low cost determination method is crucial. In this work, a rapid, sensitive and low-cost method for the simultaneous determination of formamidine pesticides (amitraz, chlordimeform, formetanate) and their main metabolites, N-(2,4-dimethylphenyl)-N-methyl-formamidine, 2,4-dimethylformamidine, 2,4-dimethylaniline, 4-chloro-2-methylaniline and 3-hydroxyacetanilide in human blood by high-performance liquid chromatography with tandem mass spectrometry is developed. The application of columns with core-shell particles significantly reduced the analysis time. Very low LODs (0.01-0.04 µg L-1) were obtained for formamidine pesticides and their metabolites. The method was successfully applied to the analysis of human blood samples from a real forensic case. The significantly reduced analysis time, high sensitivity and low cost are the primary advantages of the developed method. This methodology provides important value for sensitive and rapid determination of residue pesticides and metabolites, study of residue pesticides behavior in human body, as well as application in real forensic cases.

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.

A semi-mechanistic modeling strategy for characterization of regional absorption properties and prospective prediction of plasma concentrations following administration of new modified release formulations.

PURPOSE: To outline and test a new modeling approach for prospective predictions of absorption from newly developed modified release formulations based on in vivo studies of gastro intestinal (GI) transit, drug release and regional absorption for the investigational drug AZD0837. METHODS: This work was a natural extension to the companion article "A semi-mechanistic model to link in vitro and in vivo drug release for modified release formulations". The drug release model governed the amount of substance released in distinct GI regions over time. GI distribution of released drug substance, region specific rate and extent of absorption and the influence of food intake were estimated. The model was informed by magnetic marker monitoring data and data from an intubation study with local administration in colon. RESULTS: Distinctly different absorption properties were characterized for different GI regions. Bioavailability over the gut-wall was estimated to be high in duodenum (70%) compared to the small intestine (25%). Colon was primarily characterized by a very slow rate of absorption. CONCLUSIONS: The established model was largely successful in predicting plasma concentration following administration of three newly developed formulations for which no clinical data had been applied during model building.

Effects of ketoconazole on the in vivo biotransformation and hepatobiliary transport of the thrombin inhibitor AZD0837 in pigs.

Ketoconazole has been shown in clinical trials to increase the plasma exposure of the oral anticoagulant prodrug AZD0837 [(2S)-N-{4- [(Z)-amino(methoxyimino)methyl]benzyl}-1-{(2R)-2-[3-chloro-5-(difluoromethoxy)phenyl]-2-hydroxyethanoyl}-azetidine-2-carboxamide] and its active metabolite, AR-H067637 [(2S)-N-{4-[amino(imino)methyl]benzyl}-1-{(2R)-2-[3-chloro-5-(difluoromethoxy)phenyl]-2-hydroxyethanoyl}-azetidine-2-carboxamide]. To investigate the biotransformation of AZD0837 and the effect of ketoconazole on this process, we used an experimental model in pigs that allows repeated sampling from three blood vessels, the bile duct, and a perfused intestinal segment. The pigs received AZD0837 (500 mg) given enterally either alone (n = 5) or together with single-dose ketoconazole (600 mg) (n = 6). The prodrug (n = 2) and its active metabolite (n = 2) were also administered intravenously to provide reference doses. The plasma data revealed considerable interindividual variation in the exposure of the prodrug, intermediate metabolite, and active metabolite. However, AR-H067637 was detected at very high concentrations in the bile with low variability (Ae(bile) = 53 ± 6% of the enteral dose), showing that the compound had indeed been formed in all of the animals and efficiently transported into the bile canaliculi. Concomitant dosing with ketoconazole increased the area under the plasma concentration-time curve for AZD0837 (by 99%) and for AR-H067637 (by 51%). The effect on the prodrug most likely arose from inhibited CYP3A-mediated metabolism. Reduced metabolism also seemed to explain the increased plasma exposure of the active compound because ketoconazole prolonged the terminal half-life with no apparent effect on the extensive biliary excretion and biliary clearance. These in vivo results were supported by in vitro depletion experiments for AR-H067637 in pig liver microsomes with and without the addition of ketoconazole.

Pharmacokinetic properties and metabolism of a new potent antimalarial N-alkylamidine compound, M64, and its corresponding bioprecursors.

Antimalarial activities and pharmacokinetics of the bis-alkylamidine, M64, and its amidoxime, M64-AH, and O-methylsulfonate, M64-S-Me, derivatives were investigated. M64 and M64-S-Me had the most potent activity against the Plasmodium falciparum growth (IC(50)<12nM). The three compounds can clear the Plasmodium vinckei infection in mice (ED(50)<10mg/kg). A liquid chromatography-mass spectrometry method was validated to simultaneously quantify M64 and M64-AH in human and rat plasma. M64 is partially metabolized to M64-monoamidoxime and M64-monoacetamide by rat and mouse liver microsomes. The amidoxime M64-AH undergoes extensive metabolism forming M64, M64-monoacetamide, M64-diacetamide and M64-monoamidoxime. Strong interspecies differences were observed. The pharmacokinetic profiles of M64, M64-AH and M64-S-Me were studied in rat after intravenous and oral administrations. M64 is partially metabolized to M64-AH; while M64-S-Me is rapidly and totally converted to M64 and M64-AH. M64-AH is mostly oxidized to the inactive M64-diacetamine while its N-reduction to the efficient M64 is a minor metabolic pathway. Oral dose of M64-AH was well absorbed (38%) and converted to M64 and M64-diacetamide. This study generated substantial information about the properties of this class of antimalarial drugs. Other routes of synthesis will be explored to prevent oxidative transformation of the amidoxime and to favour the N-reduction.

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.

Highly galloylated tannin fractions from witch hazel (Hamamelis virginiana) bark: electron transfer capacity, in vitro antioxidant activity, and effects on skin-related cells.

Witch hazel ( Hammamelis virginiana) bark is a rich source of both condensed and hydrolizable oligomeric tannins. From a polyphenolic extract soluble in both ethyl acetate and water, we have generated fractions rich in pyrogallol-containing polyphenols (proanthocyanidins, gallotannins, and gallates). The mixtures were highly active as free radical scavengers against ABTS, DPPH (hydrogen donation and electron transfer), and HNTTM (electron transfer). They were also able to reduce the newly introduced TNPTM radical, meaning that they included some highly reactive components. Witch hazel phenolics protected red blood cells from free radical-induced hemolysis and were mildly cytotoxic to 3T3 fibroblasts and HaCat keratinocytes. They also inhibited the proliferation of tumoral SK-Mel 28 melanoma cells at lower concentrations than grape and pine procyanidins. The high content in pyrogallol moieties may be behind the effect of witch hazel phenolics on skin cells. Because the most cytotoxic and antiproliferative mixtures were also the most efficient as electron transfer agents, we hypothesize that the final putative antioxidant effect of polyphenols may be in part attributed to the stimulation of defense systems by mild prooxidant challenges provided by reactive oxygen species generated through redox cycling.

Influence of the selective neuronal NO synthase inhibitor ARL 17477 on nitrergic neurotransmission in porcine stomach.

Selective neuronal NOS (nNOS) inhibitors have been developed for possible application in cerebral ischemia and neurodegenerative disorders. To investigate the degree of interference with peripheral nNOS, the influence of the selective nNOS inhibitor ARL 17477 was studied on electrically induced nitrergic relaxations in pig gastric fundus strips and on gastric fundic compliance in conscious pig. Circular muscle strips of porcine gastric fundus were electrically stimulated (10 s trains at 4 Hz, 0.1 ms and 40 V). ARL 17477 inhibited the electrically induced relaxations in a concentration-dependent way (3x10(-6) M-10(-4) M). The inhibitory effect of ARL 17477 developed more progressively than that of N(G)-nitro-L-arginine methyl ester (L-NAME; 3x10(-4) M). In conscious pigs, instrumented with a fundic cannula, L-NAME (20 mg/kg i.v.) significantly increased mean arterial blood pressure and decreased fundic compliance in the fasted state (71+/-13 ml/mm Hg versus 185+/-37 ml/mm Hg after saline; P<0.05). ARL 17477 (3 mg/kg, i.v.) did not influence blood pressure but influenced gastric fundic volume-pressure curves in a similar way as L-NAME. Plasma concentration analysis of ARL 17477 indicated a half-life of less than 30 min in pig. ARL 17477 thus inhibits the effect of nitrergic neurons in the pig gastric fundus in vitro, leading to inhibited gastric compliance in the conscious pig. The study indicates that selective nNOS inhibitors, applied for cerebral disorders, might also interfere with neuronal nitrergic regulation of gastrointestinal motility.

No influence of mild-to-moderate hepatic impairment on the pharmacokinetics and pharmacodynamics of ximelagatran, an oral direct thrombin inhibitor.

BACKGROUND: The oral direct thrombin inhibitor ximelagatran is a new class of anticoagulant currently in clinical development for the prevention and treatment of thromboembolic disease. After oral administration, ximelagatran is rapidly absorbed and bioconverted to its active form melagatran. OBJECTIVE: To investigate the influence of mild-to-moderate hepatic impairment on the pharmacokinetic and pharmacodynamic properties of ximelagatran. STUDY DESIGN: Nonblinded, nonrandomised study. PARTICIPANTS: Twelve volunteers with mild-to-moderate hepatic impairment (classified as Child-Pugh A or B) and 12 age-, weight-, and sex-matched control volunteers with normal hepatic function. METHODS: Volunteers received a single oral dose of ximelagatran 24mg. Plasma and urine samples were collected for pharmacokinetic and pharmacodynamic analyses. RESULTS: The absorption and bioconversion of ximelagatran to melagatran were rapid in both groups. The maximum plasma concentration of melagatran (Cmax) was achieved 2-3 hours after administration; the mean elimination half-life (t1/2z) was 3.6 hours for hepatically impaired volunteers and 3.1 hours for the control volunteers. The area under the plasma concentration-time curve (AUC) and Cmax of melagatran in volunteers with hepatic impairment were 11 and 25% lower than in control volunteers, respectively. However, after correcting for the higher renal function (i.e. higher calculated creatinine clearance) in the hepatically impaired volunteers, the ratio of melagatran AUC for hepatically impaired/control volunteers was 0.98 (90% CI 0.80, 1.22), suggesting that mild-to-moderate hepatic impairment had no influence on the pharmacokinetics of ximelagatran. Melagatran was the predominant compound in urine, accounting for 13-14% of the ximelagatran dose. Renal clearance of melagatran was 13% higher in hepatically impaired than in control volunteers. There were no significant differences between the two groups in the concentration-response relationship between plasma melagatran concentration and activated partial thromboplastin time (APTT). Baseline prothrombin time (PT) was slightly longer in the hepatically impaired patients than in the control volunteers, probably reflecting a slight decrease in the activity of coagulation factors. However, when concentrations of melagatran were at their peak, the increase in PT from baseline values was the same in both groups. Capillary bleeding time was measured in the hepatically impaired patients only, and was not increased by ximelagatran. Ximelagatran was well tolerated in both groups. CONCLUSION: There were no differences in the pharmacokinetic or pharmacodynamic properties of melagatran following oral administration of ximelagatran between the hepatically impaired and control volunteers. These findings suggest that dose adjustment for patients with mild-to-moderate impairment of hepatic function is not necessary.

Ximelagatran, an oral direct thrombin inhibitor, has a low potential for cytochrome P450-mediated drug-drug interactions.

BACKGROUND: Ximelagatran is an oral direct thrombin inhibitor currently in clinical development for the prevention and treatment of thromboembolic disorders. After oral administration, ximelagatran is rapidly absorbed and extensively bioconverted, via two intermediates (ethyl-melagatran and hydroxy-melagatran), to its active form, melagatran. In vitro studies have shown no evidence for involvement of cytochrome P450 (CYP) enzymes in either the bioactivation or the elimination of melagatran. OBJECTIVE: To investigate the potential of ximelagatran, the intermediates ethyl-melagatran and hydroxy-melagatran, and melagatran to inhibit the CYP system in vitro and in vivo, and the influence of three CYP substrates on the pharmacokinetics of melagatran in vivo. METHODS: The CYP inhibitory properties of ximelagatran, the intermediates and melagatran were tested in vitro by two different methods, using heterologously expressed enzymes or human liver microsomes. Diclofenac (CYP2C9), diazepam (CYP2C19) and nifedipine (CYP3A4) were chosen for coadministration with ximelagatran in healthy volunteers. Subjects received oral ximelagatran 24mg and/or diclofenac 50mg, a 10-minute intravenous infusion of diazepam 0.1 mg/kg, or nifedipine 60mg. The plasma pharmacokinetics of melagatran, diclofenac, diazepam, N-desmethyl-diazepam and nifedipine were determined when administered alone and in combination with ximelagatran. RESULTS: No inhibition, or only minor inhibition, of CYP enzymes by ximelagatran, the intermediates or melagatran was shown in the in vitro studies, suggesting that ximelagatran would not cause CYP-mediated drug-drug interactions in vivo. This result was confirmed in the clinical studies. There were no statistically significant differences in the pharmacokinetics of diclofenac, diazepam and nifedipine on coadministration with ximelagatran. Moreover, there were no statistically significant differences in the pharmacokinetics of melagatran when ximelagatran was administered alone or in combination with diclofenac, diazepam or nifedipine. CONCLUSION: As ximelagatran did not exert a significant effect on the hepatic CYP isoenzymes responsible for the metabolism of diclofenac, diazepam and nifedipine, it is reasonable to expect that it would have no effect on the metabolism of other drugs metabolised by these isoenzymes. Furthermore, the pharmacokinetics of melagatran after oral administration of ximelagatran are not expected to be altered by inhibition or induction of CYP2C9, CYP2C19 or CYP3A4. Together, the in vitro and in vivo studies indicate that metabolic drug-drug interactions involving the major human CYP enzymes should not be expected with ximelagatran.

Species differences in the susceptibility of erythrocytes exposed to free radicals in vitro.

The susceptibility of human, cow, pig, sheep and rabbit erythrocytes to free radicals (peroxyl radicals) generated in vitro by 2,2'-azo-bis(2-amidinopropane) hydrochloride (AAPH) was evaluated by means of a haemolysis test and expressed as the time to 50% of maximal haemolysis (HT50). The most sensitive to damage by free radicals appeared to be the erythrocytes of pigs and sheep, their HT50 values being (mean +/- SEM) 85.1 +/- 1.28 min and 89.0 +/- 1.31 min, respectively. Human erythrocytes and those of cows and rabbits were about twice as resistant, their HT50 values being (mean +/- SEM) 174.3 +/- 1.53 min, 181.2 +/- 1.22 min and 183.4 +/- 2.54 min, respectively. Pig and sheep erythrocytes used in the haemolysis test provided an indication of the antioxidant status in a shorter time (2.5 h versus 4.5 h) than those of the other species studied. The results indicate that the HT50 test may be a convenient alternative to the osmotic resistance test for defining the antioxidant resistance of erythrocytes.

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.

A double antibody radioimmunoassay for the determination of XV459, the active hydrolysis metabolite of roxifiban, in human plasma.

A radioimmunoassay (RIA) was developed for the determination of XV459, the active hydrolysis metabolite of the oral prodrug roxifiban (DMP 754), in human plasma. XV459 is a potent antagonist of the glycoprotein IIb/IIIa receptor. The method utilizes a competitive double antibody format employing an 125I-labeled XV459 analogue tracer which competes with XV459 for antibody binding sites and a second antibody precipitation step to separate antibody bound analyte from free analyte. The method has a validated lower quantification limit of 0.35 ng/ml (0.81 nM) using 12.5 microl of plasma and with dilution can accommodate clinical plasma samples ranging up to 48.0 ng/ml (110.7 nM). Cross-validation to an existing quantitative liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method showed good correlation (r(2)=0.98). The RIA has been successfully used to assay over 10000 clinical samples with sensitivity and specificity comparable to the LC-MS/MS method, but with faster turn around time and at greatly reduced costs.

Integrated pharmacokinetic/pharmacodynamic model of XV459, a potent and specific GPIIb/IIIa inhibitor, in healthy male volunteers.

Roxifiban is an oral prodrug of XV459, a potent and specific inhibitor of the glycoprotein (GP) IIb/IIIa receptor previously under investigation for the treatment of peripheral arterial disease and acute coronary care syndrome. The objective of the present analysis was to develop a pharmacokinetic/pharmacodynamic (PK/PD) model that would be used to guide dose selection in Phase 2. This was a randomized, sequential, rising multiple-dose study in 41 healthy male volunteers given doses of 0.5 to 1.25 mg daily for 7 to 10 days. Total XV459 was measured in plasma by a sensitive and specific LC/MS/MS method. The percent inhibition of platelet aggregation (%IPA) was evaluated in citrated plasma in response to 10 microM ADP using the initial slope of the response. The resulting PK data were fit to a two-compartment model with first-order absorption and saturable oral absorption. The pharmacodynamics was modeled using a direct sigmoidal Emax model. Modeling was performed using NONMEM V. Intersubject variability was moderate in both PK and PD (15.3%-18.5%), except for V2/F (64.8%). Residual variability was low at 11.8%. Platelet count influenced both CL/F and EC50. Age and weight did not explain any additional variability in either PK or PD. The model was shown to produce realistic data when used for simulation. Overall, the results suggest that XV459 concentrations in the range of 10 to 20 ng/ml will yield %IPA values in the range of 40% to 80% inhibition. Because of the pharmacodynamically mediated PK of XV459 (due to platelet binding), the EC50 and CL/F are negatively correlated, limiting the utility of plasma concentration monitoring.

Assessment of ex vivo pharmacodynamic markers during inhibition of thrombosis by CI-1031 (ZK-807834), a novel direct factor Xa inhibitor.

CI-1031 (ZK-807834) is a novel, synthetic factor Xa (FXa) inhibitor with a Ki of 0.11 nM against human FXa. In human plasma in vitro, CI-1031 doubled PT and aPTT at 0.23 and 0.49 microM, respectively. The in vivo antithrombotic effect of CI-1031 was evaluated in a veno-venous shunt model of thrombosis in anesthetized rabbits. After thrombus formation was verified in the first shunts, rabbits received either vehicle or CI-1031 intravenously (bolus injection of 60, 240, or 480 microg/kg followed by an infusion of 2, 8, or 16 microg/kg/min for 140 min, respectively). The second shunts were inserted after 20 min of infusion of CI-1031 or vehicle. CI-1031 dose-dependently prolonged time to occlusion (TTO) in the second shunts (35 +/- 21, 62 +/- 24, and 120 +/- 0 min for the three dose groups, respectively, vs. 10 +/- 1 min for vehicle). Thrombus mass (TM) was reduced in a dose-dependent manner by CI-1031 (42 +/- 7, 27 +/- 6, and 18 +/- 4 mg vs. 50 +/- 4 mg for vehicle). Maximal TM reduction was 70% with an IC(50) of 0.6 microg/ml. Among all the coagulation parameters tested, PT had the best correlation with plasma CI- 1031 concentration (r = 0.97). Ex vivo plasma anti-FXa activity was also well correlated with plasma concentration of CI-1031 and with PT (r = 0.96 and 0.98, respectively). These results indicate that CI-1031, which is currently undergoing clinical evaluation, is an effective antithrombotic compound with a favorable efficacy-to-bleeding ratio. In addition, CI-1031 concentration in plasma can be monitored using PT or anti-Xa assays, thereby providing reliable methods to ensure safe and accurate dose titration of CI-1031.

The antithrombotic effects of CI-1031 (ZK-807834) and enoxaparin in a canine electrolytic injury model of arterial and venous thrombosis.

Factor Xa is a serine protease positioned at the convergence point of the intrinsic and extrinsic coagulation pathways and is therefore an attractive target in the development of novel anticoagulant drugs. The objective of this study was to evaluate the efficacy of CI-1031 (N-[2-[5-amidino-2-hydroxyphenoxy]-6-[3-(1-methyl-1H-imidazolin-2-yl)-phenoxy]-3,5-difluoropyrid), a potent and selective inhibitor of Factor Xa, in a canine electrolytic injury model of arterial and venous thrombosis. Enoxaparin (enoxaparin sodium), a low molecular weight heparin currently approved for treatment and prevention of deep vein thrombosis and unstable angina, was also tested for efficacy in this model. CI-1031 was administered intravenously to anesthetized dogs at three doses: 1.25, 2.5 and 5 microg/kg/min (n=5 for each group) as a continuous infusion for 5.5 h. The control group (n=5) received a continuous infusion of vehicle (3.69 mmol citric acid and 0.9% sodium chloride solution) at a rate of 1 ml/kg/h. Ninety minutes after administration of CI-1031 prothrombin times increased 1.2-, 1.6- and 2.0-fold over baseline values in the 1.25, 2.5 and 5 microg/kg/min groups, respectively. The time to formation of an occlusive thrombus in the femoral arteries averaged 69+/-5 min in the control group compared to 127+/-19, 192+/-33 and 219+/-15 min in the low-, mid- and high-dose CI-1031 groups. In the femoral veins, occlusion time in the controls averaged 56+/-11 min compared to 153+/-22, 137+/-30 and 214+/-26 min in the three treatment groups. Thrombus weights in the control arteries averaged 51+/-4 mg compared to 45+/-5, 28+/-10 and 15+/-3 mg in the CI-1031 treated groups. On the venous side, control thrombus weights averaged 96+/-18 mg compared to 75+/-16, 51+/-16 and 25+/-4 mg in the low-, mid- and high-dose CI-1031 groups. A plasma CI-1031 concentration of approximately 400 ng/ml was associated with a 50% reduction in thrombus weight relative to control animals. Enoxaparin was administered intravenously at a loading dose of 50, 100 or 200 IU/kg for 1 h followed by a maintenance infusion of 25, 50 or 100 IU/kg/h for 4.5 h. The most dramatic changes in coagulation parameters were observed in thrombin time with virtually no changes in prothrombin time. Enoxaparin elicited a dose-dependent increase in time to thrombotic occlusion and a dose-dependent decrease in thrombus weight similar to that observed with CI-1031. Time to occlusion in the enoxaparin-treated groups averaged 117+/-33, 188+/-32 and 217+/-22 min in the low-, mid- and high-dose groups in the femoral arteries and 84+/-22, 171+/-31 and 133+/-33 min in the femoral veins. Thrombus weights averaged 33+/-10, 12+/-5 and 10+/-4 mg in the arteries and 32+/-9, 13+/-2 and 21+/-6 mg in the veins in the low-, mid- and high-dose groups. Blood loss with CI-1031 tended to be less than enoxaparin at doses that provided comparable efficacy. These results demonstrate that CI-1031, like enoxaparin, is an effective antithrombotic agent in an established canine model of arterial and venous thrombosis. CI-1031 provided dose-dependent efficacy with minimal changes in ex vivo coagulation parameters, suggesting it may be a safe and effective antithrombotic agent for both arterial and venous indications.

Population pharmacokinetics and pharmacodynamics of TS-943 for selective nonpeptide platelet glycoprotein IIb/IIIa receptor antagonist in normal healthy subjects.

The pharmacokinetics and pharmacodynamics of TS-943 were evaluated with use of NONMEM in 36 healthy male subjects after constant infusion of five different single-dose regimens. Population analysis showed the plasma concentration-time profiles of TS-943 to be best-fit characterized by a two-compartment open model with constant infusion and first-order elimination. The pharmacodynamic model that best fitted the platelet aggregation was a sigmoid Emax model. The final estimates for baseline effect, 50% inhibitory concentration (IC50), and the Hill coefficient were 79.4%, 23.4 ng/mL and 1.63, respectively. The maximum effect (Emax) was fixed at 80% (submaximal aggregation response). In addition, correlations between TS-943 plasma concentration and extension of template bleeding time were examined by fitting with an exponential model. The model estimates that the TS-943 plasma concentration necessary to double template bleeding time is approximately 63 ng/mL (ie, 2.7-fold greater than the IC50). The population approaches for pharmacokinetic-pharmacodynamic investigation can be useful for the analysis of concentration-effect relationships and concentration-adverse event relationships for a platelet glycoprotein IIb/IIIa receptor antagonist.

Intravenous and oral antithrombotic efficacy of the novel platelet GPIIb/IIIa antagonist roxifiban (DMP754) and its free acid form, XV459.

Currently used antiplatelet drugs, including aspirin, ticlopidine, and others, are effective against certain but not all of the many endogenous platelet activators. Because of their limited efficacy, a significant number of serious thromboembolic complications still occur, highlighting the need for a more effective therapy. DMP754 (roxifiban), a prodrug of XV459, is a recently discovered, potent antiplatelet agent with high affinity and specificity for platelet GPIIb/IIIa receptors that blocks platelet aggregate formation regardless of the agonist (IC(50)=0.030 to 0.05 micromol/L) or anticoagulant used for blood collection. DMP754 rapidly converts to its active free-acid form, XV459, which has a comparable high affinity for both resting and activated platelets (K(d)=1 to 2 nmol/L) and a relatively slow rate of dissociation from resting platelets. The present study was undertaken to determine intravenous and oral antithrombotic efficacies of DMP754 and XV459 and to compare them with those of other antiplatelet and anticoagulant agents in canine models of arterial thrombosis. In these models, thrombosis was induced either electrolytically (200-microA anodal current) in the carotid artery or mechanically by external clamping of the femoral artery along with stenosis, which resulted in either total occlusive thrombus formation or cyclic flow reduction, respectively. DMP754 and XV459 were given either intravenously (0.1 mg/kg bolus) or orally (0.1 to 0.4 mg/kg). Additionally, the antithrombotic efficacies of DMP754, aspirin, heparin, and ticlopidine in the canine carotid artery electrolytic injury model were compared. DMP754 demonstrated oral bioavailability of 20.8% in dogs after administration at different doses and prevented cyclic flow reduction (ED(90-100)=<0.1 mg/kg IV or PO). Additionally, both DMP754 and XV459 (0.1 mg/kg IV or 0.3 to 0.4 mg/kg PO) demonstrated maximal antithrombotic efficacy in preventing electrically induced carotid and coronary artery thrombosis and significant antithrombotic efficacy (P<0.001) at relatively low doses in different settings of arterial thrombosis in the canine model. DMP754 resulted in a significant reduction in thrombus mass and sustained arterial blood flow with 100% prevention of occlusive and nonocclusive thrombosis. In contrast, administration of aspirin (10 mg/kg PO for 2 days), heparin (10 IU/kg IV bolus followed by 90 IU/kg IV infusion over 3 hours), or ticlopidine (300 mg/kg PO for 3 days) before initiation of arterial thrombosis did not reduce the incidence of electrolytic injury-induced occlusive arterial thrombosis. These studies demonstrated a distinct antithrombotic efficacy of DMP754 as compared with existing strategies and suggest potential intravenous and oral antithrombotic uses of DMP754 in the prevention and treatment of thromboembolic disorders.

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.