Exposure to the pharmaceutical buspirone alters locomotor activity, anxiety-related behaviors, and transcripts related to serotonin signaling in larval zebrafish (Danio rerio).

Buspirone is a pharmaceutical used to treat general anxiety disorder by acting on the dopaminergic and serotoninergic system. Buspirone, like many human pharmaceuticals, has been detected in municipal wastewater; however, the environmental exposure risks are unknown for this psychoactive compound. We studied the effects of buspirone on the behavior of zebrafish, focusing on locomotor and anxiolytic behavior. We also measured transcripts associated with oxidative stress, neurotoxicity, and serotonin signaling to identify potential mechanisms underlying the behavioral changes. Concentrations ranged from environmentally relevant (nM) to physiologically active concentrations typical of human pharmaceuticals (µM). Buspirone treatment did not impact survival, nor did it induce deformities in zebrafish treated for 7 days up to 10 µM. There was a positive relationship between locomotor activity and buspirone concentration in dark periods of the visual motor response test. In the light-dark preference test, both the average time per visit to the dark zone and the percent cumulative duration in the dark zone were increased by 1 µM buspirone. Transcript levels of ache, manf, and mbp were decreased in larvae, while the expression of gap43 was increased following exposure to buspirone, indicating potential neurotoxic effects. There was also reduced expression of serotonin-related genes encoding receptors, transporters, and biosynthesis enzymes (i.e., 5ht1aa, sertb, and tph1a). These data increase understanding of the behavioral and molecular responses in zebrafish following waterborne exposure to neuroactive pharmaceuticals like buspirone.

Inhibition of Axitinib on Buspirone Metabolism in vitro and in vivo.

Objective: To evaluate the effect of axitinib on buspirone metabolism in vitro and in vivo. Methods: A microsome incubation assay was performed to study the effect and mechanism of axitinib on buspirone metabolizing. In vivo, buspirone was administered with or without axitinib to Sprague-Dawley rats. Plasma samples were collected and subjected to ultra-performance liquid chromatography-tandem mass spectrometry. Results: In both human liver microsomes (HLMs) and rat liver microsomes (RLMs), axitinib (100 µM) decreased buspirone hydroxylation and N-dealkylation by >85%. Axitinib inhibited buspirone hydroxylation and N-dealkylation, with an IC50 of 15.76 and 9.74 for RLMs, and 10.63 and 9.902 for HLMs. Axitinib showed noncompetitive inhibition of both 6'-hydroxylation and N-dealkylation. Moreover, coadministration of axitinib and buspirone led to an increase in the maximum plasma concentration (C max ) and area under the plasma concentration-time curve (AUC) of buspirone by 4.3- and 5.3-fold, respectively, compared with the control group. Conclusion: Axitinib inhibited buspirone metabolism in vivo and in vitro, which increases the risk of the side effects of buspirone in the clinic. When coadministered with axitinib, a lower dosage of buspirone should be defined to avoid a toxic response. Axitinib is suspected to function as an inhibitor of CYP3A4.

A New Workflow for Drug Metabolite Profiling by Utilizing Advanced Tribrid Mass Spectrometry and Data-Processing Techniques.

Drug metabolite profiling utilizes liquid chromatography with tandem mass spectrometry (LC/MS/MS) to acquire ample information for metabolite identification and structural elucidation. However, there are still challenges in detecting and characterizing all potential metabolites that can be masked by a high biological background, especially the unknown and uncommon ones. In this work, a novel metabolite profiling workflow was established on a platform using a state-of-the-art tribrid high-resolution mass spectrometry (HRMS) system. Primarily, an instrumental method was developed based on the novel design of the tribrid system that facilitates in-depth MSn scans with two fragmentation devices. Additionally, different advanced data acquisition techniques were assessed and compared, and automatic background exclusion and deep-scan approaches were adopted to promote assay efficiency and metabolite coverage. Finally, different data-analysis techniques were explored to fully extract metabolite data from the information-rich MS/MS data sets. Overall, a workflow combining tribrid mass spectrometry and advanced acquisition methodology has been developed for metabolite characterization in drug discovery and development. It maximizes the tribrid HRMS platform's utility and enhances the coverage, efficiency, quality, and speed of metabolite profiling assays.

Comparison of TiO2 photocatalysis, electrochemically assisted Fenton reaction and direct electrochemistry for simulation of phase I metabolism reactions of drugs.

The feasibility of titanium dioxide (TiO2) photocatalysis, electrochemically assisted Fenton reaction (EC-Fenton) and direct electrochemical oxidation (EC) for simulation of phase I metabolism of drugs was studied by comparing the reaction products of buspirone, promazine, testosterone and 7-ethoxycoumarin with phase I metabolites of the same compounds produced in vitro by human liver microsomes (HLM). Reaction products were analysed by UHPLC-MS. TiO2 photocatalysis simulated the in vitro phase I metabolism in HLM more comprehensively than did EC-Fenton or EC. Even though TiO2 photocatalysis, EC-Fenton and EC do not allow comprehensive prediction of phase I metabolism, all three methods produce several important metabolites without the need for demanding purification steps to remove the biological matrix. Importantly, TiO2 photocatalysis produces aliphatic and aromatic hydroxylation products where direct EC fails. Furthermore, TiO2 photocatalysis is an extremely rapid, simple and inexpensive way to generate oxidation products in a clean matrix and the reaction can be simply initiated and quenched by switching the UV lamp on/off.

Systematic evaluation of commercially available ultra-high performance liquid chromatography columns for drug metabolite profiling: optimization of chromatographic peak capacity.

The present study investigated the practical use of modern ultra-high performance liquid chromatography (UHPLC) separation techniques for drug metabolite profiling, aiming to develop a widely applicable, high-throughput, easy-to-use chromatographic method, with a high chromatographic resolution to accommodate simultaneous qualitative and quantitative analysis of small-molecule drugs and metabolites in biological matrices. To this end, first the UHPLC system volume and variance were evaluated. Then, a mixture of 17 drugs and various metabolites (molecular mass of 151-749Da, logP of -1.04 to 6.7), was injected on six sub-2µm particle columns. Five newest generation core shell technology columns were compared and tested against one column packed with porous particles. Two aqueous (pH 2.7 and 6.8) and two organic mobile phases were evaluated, first with the same flow and temperature and subsequently at each column's individual limit of temperature and pressure. The results demonstrated that pre-column dead volume had negligible influence on the peak capacity and shape. In contrast, a decrease in post-column volume of 57% resulted in a substantial (47%) increase in median peak capacity and significantly improved peak shape. When the various combinations of stationary and mobile phases were used at the same flow rate (0.5mL/min) and temperature (45°C), limited differences were observed between the median peak capacities, with a maximum of 26%. At higher flow though (up to 0.9mL/min), a maximum difference of almost 40% in median peak capacity was found between columns. The finally selected combination of solid-core particle column and mobile phase composition was chosen for its selectivity, peak capacity, wide applicability and peak shape. The developed method was applied to rat hepatocyte samples incubated with the drug buspirone and demonstrated to provide a similar chromatographic resolution, but a 6 times higher signal-to-noise ratio than a more traditional UHPLC metabolite profiling method using a fully porous particle packed column, within one third of the analysis time. In conclusion, a widely applicable, selective and fast chromatographic method was developed that can be applied to perform drug metabolite profiling in the timeframe of a quantitative analysis. It is envisioned that this method will in future be used for simultaneous qualitative and quantitative analysis and can therefore be considered a first important step in the Quan/Qual workflow.

Conformational analysis and geometry optimization of buspirone-A 5-HT1A receptor agonist.

Buspirone, a partial 5-HT1A receptor agonist, is a clinically prescribed anxiolytic. In the present study, conformational analysis and geometry optimization of buspirone were done as per Hartree-Fock (HF) calculation method by Argus Lab 4.0.1 software. The minimum potential energy was calculated by geometry convergence function by Argus Lab software. The results indicate that the best conformation of molecule is present at minimum potential energy of-100679.5513 kcal/mol. At this point, buspirone will be more active.

The exposure of highly toxic aconitine does not significantly impact the activity and expression of cytochrome P450 3A in rats determined by a novel ultra performance liquid chromatography-tandem mass spectrometric method of a specific probe buspirone.

Aconitum species are widely used to treat rheumatism, cardiovascular diseases, and tumors in China and other Asian countries. The herbs are always used with drugs such as paclitaxel. Aconitine (AC) is one of the main bioactive/high-toxic alkaloids of Aconitum roots. AC is metabolized by cytochrome P450 (CYP) 3A. However, whether AC inhibits/induces CYP3A, which causes drug-drug interaction (DDI) is unclear. Our study aims to explore the potent effects of AC, as a marker component of Aconitum, on CYP3A using the probe buspirone in rats. The effects of oral AC on pharmacokinetics of buspirone were evaluated. CYP3A activity and protein levels in rat liver microsomes pretreated with oral AC were also measured using in vitro buspirone metabolism and Western blot. Buspirone and its major metabolites 1-(2-pyrimidinyl)piperazine and 6'-hydroxybuspirone were determined using a newly validated UPLC-MS/MS method. Single dose and 7-day AC administration at 0.125mg/kg had no effect on CYP3A activity since no change in the formation of 1-(2-pyrimidinyl)piperazine and 6'-hydroxybuspirone. CYP3A activity and protein levels in liver microsomes were also not affected by 7-day AC pretreatment at 0.125mg/kg. Therefore, AC neither inhibits nor induces CYP3A in rats, indicating AC does not cause CYP3A-related DDI in the liver.

Quantitative estimation of circulating metabolites without synthetic standards by ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry in combination with UV correction.

An early assessment of metabolite exposure in preclinical species can provide quantitative estimation on possible active or toxic metabolites. Frequently, synthetic metabolite standards are not available at the preclinical stage, precluding the quantitation of metabolites by means of calibration curves and quality control (QC) samples. We present here an approach to determine the extent of circulating metabolites using 'metabolite standards' generated by in vitro incubations in combination with the correction for mass spectrometry response based on UV response. The study was done by coupling ultra-high-performance liquid chromatography (UHPLC) to LTQ-Orbitrap high-resolution mass spectrometry, and the quantitation was based on full scan high-resolution accurate mass analysis in combination with retention time. First, we investigated the separation capacity of a 10.5 min UHPLC method and the quantitative capability of an LTQ-Orbitrap for full scan accurate mass quantitation by spiking chemical standards of buspirone and its six metabolites in blank plasma. Then we demonstrated the use of a UV correction approach to quantitatively estimate buspirone and its metabolites in plasma samples from a rat pharmacokinetics study. We compared the concentration versus time profiles of buspirone and its six metabolites in rat plasma samples obtained using three different approaches, including using UV correction, using individual standard curves for each metabolite prepared from the synthetic standard, and using a calibration curve of the parent compound buspirone. We demonstrated the estimated metabolite exposure of buspirone using this UV correction approach resulted in rank ordering of metabolite exposure within three-fold of the value obtained with metabolite standards, in contrast to eight-fold without UV correction. The approach presented in this paper provides a practical solution to an unmet bioanalytical need for quantitative information on metabolites without standards in preclinical in vivo studies.

Application of a fluorescence-based continuous-flow bioassay to screen for diversity of cytochrome P450 BM3 mutant libraries.

A fluorescence-based continuous-flow enzyme affinity detection (EAD) setup was used to screen cytochrome P450 BM3 mutants on-line for diversity. The flow-injection screening assay is based on the BM3-mediated O-dealkylation of alkoxyresorufins forming the highly fluorescent product resorufin, and can be used in different configurations, namely injection of ligands, enzymes and substrates. Screening conditions were optimized and the activity of a library of 32 BM3 mutants towards the recently synthesized new probe substrate allyloxyresorufin was measured in flow-injection analysis (FIA) mode and it was shown that large activity differences between the mutants existed. Next, six BM3 mutants containing mutations at different positions in the active site were selected for which on-line enzyme kinetics were determined. Subsequently, for these six BM3 mutants affinity towards a set of 30 xenobiotics was determined in FIA EAD mode. It was demonstrated that significant differences existed for the affinity profiles of the mutants tested and that these differences correlated to alterations in the BM3 mutant-generated metabolic profiles of the drug buspirone. In conclusion, the developed FIA EAD approach is suitable to screen for diversity within BM3 mutants and this alternative screening technology offers new perspectives for rapid and sensitive screening of compound libraries towards BM3 mutants.

Development and evaluation of a multiple-plate fraction collector for sample processing: application to radioprofiling in drug metabolism studies.

Microplate scintillation counters are utilized routinely in drug metabolism laboratories for the off-line radioanalysis of fractions collected during HPLC radioprofiling. In this process, the current fraction collection technology is limited by the number of plates that can be used per injection as well as the potential for sample loss due to dripping or spraying as the fraction collector head moves from well to well or between plates. More importantly, sample throughput is limited in the conventional process, since the collection plates must be manually exchanged after each injection. The Collect PAL, an innovative multiple-plate fraction collector, was developed to address these deficiencies and improve overall sample throughput. It employs a zero-loss design and has sub-ambient temperature control. Operation of the system is completely controlled with software and up to 24 (96- or 384-well) fraction collection plates can be loaded in a completely automated run. The system may also be configured for collection into various-sized tubes or vials. At flow rates of 0.5 or 1.0 mL/min and at collection times of 10 or 15s, the system precisely delivered 83-µL fractions (within 4.1% CV) and 250-µL fractions (within 1.4% CV), respectively, of three different mobile phases into 12 mm × 32 mm vials. Similarly, at a flow rate of 1 mL/min and 10s collection times, the system precisely dispensed mobile phase containing a [(14)C]-radiolabeled compound across an entire 96-well plate (% CV was within 5.3%). Triplicate analyses of metabolism test samples containing [(14)C]buspirone and its metabolites, derived from three different matrices (plasma, urine and bile), indicated that the Collect PAL produced radioprofiles that were reproducible and comparable to the current technology; the % CV for 9 selected peaks in the radioprofiles generated with the Collect PAL were within 9.3%. Radioprofiles generated by collecting into 96- and 384-well plates were qualitatively comparable; however, the peak resolution was greater in the profiles that were collected in 384-well plates due to the collection of a larger number of fractions per minute. In conclusion, this new and innovative fraction collector generated radioprofile results that were comparable to current technology and should provide a major improvement in capacity and throughput for radioprofiling studies.

Two-injection workflow for a liquid chromatography/LTQ-Orbitrap system to complete in vivo biotransformation characterization: demonstration with buspirone metabolite identification.

The relatively high background matrix in in vivo samples typically poses difficulties in drug metabolite identification, and causes repeated analytical runs on unit resolution liquid chromatography/mass spectrometry (LC/MS) systems before the completion of biotransformation characterization. Ballpark parameter settings for the LTQ-Orbitrap are reported herein that enable complete in vivo metabolite identification within two HPLC/MS injections on the hybrid LTQ-Orbitrap data collection system. By setting the FT survey full scan at 60K resolution to trigger five dependent LTQ MS(2) scans, and proper parameters of Repeat Duration, Exclusion Duration and Repeat Count for the first run (exploratory), the Orbitrap achieved the optimal parallel data acquisition capability and collected maximum number of product ion scans. Biotransformation knowledge based prediction played the key role in exact mass ion extraction and multiple mass defect filtration when the initial data was processed. Meanwhile, product ion extraction and neutral loss extraction of the initial dependent data provided additional bonus in identifying metabolites. With updated parent mass list and the data-dependent setting to let only the ions on the parent mass list trigger dependent scans, the second run (confirmatory) ensures that all precursor ions of identified metabolites trigger not only dependent product ion scans, but also at or close to the highest concentration of the eluted metabolite peaks. This workflow has been developed for metabolite identification of in vivo or ADME studies, of which the samples typically contain a high level of complex matrix. However, due to the proprietary nature of the in vivo studies, this workflow is presented herein with in vitro buspirone sample incubated with human liver microsomes (HLM). The major HLM-mediated biotransformation on buspirone was identified as oxidation or hydroxylation since five mono- (+16 Da), seven di- (+32 Da) and at least three tri-oxygenated (+48 Da) metabolites were identified. Besides the metabolites 1-pyrimidinylpiperazine (1-PP) and hydroxylated 1-PP that formed by N-dealkylation, a new metabolite M308 was identified as the result of a second N-dealkylation of the pyrimidine unit. Two new metabolites containing the 8-butyl-8-azaspiro[4,5]decane-7,9-dione partial structure, M240 and M254, were also identified that were formed apparently due to the first N-dealkylation of the 1-PP moiety.

Porcine buccal mucosa as an in vitro model: relative contribution of epithelium and connective tissue as permeability barriers.

Porcine buccal mucosa has been extensively used as an in vitro model to study the permeability of various diffusants and to assess their potential to be delivered through the buccal route. The relative contribution of each component of the buccal mucosa on drug permeability was assessed in this study. The permeability of model diffusants decreased significantly with an increase in the mucosal thickness. A bilayer membrane model was developed to delineate the relative contribution to the barrier function offered by the epithelium and the connective tissue region. The decrease in permeability with mucosal thickness was attributed to the increase in the thickness of connective tissue. However, the epithelium acted as a primary barrier to permeation of all diffusants studied at a mucosal thickness up to 500 microm. In addition, the epithelium exhibited higher resistance to the permeation of hydrophilic diffusants than to lipophilic diffusants. With an increase in buccal mucosal membrane thickness, the lag time for the diffusants increased. Based on the analysis of permeation data, the buccal membrane, as a composite of epithelium and connective tissue, is considered as a heterogeneous permeation barrier. A mucosal tissue thickness of about 500 microm is recommended for in vitro transbuccal permeation studies since the epithelium remained the major permeability barrier for all diffusants at this thickness.

5-HT(1A) receptors in the frontal cortical brain areas in Cloninger type 1 and 2 alcoholics measured by whole-hemisphere autoradiography.

AIMS: The Cloninger type 1 alcoholics are prone to anxiety, and in many cases patients have begun to use alcohol in order to relieve their anxiety. We have previously reported a decrease of the serotonin transporter density in the perigenual anterior cingulate cortex (pACC) in type 1 alcoholics. The 5-HT(1A) receptors are the binding sites for anxiolytic drug buspirone. We aimed to investigate the alteration in the density of 5-HT(1A) receptors, that may also alter the effect of serotonin in the pACC in alcoholics. METHODS: The density of the serotonin receptor 5-HT(1A) among Cloninger type 1 and 2 alcoholics (nine and eight subjects, respectively) and 10 control subjects were determined by postmortem whole-hemisphere autoradiography with WAY-100635. RESULTS: Substantially sparser 5-HT(1A) (by -31%, P = 0.010) density was observed in the pACC of alcoholic subjects in relation to non-alcoholic comparison subjects. In a secondary analysis for the difference between the alcoholic subtypes and controls, the 5-HT(1A) density was decreased significantly by -32% (P = 0.015) in the upper level of pACC in type 1 alcoholics. CONCLUSIONS: The detected decrease of 5-HT(1A) receptor density on the pACC suggests further that the serotoninergic system is defected in the so-called affect region, especially in the type 1 alcoholics.

The molecular interactions of buspirone analogues with the serotonin transporter.

A major problem with the selective serotonin reuptake inhibitors (SSRIs) is the delayed onset of action. A reason for that may be that the initial SSRI-induced increase in serotonin levels activates somatodendritic 5-HT(1A) autoreceptors, causing a decrease in serotonin release in major forebrain areas. It has been suggested that compounds combining inhibition of the serotonin transport protein with antagonistic effects on the 5-HT(1A) receptor will shorten the onset time. The anxiolytic drug buspirone is known as 5-HT(1A) partial agonist. In the present work, we are studying the inhibition of the serotonin transporter protein by a series of buspirone analogues by molecular modelling and by experimental affinity measurements. Models of the transporter protein were constructed using the crystal structure of the Escherichia coli major facilitator family transporter-LacY and the X-ray structure of the neurotransmitter symporter family (NSS) transporter-LeuT(Aa) as templates. The buspirone analogues were docked into both SERT models and the interactions with amino acids within the protein were analyzed. Two putative binding sites were identified on the LeuT(Aa) based model, one suggested to be a high-affinity site, and the other suggested to be a low-affinity binding site. Molecular dynamic simulations of the LacY based model in complex with ligands did not induce a helical architecture of the LacY based model into an arrangement more similar to that of the LeuT(Aa) based model.

Can in vitro metabolism-dependent covalent binding data in liver microsomes distinguish hepatotoxic from nonhepatotoxic drugs? An analysis of 18 drugs with consideration of intrinsic clearance and daily dose.

In vitro covalent binding assessments of drugs have been useful in providing retrospective insights into the association between drug metabolism and a resulting toxicological response. On the basis of these studies, it has been advocated that in vitro covalent binding to liver microsomal proteins in the presence and the absence of NADPH be used routinely to screen drug candidates. However, the utility of this approach in predicting toxicities of drug candidates accurately remains an unanswered question. Importantly, the years of research that have been invested in understanding metabolic bioactivation and covalent binding and its potential role in toxicity have focused only on those compounds that demonstrate toxicity. Investigations have not frequently queried whether in vitro covalent binding could be observed with drugs with good safety records. Eighteen drugs (nine hepatotoxins and nine nonhepatotoxins in humans) were assessed for in vitro covalent binding in NADPH-supplemented human liver microsomes. Of the two sets of nine drugs, seven in each set were shown to undergo some degree of covalent binding. Among hepatotoxic drugs, acetaminophen, carbamazepine, diclofenac, indomethacin, nefazodone, sudoxicam, and tienilic acid demonstrated covalent binding, while benoxaprofen and felbamate did not. Of the nonhepatotoxic drugs evaluated, buspirone, diphenhydramine, meloxicam, paroxetine, propranolol, raloxifene, and simvastatin demonstrated covalent binding, while ibuprofen and theophylline did not. A quantitative comparison of covalent binding in vitro intrinsic clearance did not separate the two groups of compounds, and in fact, paroxetine, a nonhepatotoxin, showed the greatest amount of covalent binding in microsomes. Including factors such as the fraction of total metabolism comprised by covalent binding and the total daily dose of each drug improved the discrimination between hepatotoxic and nontoxic drugs based on in vitro covalent binding data; however, the approach still would falsely identify some agents as potentially hepatotoxic.

Rapid and sensitive characterization of the metabolite formation enzyme kinetics of radiolabeled drugs using stop-flow liquid radiochromatography.

This study evaluated the reproducibility, sensitivity, accuracy, and precision of stop-flow liquid radiochromatographic detection (RFD). Stop-flow RFD was about 10-fold more sensitive compared with traditional RFD and had a good reproducibility for quantification and HPLC retention time. Stop-flow RFD was applied to determine enzyme kinetics of radiolabeled drugs. The enzyme kinetic parameters of muraglitazar glucuronidation determined by stop-flow RFD were comparable with those determined by microplate scintillation counting (MSC).

Pharmacokinetics of 6-hydroxybuspirone and its enantiomers administered individually or following buspirone administration in humans.

The objective of this study was to assess the pharmacokinetics of 6-hydroxybuspirone (6OHB) when given orally via three forms: racemate (BMS-528215), S-enantiomer (BMS-442606) and R-enantiomer (BMS-442608), versus following the administration of buspirone. A double-blind, randomized, four-period, four-treatment, crossover study balanced for residual effects in healthy subjects was conducted (n=20). Subjects received single 10 mg doses of each compound in a randomized fashion with pharmacokinetics determined over a 24 h period. There was a 4-day washout between each dosing period. All three forms of 6OHB (racemate, S-enantiomer and R-enantiomer) were well tolerated. There was nterconversion between enantiomers. The dominant enantiomer was the S-enantiomer no matter which form of 6OHB was administered. All three forms of 6OHB produced approximately 2- to 3-fold greater exposure to total 6OHB than did buspirone. All three forms produced equal exposure to 1-(2-pyrimidinyl)-piperazine (1-PP) which was approximately 30% less than the 1-PP exposure derived from buspirone administration. All three forms of 6OHB produced approximately 3-fold higher 6OHB:1-PP ratios and approximately 2.5-fold higher total 6OHB exposures than did buspirone administration. All compounds were well tolerated. There seemed to be no advantage of one of the enantiomers of 6OHB over the racemate. Therefore, the racemate was chosen for further clinical development.

6-Hydroxybuspirone is a major active metabolite of buspirone: assessment of pharmacokinetics and 5-hydroxytryptamine1A receptor occupancy in rats.

The pharmacokinetics and in vivo potency of 6-hydroxybuspirone (6-OH-buspirone), a major metabolite of buspirone, were investigated. The plasma clearance (47.3 +/- 3.5 ml/min/kg), volume of distribution (2.6 +/- 0.3 l/kg), and half-life (1.2 +/- 0.2 h) of 6-OH-buspirone in rats were similar to those for buspirone. Bioavailability was higher for 6-OH-buspirone (19%) compared with that for buspirone (1.4%). After intravenous infusions to steady-state levels in plasma, 6-OH-buspirone and buspirone increased 5-hydroxytryptamine (HT)(1A) receptor occupancy in a concentration-dependent manner with EC(50) values of 1.0 +/- 0.3 and 0.38 +/- 0.06 microM in the dorsal raphe and 4.0 +/- 0.6 and 1.5 +/- 0.3 microM in the hippocampus, respectively. Both compounds appeared to be approximately 4-fold more potent in occupying presynaptic 5-HT(1A) receptors in the dorsal raphe than the postsynaptic receptors in the hippocampus. Oral dosing of buspirone in rats resulted in exposures (area under the concentration-time profile) of 6-OH-buspirone and 1-(2-pyrimidinyl)-piperazine (1-PP), another major metabolite of buspirone, that were approximately 12 (6-OH-buspirone)- and 49 (1-PP)-fold higher than the exposure of the parent compound. As a whole, these preclinical data suggest that 6-OH-buspirone probably contributes to the clinical efficacy of buspirone as an anxiolytic agent.

Simultaneous determination of metabolic stability and identification of buspirone metabolites using multiple column fast liquid chromatography time-of-flight mass spectrometry.

A recent trend in the drug discovery and development process is to shift the starting point of drug metabolism and pharmacokinetic (DMPK) studies to a time as early as possible in the development chain to address potential issues in parallel with the optimization of the drug's lead structure. Therefore, it is necessary to develop assay methods to determine early adsorption, distribution, metabolism and excretion (ADME) parameters like metabolic stability and metabolite identification. For metabolite identification it is of crucial importance to work with fast liquid chromatography/mass spectrometry (LC/MS) systems, which provide the necessary high throughput functionalities to handle a large number of samples in combination with high speed and high resolution chromatography as well as mass accuracy. In this study a fast two-column liquid chromatography (LC) method will be used to simultaneously determine metabolic stability and to identify metabolites of buspirone using highly accurate mass measurement by means of an electrospray time-of-flight (ESI-TOF) mass spectrometer. Whereby, the metabolic stability will be determined on a short sub-two micron column, the main metabolites will be identified in the same experiment by the automated use of a long sub-two micron column, which provides the necessary high resolution.

Automated software-guided identification of new buspirone metabolites using capillary LC coupled to ion trap and TOF mass spectrometry.

The identification and structure elucidation of drug metabolites is one of the main objectives in in vitro ADME studies. Typical modern methodologies involve incubation of the drug with subcellular fractions to simulate metabolism followed by LC-MS/MS or LC-MS(n) analysis and chemometric approaches for the extraction of the metabolites. The objective of this work was the software-guided identification and structure elucidation of major and minor buspirone metabolites using capillary LC as a separation technique and ion trap MS(n) as well as electrospray ionization orthogonal acceleration time-of-flight (ESI oaTOF) mass spectrometry as detection techniques. Buspirone mainly underwent hydroxylation, dihydroxylation and N-oxidation in S9 fractions in the presence of phase I co-factors and the corresponding glucuronides were detected in the presence of phase II co-factors. The use of automated ion trap MS/MS data-dependent acquisition combined with a chemometric tool allowed the detection of five small chromatographic peaks of unexpected metabolites that co-eluted with the larger chromatographic peaks of expected metabolites. Using automatic assignment of ion trap MS/MS fragments as well as accurate mass measurements from an ESI oaTOF mass spectrometer, possible structures were postulated for these metabolites that were previously not reported in the literature.