Unbound Fraction of Clozapine Significantly Decreases with Elevated Plasma Concentrations of the Inflammatory Acute-Phase Protein Alpha-1-Acid Glycoprotein.

BACKGROUND: During inflammation, elevated total (unbound plus protein-bound) clozapine plasma concentrations have been observed. Elevated alpha-1-acid glycoprotein concentrations during inflammation are suggested to cause increased plasma clozapine-alpha-1-acid glycoprotein binding, resulting in elevated total clozapine plasma concentrations without significant changes in unbound concentrations. Here, we investigated the association between alpha-1-acid glycoprotein plasma concentrations and clozapine unbound fraction. METHODS: First, 25 and 60 µL of alpha-1-acid glycoprotein solution (20 mg/mL) were added to plasma samples (n = 3) of clozapine users (spiking experiment). Second, the association between alpha-1-acid glycoprotein plasma concentration and clozapine unbound fraction was assessed in patient samples (patient study). Samples were determined by liquid chromatography-tandem mass spectrometry. Data were analyzed with a paired t test (spiking experiment) and an unpaired t test (patient study). RESULTS: The spiking experiment showed significantly lower mean unbound fractions following 25- and 60-µL alpha-1-acid glycoprotein spikes (relative reductions of 28.3%, p = 0.032 and 43.4%, p = 0.048). In the patient study, total clozapine plasma concentrations were 10% higher in elevated (n = 6) compared with normal alpha-1-acid glycoprotein (n = 20) samples [525 µg/L vs. 479 µg/L, mean difference = 47 µg/L (95% confidence interval -217 to 310), p = 0.72]. Elevated alpha-1-acid glycoprotein samples had a 26% lower mean unbound fraction compared with normal samples [1.22% vs. 1.65%, mean difference = -0.43% (95% confidence interval -0.816 to -0.0443), p = 0.03]. CONCLUSIONS: Both the spiking experiment and patient study showed a significant association between elevated alpha-1-acid glycoprotein plasma concentrations and a lower clozapine unbound fraction. Future studies should include clinical data to examine whether this association is clinically relevant, suggesting any clozapine dose adjustments.

A definite measure of occupancy exposures, seeking with non-radiolabeled in vivo 5-HT2A receptor occupancy and in vitro free fractions.

Unbound drug concentration in the brain would be the true exposure responsible for specific target occupancy. Drug exposures from preclinical are total concentrations of those over/underestimate the clinical dose projection. With the application of mass spectrometry, the current work proposes a definite measure of test drug exposures at serotonin-2A occupancy. The 5-HT2A occupancy of antagonist in the rat brain has determined with non-radiolabeled tracer MDL-100,907 at an optimized dose (3 µg/kg) and treatment time (30 min). Equilibrium dialysis method determines the in vitro free fraction of the test antagonist in untreated rat brain homogenates and plasma. Drug-free fractions derived the unbound concentration (EC50) in plasma and brain at test doses. The corresponding binding affinities (Ki) correlated with the unbound concentrations. Except for quetiapine, the ED50 values in the dose-occupancy curves of antagonists are close and ranged from 1 to 3 mg/kg. The test drug quetiapine, eplivanserin, and clozapine showed high free fractions in plasma, but for ketanserin and olanzapine, the brain free fraction was higher. The correlation between the unbound EC50 of the antagonists and corresponding Ki values was good (r2=0.828). The improved EC50 accuracy with unbound concentrations was 10-250 folds in plasma and 10-170 folds in the brain. Further, the free fractions (fu, plasma/fu, brain) of test drugs had shown a correlation of ∼83% with brain permeability (Ctotal brain/Ctotal plasma), a limiting factor. Thus, correlating the occupancy with unbound exposure and pharmacology would result in an accurate measurement of drug potency and optimizes in selecting the clinical dose.

Single Administration of HBK-15-a Triple 5-HT1A, 5-HT7, and 5-HT3 Receptor Antagonist-Reverses Depressive-Like Behaviors in Mouse Model of Depression Induced by Corticosterone.

Studies suggest that the blockade of 5-HT1A, 5-HT7, and 5-HT3 receptor may increase the speed of antidepressant response. 1-[(2,6-Dimethylphenoxy)ethoxyethyl]-4-(2-methoxyphenyl)piperazine hydrochloride (HBK-14) and 1-[(2-chloro-6-methylphenoxy)ethoxyethyl]-4-(2-methoxyphenyl)piperazine hydrochloride (HBK-15), dual 5-HT1A and 5-HT7 antagonists, showed significant antidepressant- and anxiolytic-like properties in our previous tests in rodents. In this study, we aimed to investigate their antidepressant potential using mouse model of corticosterone-induced depression. We chose sucrose preference test, forced swim test, and elevated plus maze to determine anhedonic-, antidepressant-, and anxiolytic-like activities. We also evaluated the influence of the active compound on brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) levels in the hippocampus. Moreover, for both compounds, we performed biofunctional (5-HT3 receptor) and pharmacokinetic studies. We found that HBK-14 and HBK-15 were potent 5-HT3 receptor antagonists. HBK-14 (2.5 mg/kg) and HBK-15 (1.25 mg/kg) after intravenous (i.v.) and intraperitoneal (i.p.) administration permeated the blood-brain barrier with brain/plasma ratio lower than 1. The bioavailability of studied compounds after i.p. administration was 15% for HBK-14 and 54% for HBK-15. Chronic administration of HBK-15 (1.25 mg/kg) and fluoxetine (10 mg/kg) protected corticosterone-treated mice from anhedonic-, depressive-, and anxiety-like behaviors, as well as decreases in BDNF and NGF levels in the hippocampus. HBK-14 (2.5 mg/kg) counteracted anxiety-like behaviors in corticosterone-treated mice. Single administration of HBK-15 (1.25 mg/kg) and ketamine (1 mg/kg) reversed depression-like behavior and regulated decreased BDNF level in the hippocampus in corticosterone-treated mice. Our results suggest that simultaneous blockade of serotonergic 5-HT1A, 5-HT7, and 5-HT3 receptors might accelerate antidepressant response.

New evidence for oxetorone toxicity.

CONTEXT: Oxetorone is a serotonin antagonist antimigraine drug but literature relating to its toxic properties is poor. The aim of this study is to describe the toxicological profile of oxetorone and to highlight any relationship between clinical and analytical findings. MATERIALS AND METHODS: This is a retrospective and observational study of cases exposure to oxetorone, reported to the Angers Poison and Toxicovigilance Centre between January 2002 and May 2016. Severity was assessed using the Poisoning Severity Score (PSS). Cases where data were incomplete, where oxetorone was deemed not accountable, where clinical signs were linked mainly to a co-ingested drug or where the plasma concentration of oxetorone was negative were all excluded. RESULTS: We included 43 cases of exposure, 31 of whom were suicide attempts. The assumed ingested dose (60-3600 mg) was correlated to severity (rs = 0.45, p = 0.01). Symptoms of moderate severity (PSS2 = drowsiness, hypertonia, myosis, convulsions, arterial hypotension, QRS widening, QTc prolongation) were observed following ingestion of more than 600 mg of oxetorone (median dose =1200 mg) and severe symptoms (PSS 3 = coma, convulsions, QTc prolongation, QRS widening, ventricular tachycardia, arterial hypotension, cardiogenic shock) were observed starting from 1800 mg (median dose =2700 mg). In four cases, a secondary worsening of symptoms 10-48 h following ingestion was observed. Plasma oxetorone was measured in four patients. Severe symptoms were observed in the event of a concentration over 0.3 mg/L and the highest measured serum oxetorone level was delayed by 20-48 h following the ingestion for two cases. CONCLUSIONS: Several clinical and paraclinical parameters strongly point towards membrane-stabilising properties of the molecule and the risk of a delayed occurrence of symptoms or a secondary worsening.

Development and validation of a rapid LC-MS/MS method for simultaneous determination of netupitant and palonosetron in human plasma and its application to a pharmacokinetic study.

A simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was firstly developed and validated for simultaneous determination of netupitant and palonosetron in human plasma using ibrutinib as the internal standard (IS). Following liquid-liquid extraction, the compounds were eluted isocratically on a Phenomenex C18 column (50mm×2.0mm, 3µm) with the mobile phase consisting of acetonitrile and 10mM ammonium acetate buffer (pH 9.0) (89:11, v/v) at the flow rate of 0.3mL/min. The monitored ion transitions were m/z 579.5→522.4 for netupitant, m/z 297.3→110.2 for palonosetron and m/z 441.2→138.1 for IS. Chromatographic run time was 2.5min per injection, which made it possible to analyze more than 300 of samples per day. The assay exhibited a linear dynamic range of 5-1000ng/mL for netupitant and 0.02-10ng/mL for palonosetron in plasma. The values for both within- and between-day precision and accuracy were well within the generally accepted criteria for analytical methods (<15%). Selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, stability, matrix effect, recovery and carry-over effect were evaluated for all analytes. The method is simple, rapid, and has been applied successfully to a pharmacokinetic study of netupitant and palonosetron in healthy volunteers.

Sustained release of risperidone from biodegradable microspheres prepared by in-situ suspension-evaporation process.

Risperidone-loaded poly (D,L-lactide-co-glycolide) (PLGA) microspheres were prepared with a suspension-evaporation process with an aqueous suspension containing an in situ-formed aluminum hydroxide inorganic gel (SEP-AL process) and evaluated for encapsulation efficiency, particle size, surface morphology, glass transition temperature, in vitro drug release profile, and in vivo behavior. The SEP-AL microspheres were compared with conventional oil-in-water (O/W) emulsion solvent evaporation method using polyvinylalcohol (PVA) as an emulsifier (CP-PVA process). The microspheres were spherical in shape. DSC measurements showed that risperidone crystallinity was greatly reduced due to the homogeneous distribution of risperidone in PLGA microspheres. In vitro drug release profile from the microspheres showed a sigmoidal pattern of negligible initial burst up to 24h and minimal release (time-lag) for 7 days. After the lag phase, slow release took a place up to 25 days and then rapid release occurred sharply for 1 week. In vivo rat pharmacokinetic profile from the microspheres showed very low blood concentration level at the initial phase (up to 24h) followed by the latent phase up to 21 days. At the 3rd week, main phase started and the blood concentration of the drug increased up to the 5th week, and then gradually decreased. The risperidone-loaded PLGA microspheres produced by SEP-AL process showed excellent controlled release characteristics for the effective treatment of schizophrenia patients.

Pharmacokinetics and bioavailability study of two ondansetron oral soluble film formulations in fasting healthy male Chinese volunteers.

BACKGROUND: Ondansetron oral soluble film is designed to be applied on top of the tongue without requiring water to aid dissolution or swallowing, which is especially fitting for nausea and vomiting patients. PURPOSE: This study was conducted to compare the bioavailability of two 8 mg ondansetron oral soluble film formulations. PATIENTS AND METHODS: This randomized, open-label, two-period crossover study was performed under fasting conditions. A total of ten eligible subjects were randomly assigned at a 1:1 ratio to receive a single 8 mg dose of the test and reference ondansetron oral soluble film formulations, followed by a 1-week washout period and administration of the alternate formulation. The concentrations of ondansetron were assayed using an liquid chromatograph-mass spectrometer/mass spectrometer (LC-MS/MS) method. For analysis of pharmacokinetic properties, including the peak concentration of T max (C max), AUC from time 0 (baseline) to t hours (AUC0- t ), and AUC from baseline to infinity (AUC0-∞), blood samples were obtained at intervals over the 24-hour period after studying drug administration. Tolerability was assessed by monitoring vital signs and laboratory tests (hematology, blood biochemistry, hepatic function, and urinalysis) and by questioning subjects about adverse events. RESULTS: The mean (standard derivation [SD]) relative bioavailability was 96.5 (23.7%). The 90% confidence intervals (CIs) for the log-transformed ratios of C max and AUC0- t were 84.71%-103.28% and 91.38%-108.60%, respectively (P>0.05). Similar results were found for the data without log-transformation. No statistically significant differences were found based on analysis of variance. No significant adverse events occurred or were reported during the study. CONCLUSION: As the 90% CIs based on the differences between the test and reference formulation were within the 80%-125% range for both the C max and AUC0- t , we concluded that the two formulations were bioequivalent with respect to the rate or the extent of absorption. Both formulations are well tolerated.

Simultaneous determination of sarpogrelate and its active metabolite in human plasma by liquid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study.

We established a rapid and simple liquid chromatography with tandem mass spectrometry method for the simultaneous determination of sarpogrelate and its active metabolite, M-1, in human plasma. Sarpogrelate, M-1, and the internal standard, ketanserin, were extracted from a 50 µL aliquot of human plasma by protein precipitation using acetonitrile. Chromatographic separation was performed on a Shim-pack GIS ODS C18 column (100 × 3.0 mm; 3 µm) with an isocratic mobile phase consisting of 10 mM ammonium acetate and acetonitrile (70:30, v/v) at a flow rate of 0.6 mL/min; the total run time was <2.5 min. Mass spectrometric detection was conducted in selected reaction-monitoring mode with positive electrospray ionization at m/z 430.35 → 135.10 for sarpogrelate, m/z 330.30 → 58.10 for M-1, and m/z 395.70 → 188.85 for ketanserin. The linear ranges of concentration for sarpogrelate and M-1 were 1-1000 and 0.5-500 ng/mL, respectively. The coefficient of variation for the assay's precision was ≤9.95%, and the accuracy was 90.6-107%. All analytes were stable under various storage and handling conditions, and no relevant crosstalk and matrix effect was observed. This method was successfully applied to a pharmacokinetic study after oral administration of a 100 mg sarpogrelate tablet to healthy male Korean volunteers.

In vivo occupancy of the 5-HT1A receptor by a novel pan 5-HT1(A/B/D) receptor antagonist, GSK588045, using positron emission tomography.

5-hydroxytryptamine 1 (5-HT1) receptor blockade in combination with serotonin reuptake inhibition may provide a more rapid elevation of synaptic 5-HT compared to serotonin reuptake alone, by blocking the inhibitory effect of 5-HT1 receptor activation on serotonin release. GSK588045 is a novel compound with antagonist activity at 5-HT1A/1B/1D receptors and nanomolar affinity for the serotonin transporter, which was in development for the treatment of depression and anxiety. Here we present the results of an in vivo assessment of the relationship between plasma exposure and 5-HT1A receptor occupancy. Six Cynomolgus monkeys (Macaca fascicularis) were scanned using the PET ligand [(11)C]WAY100635 before and after dosing with GSK588045 (0.03, 0.1 and 0.3 mg/kg 60 min i.v. infusion). Data was quantified using a simplified reference tissue model, with the cerebellar time-activity curve used as an input function. Plasma levels of GSK588045 were measured, and the EC50 of GSK588045 for 5-HT1A receptor occupancy was estimated. An Emax model described the relationship between the GSK588045 plasma concentration and 5-HT1A receptor occupancy data well. EC50 estimates (and 95% confidence intervals) for raphe nuclei and the frontal cortex were 6.99 (2.48 to 11.49) and 7.80 (2.84 to 12.76) ng/ml respectively. GSK588045 dose dependently blocked the signal of the PET ligand [(11)C]WAY100635, confirming its brain entry and occupancy of 5-HT1A receptors in the primate brain. The estimated EC50 at the post-synaptic heteroreceptors and somatodendritic autoreceptors is similar. 5-HT1 receptor blockade by compounds such as GSK588045 may provide a faster alternate mechanism of antidepressant and anxiolytic action than standard SSRI treatment.

ADN-1184 a monoaminergic ligand with 5-HT(6/7) receptor antagonist activity: pharmacological profile and potential therapeutic utility.

BACKGROUND AND PURPOSE: Many dementia patients exhibit behavioural and psychological symptoms (BPSD) that include psychosis, aggressivity, depression and anxiety. Antipsychotic drugs are frequently prescribed but fail to significantly attenuate mood deficits, may interfere with cognitive function and are associated with motor and cardiac side effects, which are problematic in elderly patients. A need therefore exists for drugs that are better suited for the treatment of BPSD. EXPERIMENTAL APPROACH: We used in vitro cellular and in vivo behavioural tests to characterize ADN-1184, a novel arylsulfonamide ligand with potential utility for treatment of BPSD. KEY RESULTS: ADN-1184 exhibits substantial 5-HT6 /5-HT7 /5-HT2A /D2 receptor affinity and antagonist properties in vitro. In tests of antipsychotic-like activity, it reversed MK-801-induced hyperactivity and stereotypies and inhibited conditioned avoidance response (MED = 3 mg·kg(-1) i.p.). Remarkably, ADN-1184 also reduced immobility time in the forced swim test at low doses (0.3 and 1 mg·kg(-1) i.p.; higher doses were not significantly active). Notably, up to 30 mg·kg(-1) ADN-1184 did not impair memory performance in the passive avoidance test or elicit significant catalepsy and only modestly inhibited spontaneous locomotor activity (MED = 30 mg·kg(-1) i.p.). CONCLUSIONS AND IMPLICATIONS: ADN-1184 combines antipsychotic-like with antidepressant-like properties without interfering with memory function or locomotion. This profile is better than that of commonly used atypical antipsychotics tested under the same conditions and suggests that it is feasible to identify drugs that improve BPSD, without exacerbating cognitive deficit or movement impairment, which are of particular concern in patients with dementia.

Determination of 5-HT receptor antagonists, MEFWAY and MPPF using liquid chromatography electrospray ionization tandem mass spectrometry in rat plasma and brain tissue.

A simple, selective, and sensitive liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was validated for the determination of 4-fluoromethyl-N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridyl)cyclohexane-1-carboxamide (MEFWAY) and 4-fluoro-N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridyl)benzamide (MPPF) in rat plasma and brain samples, respectively. Plasma and brain samples were extracted with a mixture of acetonitrile and methanol (1:1, v/v) and then separated on a C(18) column (Gemini 3µm 110Å, 50×2.00mm ID, Phenomenex, USA). Quantitation was performed using LC-ESI-MS/MS in multiple-reaction monitoring (MRM) mode with positive ion electrospray ionization (ESI). The limit of quantification (LOQ) of 5ng/mL and 1ng/mL were obtained in 50µL brain homogenate and plasma, respectively. The analytical linear ranges of this method were 1-4000ng/mL in plasma and 5-4000ng/mL in brain homogenate with a correlation coefficients (R(2)) greater than 0.9993. The intra- and inter-day precision and accuracy values were within the assay validation guideline (lower than 13.0%). The analytes in plasma and brain samples were stable after three freeze-thaw cycles, long-term storage (one month at -80°C), and short-term (4h) storage at room temperature. The present method was successfully applied to plasma-brain pharmacokinetic studies to investigate brain penetration of a single dose of MEFWAY and MPPF in rats.

Outcome definitions and clinical predictors influence pharmacogenetic associations between HTR3A gene polymorphisms and response to clozapine in patients with schizophrenia.

RATIONALE: Pharmacogenetics of schizophrenia has not yet delivered anticipated clinical dividends. Clinical heterogeneity of schizophrenia contributes to the poor replication of the findings of pharmacogenetic association studies. Functionally important HTR3A gene single-nucleotide polymorphisms (SNPs) were reported to be associated with response to clozapine. OBJECTIVE: The aim of this study was to investigate how the association between HTR3A gene SNP and response to clozapine is influenced by various clinical predictors and by differing outcome definitions in patients with treatment-resistant schizophrenia (TRS). METHODS: We recruited 101 consecutive patients with TRS, on stable doses of clozapine, and evaluated their HTR3A gene SNP (rs1062613 and rs2276302), psychopathology, and serum clozapine levels. We assessed their socio-demographic and clinical profiles, premorbid adjustment, traumatic events, cognition, and disability using standard assessment schedules. We evaluated their response to clozapine, by employing six differing outcome definitions. We employed appropriate multivariate statistics to calculate allelic and genotypic association, accounting for the effects of various clinical variables. RESULTS: T allele of rs1062613 and G allele of rs2276302 were significantly associated with good clinical response to clozapine (p = 0.02). However, varying outcome definitions make these associations inconsistent. rs1062613 and rs2276302 could explain only 13.8 % variability in the responses to clozapine, while combined clinical predictors and HTR3A pharmacogenetic association model could explain 38 % variability. CONCLUSIONS: We demonstrated that the results of pharmacogenetic studies in schizophrenia depend heavily on their outcome definitions and that combined clinical and pharmacogenetic models have better predictive values. Future pharmacogenetic studies should employ multiple outcome definitions and should evaluate associated clinical variables.

Determination of palonosetron in human plasma by ultra performance liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study.

A rapid, sensitive and selective ultra performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method was developed for the determination of palonosetron (PALO) in human plasma. Verapamil was used as the internal standard (I.S.). Sample pretreatment involved liquid-liquid extraction with diethyl ether under alkaline condition. Chromatographic separation was carried out on an ACQUITY UPLC™ HSS T(3) column with mobile phase consisting of methanol-water containing 0.1% formic acid (80:20, v/v) at a flow-rate of 0.20mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The ion transitions of PALO and I.S. were m/z 297.3→109.8 and m/z 455.1→164.9, respectively. Each plasma sample was chromatographed within 1.2min. The linear calibration curves were obtained in the concentration range of 0.0190-3.80ng/mL (r(2)≥0.99) with a lower limit of quantification (LLOQ) of 0.0190ng/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were all less than 11% and accuracy (relative error, R.E.) was from 4.3% to 6.1% at all quality control (QC) levels. The method has been successfully applied to determine the plasma concentration of PALO in healthy Chinese volunteers after intravenous administration of a single dose of 0.125mg palonosetron hydrochloride.

Effects of high-dose large neutral amino acid supplementation on exercise, motor skill, and mental performance in Australian Rules Football players.

This study investigated the effects of high-dose large neutral amino acid (LNAA) supplementation on attenuating fatigue-induced decrements in exercise and motor skill performance in Australian Rules Football (ARF) players. Fifteen subelite ARF players participated in 3 testing sessions separated by 7 days. Players completed an initial control trial involving a reactive motor skills test (RMST) and a reactive agility test (RAT) carried out before and after fatiguing exercise. In the subsequent experimental trials, players ingested a serotonin-depleting or protein control (PC) LNAA mixture 3 h before testing, allocated in a double-blind randomized cross-over design. Blood samples were taken at presupplementation and pre- and postexercise for analysis of plasma amino acid, insulin, and metabolite concentrations. The effect of the LNAA was established as the difference in the change in the mean RMST and RAT test scores among the depleting, PC, and baseline (BL) trials. Mean overall repetition time of the RAT was moderately improved by -5.2% ± 3.4% (mean ± 90% confidence limits; effect size -0.45 ± 0.28) after ingestion of the serotonin-depleting mixture compared with the BL trial. Serotonin-depleting and PC supplements had a divergent effect on mean repetition time after fatiguing exercise in RMST: depleting serotonin elicited a small improvement (-3.0% ± 2.7%) in motor skill performance in contrast to a small decrement (2.4% ± 2.7%) after ingestion of the PC mixture, when compared to the BL. High-dose serotonin-"depleting" LNAA supplementation given 3 h prior to intermittent high-intensity exercise improved reactive motor skill and agility performance in ARF players.

Simple determination of azasetron in rat plasma by column-switching high-performance liquid chromatography.

For the quantification of azasetron in rat plasma samples, a column-switching HPLC method was developed and validated. Following dilution of plasma samples with mobile phase A (17 mM potassium phosphate buffer (pH 3.0)) and simple protein precipitation by addition of perchloric acid (60%), the mixture was directly injected onto the pre-column. After endogenous plasma substances were eluted to waste, the analyte was transferred to the trap column by switching the system. Then, the analyte was back-flushed to the analytical column for separation with mobile phase B (a 22:78 v/v mixture of acetonitrile and 17 mM potassium phosphate buffer (pH 3.0)) and detected at 250 nm using a photodiode array detector. A linear standard curve was obtained in the concentration range of 10-800 ng/mL with the correlation coefficient (r) of 0.9998. The intra- and inter-day precision and accuracy values for azasetron were in the ranges of 0.3-12.9% and 89.7-101.4%, respectively. The method was valid in terms of specificity, precision, and accuracy. In addition, this efficient analytical method was successfully applied to determine plasma concentrations of azasetron following oral administration of azasetron at a dose of 4.0 mg/kg to rats.

Validated LC-MS/MS method for the determination of sarpogrelate in human plasma: application to a pharmacokinetic and bioequivalence study in Chinese volunteers.

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for the determination of sarpogrelate in human plasma. One-step protein precipitation with acetonitrile was used to extract the analytes from the plasma. Sarpogrelate and tramadol (internal standard, I.S.) were separated on a Venusil MP-C(18) column within 1.7 min, using acetonitrile:ammonium acetate (10 mM, pH 6.8) (55:45, v/v) as mobile phase at a flow rate of 1.2 mL/min with an approximately 1:1 split entering the mass spectrometer. Detection was performed on electrospray positive ionization mass spectrometry by multiple reaction monitoring of the transitions of sarpogrelate at m/z 430.3-->135.3 and of I.S. at m/z 264.1-->58.0. The assay was validated over the concentration range of 1-1000 ng/mL with a lower limit of quantitation (LLOQ) of 1 ng/mL using 50 microL of plasma. The intra- and inter-day precision (relative standard deviation, R.S.D.) were

Rapidfilm: an innovative pharmaceutical form designed to improve patient compliance.

The aim of the research was to assess the bioequivalence between Rapidfilm, a new patented delivery system, versus the traditional orodispersible tablet (ODT). A randomized, two-way, single dose, crossover, bioequivalence study was conducted in 24 fasting, healthy volunteers with two formulations of ondansetron (Ondansetron Rapidfilm vs. Zofran Zydis Lingual ODT by GlaxoSmithKline GmbH & Co. KG). Plasma samples were analysed by a validated LC-MS/MS method during a collection period of 24 h post-dosing. The analysis of variance (ANOVA) on the targeted pharmacokinetic parameters did not show any significant difference between the two formulations and 90% confidence intervals (CIs) fell within the common acceptance range of 80-125%, satisfying the bioequivalence criteria. These results allow Rapidfilm to claim the same panel of indications of the conventional immediate release oral solid dosage forms, but offering several advantages also over the ODT: it can result in higher patient convenience for several applications.

Targeted inhibition of the serotonin 5HT2A receptor improves coronary patency in an in vivo model of recurrent thrombosis.

BACKGROUND: Release of serotonin and activation of serotonin 5HT2A receptors on platelet surfaces is a potent augmentative stimulus for platelet aggregation. However, earlier-generation serotonin receptor antagonists were not successfully exploited as antiplatelet agents, possibly owing to their lack of specificity for the 5HT2A receptor subtype. OBJECTIVE: To assess whether targeted inhibition of the serotonin 5HT2A receptor attenuates recurrent thrombosis and improves coronary patency in an in vivo canine model mimicking unstable angina. METHODS: In protocol 1, anesthetized dogs were pretreated with a novel, selective inverse agonist of the 5HT2A receptor (APD791) or saline. Recurrent coronary thrombosis was then initiated by coronary artery injury+stenosis, and coronary patency was monitored for 3 h. Protocol 2 was similar, except that: (i) treatment with APD791 or saline was begun 1 h after the onset of recurrent thrombosis; (ii) template bleeding time was measured; and (iii) blood samples were obtained for in vitro flow cytometric assessment of platelet responsiveness to serotonin. RESULTS: APD791 attenuated recurrent thrombosis, irrespective of the time of treatment: in both protocols, flow-time area (index of coronary patency; normalized to baseline coronary flow) averaged 58-59% (P<0.01) following administration of APD791 vs. 21-28% in saline controls. Moreover, the in vivo antithrombotic effect of APD791 was not accompanied by increased bleeding, but was associated with significant and selective inhibition of serotonin-mediated platelet activation. CONCLUSION: 5HT2A receptor inhibition with APD791, even when initiated after the onset of recurrent thrombosis, improves coronary patency in the in vivo canine model.

APD791, 3-methoxy-n-(3-(1-methyl-1h-pyrazol-5-yl)-4-(2-morpholinoethoxy)phenyl)benzamide, a novel 5-hydroxytryptamine 2A receptor antagonist: pharmacological profile, pharmacokinetics, platelet activity and vascular biology.

We have evaluated the receptor pharmacology, antiplatelet activity, and vascular pharmacology of APD791 [3-methoxy-N-(3-(1-methyl-1H-pyrazol-5-yl)-4-(2-morpholinoethoxy)phenyl)benzamide] a novel 5-hydroxytryptamine 2A (5-HT(2A)) receptor antagonist. APD791 displayed high-affinity binding to membranes (K(i) = 4.9 nM) and functional inverse agonism of inositol phosphate accumulation (IC(50) = 5.2 nM) in human embryonic kidney cells stably expressing the human 5-HT(2A) receptor. In competition binding assays, APD791 was greater than 2000-fold selective for the 5-HT(2A) receptor versus 5-HT(2C) and 5-HT(2B) receptors, and was inactive when tested against a wide panel of other G-protein-coupled receptors. APD791 inhibited 5-HT-mediated amplification of ADP-stimulated human and dog platelet aggregation (IC(50) = 8.7 and 23.1 nM, respectively). Similar potency was observed for inhibition of 5-HT-stimulated DNA synthesis in rabbit aortic smooth muscle cells (IC(50) = 13 nM) and 5-HT-mediated vasoconstriction in rabbit aortic rings. Oral administration of APD791 to dogs resulted in acute (1-h) and subchronic (10-day) inhibition of 5-HT-mediated amplification of collagen-stimulated platelet aggregation in whole blood. Two active metabolites, APD791-M1 and APD791-M2, were generated upon incubation of APD791 with human liver microsomes and were also indentified in dogs after oral administration of APD791. The affinity and selectivity profiles of both metabolites were similar to APD791. These results demonstrate that APD791 is an orally available, high-affinity 5-HT(2A) receptor antagonist with potent activity on platelets and vascular smooth muscle.

Determination of tropisetron in human plasma by liquid chromatography-tandem mass spectrometry.

A sensitive and selective liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for the determination of tropisetron in human plasma was developed and validated over the concentration range of 0.100-100 ng/mL. Diphenhydramine was used as the internal standard (IS). The tropisetron and the IS were extracted from alkalized plasma samples into diethyl ether-dichloromethane (2:1, v/v) and the LC separation was performed by a Diamonsil C18 column (150 mm x 4.6 mm, i.d., 5 microm). The mobile phase was methanol:water (80:20, v/v) containing 0.2% formic acid delivered at a flow rate of 0.5 mL/min. The total chromatographic run time was 4.5 min. The MS data acquisition was accomplished by selected reaction monitoring (SRM) mode with positive atmospheric pressure chemical ionization (APCI) interface. The lower limit of quantification (LLOQ) achieved was 0.100 ng/mL with precision (RSD) of 3.1% and accuracy (RE) of -0.7%. For both inter-batch and intra-batch tests, the precision (RSD) for the entire validation was less than 6.0%, and the accuracy (RE) was within the -0.5% to 0.2% range. This validated LC-MS/MS method was later used to characterize the pharmacokinetics as well as the bioequivalence of tropisetron formulations.