Safety and cardiovascular effects of multiple-dose administration of aripiprazole and olanzapine in a randomised clinical trial.

OBJECTIVE: To assess adverse events (AEs) and safety of aripiprazole (ARI) and olanzapine (OLA) treatment. METHODS: Twenty-four healthy volunteers receiving five daily oral doses of 10 mg ARI and 5 mg OLA in a crossover clinical trial were genotyped for 46 polymorphisms in 14 genes by qPCR. Drug plasma concentrations were measured by high-performance liquid chromatography tandem mass spectrometry. Blood pressure (BP) and 12-lead electrocardiogram were measured in supine position. AEs were also recorded. RESULTS: ARI decreased diastolic BP on the first day and decreased QTc on the third and fifth day. OLA had a systolic and diastolic BP, heart rate and QTc lowering effect on the first day. Polymorphisms in ADRA2A, COMT, DRD3 and HTR2A genes were significantly associated to these changes. The most frequent adverse drug reactions (ADRs) to ARI were somnolence, headache, insomnia, dizziness, restlessness, palpitations, akathisia and nausea while were somnolence, dizziness, asthenia, constipation, dry mouth, headache and nausea to OLA. Additionally, HTR2A, HTR2C, DRD2, DRD3, OPRM1, UGT1A1 and CYP1A2 polymorphisms had a role in the development of ADRs. CONCLUSIONS: OLA induced more cardiovascular changes; however, more ADRs were registered to ARI. In addition, some polymorphisms may explain the difference in the incidence of these effects among subjects.

The effects of aripiprazole and olanzapine on pupillary light reflex and its relationship with pharmacogenetics in a randomized multiple-dose trial.

AIMS: Pupillography is a noninvasive and cost-effective method to determine autonomic nerve activity. Genetic variants in cytochrome P450 (CYP), dopamine receptor (DRD2, DRD3), serotonin receptor (HTR2A, HTR2C) and ATP-binding cassette subfamily B (ABCB1) genes, among others, were previously associated with the pharmacokinetics and pharmacodynamics of antipsychotic drugs. Our aim was to evaluate the effects of aripiprazole and olanzapine on pupillary light reflex related to pharmacogenetics. METHODS: Twenty-four healthy volunteers receiving 5 oral doses of 10 mg aripiprazole and 5 mg olanzapine tablets were genotyped for 46 polymorphisms by quantitative polymerase chain reaction. Pupil examination was performed by automated pupillometry. Aripiprazole, dehydro-aripiprazole and olanzapine plasma concentrations were measured by high-performance liquid chromatography-tandem mass spectrometry. RESULTS: Aripiprazole affected pupil contraction: it caused dilatation after the administration of the first dose, then caused constriction after each dosing. It induced changes in all pupillometric parameters (P < .05). Olanzapine only altered minimum pupil size (P = .046). Polymorphisms in CYP3A, HTR2A, UGT1A1, DRD2 and ABCB1 affected pupil size, the time of onset of constriction, pupil recovery and constriction velocity. Aripiprazole, dehydro-aripiprazole and olanzapine pharmacokinetics were significantly affected by polymorphisms in CYP2D6, CYP3A, CYP1A2, ABCB1 and UGT1A1 genes. CONCLUSIONS: In conclusion, aripiprazole and its main metabolite, dehydro-aripiprazole altered pupil contraction, but olanzapine did not have such an effect. Many polymorphisms may influence pupillometric parameters and several polymorphisms had an effect on aripiprazole, dehydro-aripiprazole and olanzapine pharmacokinetics. Pupillography could be a useful tool for the determination of autonomic nerve activity during antipsychotic treatment.

The Adrenal Medulla Modulates Mechanical Allodynia in a Rat Model of Neuropathic Pain.

We have investigated whether the stress response mediated by the adrenal medulla in rats subjected to chronic constriction injury of the sciatic nerve (CCI) modulates their nocifensive behavior. Treatment with SK29661 (300 mg/kg; intraperitoneal (I.P.)), a selective inhibitor of phenylethanolamine N-methyltransferase (PNMT) that converts noradrenaline (NA) into adrenaline (A), fully reverted mechanical allodynia in the injured hind paw without affecting mechanical sensitivity in the contralateral paw. The effect was fast and reversible and was associated with a decrease in the A to NA ratio (A/NA) in the adrenal gland and circulating blood, an A/NA that was elevated by CCI. 1,2,3,4-tetrahydroisoquinoline-7-sulfonamide (SKF29661) did not affect exocytosis evoked by Ca2+ entry as well as major ionic conductances (voltage-gated Na+, Ca2+, and K+ channels, nicotinic acetylcholine receptors) involved in stimulus-secretion coupling in chromaffin cells, suggesting that it acted by changing the relative content of the two adrenal catecholamines. Denervation of the adrenal medulla by surgical splanchnectomy attenuated mechanical allodynia in neuropathic animals, hence confirming the involvement of the adrenal medulla in the pathophysiology of the CCI model. Inhibition of PNMT appears to be an effective and probably safe way to modulate adrenal medulla activity and, in turn, to alleviate pain secondary to the injury of a peripheral nerve.

Simultaneous Determination of Imatinib, Dasatinib, and Nilotinib by Liquid Chromatography-Tandem Mass Spectrometry and Its Application to Therapeutic Drug Monitoring.

BACKGROUND: Imatinib, dasatinib, and nilotinib are tyrosine kinase inhibitors (TKIs) used as first-line treatment of chronic myeloid leukemia. Therapeutic drug monitoring is important to achieve treatment efficacy in the case of imatinib and nilotinib, and to control toxicity in the case of dasatinib. New high-sensitivity methods to monitor those drugs are needed, especially for dasatinib. Thus, a simple method to determine plasma levels of imatinib, dasatinib, and nilotinib for application in clinical practice was developed. METHODS: TKIs were eluted with a Poroshell 120 EC-C18 column (2.1 × 75 mm, 2.7 µm) at 0.5 mL/min and 60°C, under gradient conditions through a mobile phase consisting of 4 mmol/L ammonium formate, pH 3.2 (65%), and acetonitrile (35%). TKIs were detected and quantified by liquid chromatography in tandem with mass spectrometry (LC/MS-MS) with positive electrospray ionization and analytes were extracted using solid phase extraction (Versaplate-SCX). Internal standards were isotope-labeled for each analyte. RESULTS: The method was linear in the range of 2.5-5000 ng/mL for imatinib, 0.75-400 ng/mL for dasatinib, and 2-4000 ng/mL for nilotinib. The validation assays for accuracy and precision, matrix effect, extraction recovery, carryover, and stability of the samples for all the TKIs were appropriate according to regulatory agencies. Furthermore, imatinib plasma samples, stored for 4 years at -80°C were quite stable in approximately half of the samples. CONCLUSIONS: The method enables rapid quantification of TKI concentrations and is being applied to therapeutic drug monitoring to adjust dose and to manage adverse reactions in clinical practice.

Mass Spectrometry-Based Visualization of Molecules Associated with Human Habitats.

The cars we drive, the homes we live in, the restaurants we visit, and the laboratories and offices we work in are all a part of the modern human habitat. Remarkably, little is known about the diversity of chemicals present in these environments and to what degree molecules from our bodies influence the built environment that surrounds us and vice versa. We therefore set out to visualize the chemical diversity of five built human habitats together with their occupants, to provide a snapshot of the various molecules to which humans are exposed on a daily basis. The molecular inventory was obtained through untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of samples from each human habitat and from the people that occupy those habitats. Mapping MS-derived data onto 3D models of the environments showed that frequently touched surfaces, such as handles (e.g., door, bicycle), resemble the molecular fingerprint of the human skin more closely than other surfaces that are less frequently in direct contact with humans (e.g., wall, bicycle frame). Approximately 50% of the MS/MS spectra detected were shared between people and the environment. Personal care products, plasticizers, cleaning supplies, food, food additives, and even medications that were found to be a part of the human habitat. The annotations indicate that significant transfer of chemicals takes place between us and our built environment. The workflows applied here will lay the foundation for future studies of molecular distributions in medical, forensic, architectural, space exploration, and environmental applications.

Improvement and validation of a high-performance liquid chromatography in tandem mass spectrometry method for monitoring of omeprazole in plasma.

BACKGROUND: Omeprazole (OME) is a proton pump inhibitor with a 58% bioavailability after a single oral dose. It is subject to marked interindividual variations and significant drug-drug interactions. The authors developed a simple and rapid method based on liquid chromatography in tandem with mass spectrometry with solid phase extraction and isotope-labeled internal standard to monitor plasma levels of OME in pharmacokinetics and drug-drug interaction studies. METHODS: OME and its internal standard (OME-D3) were eluted with a Zorbax Extend C-18 rapid resolution column (4.6 × 50 mm, 3.5 µm) at 25°C, under isocratic conditions through a mobile phase consisting of 1 mM ammonium acetate, pH 8.5 (55%), and acetonitrile (45%). The flow rate was 0.8 mL/min, and the chromatogram run time was 1.2 minutes. OME was detected and quantified by liquid chromatography in tandem with mass spectrometry with positive electrospray ionization, which operates in multiple-reaction monitoring mode. RESULTS: The method was linear in the range of 1.5-2000 ng/mL for OME. The validation assays for accuracy and precision, matrix effect, extraction recovery, and stability of the samples for OME did not deviate more than 20% for the lower limit of quantification and no more than 15% for other quality controls. CONCLUSIONS: These findings are consistent with the requirements of regulatory agencies. The method enables rapid quantification of OME concentrations and can be used in pharmacokinetic and drug-drug interaction studies.

Evaluation of the relationship between sex, polymorphisms in CYP2C8 and CYP2C9, and pharmacokinetics of angiotensin receptor blockers.

Angiotensin II receptor blockers (ARBs) are used to treat hypertension. Most ARBs are metabolized by CYP2C9. The aim of this study is to evaluate the possible association between sex, polymorphisms in the CYP2C8 and CYP2C9 genes, and the pharmacokinetics of losartan, valsartan, candesartan, and telmisartan. The study population comprised 246 healthy volunteers from seven single-dose clinical trials: 64 from two candesartan studies, 43 from a telmisartan study, 36 from a losartan study, and 103 from three valsartan studies. DNA was extracted from blood samples and single-nucleotide polymorphisms in the CYP2C8 (CYP2C8*2, CYP2C8*3, CYP2C8*4, CYP2C8*5) and CYP2C9 (CYP2C9*2, CYP2C9*3) genes were evaluated using real-time polymerase chain reaction. Sex only affected telmisartan pharmacokinetics, since women showed a higher telmisartan C(max) than men (590.5 ± 75.8 ng/ml versus 282.1 ± 30.8 ng/ml; P ≤ 0.01). CYP2C9 variants were associated only with losartan pharmacokinetics: the half-life of losartan was higher in CYP2C9*3 allele carriers (3.1 ± 0.4 hours) than in volunteers with the wild-type genotype (2.3 ± 0.1 hours) (P ≤ 0.05). CYP2C8 polymorphisms were associated only with valsartan pharmacokinetics, since *2 allele carriers showed faster clearance (1.07 ± 0.57 l/h·kg) than those with the wild-type genotype (0.48 ± 0.72 l/h·kg; P ≤ 0.01) and carriers of the *3 allele (0.35 ± 0.49 l/h·kg; P ≤ 0.001). These results suggest that genotypes for CYP2C9 and CYP2C8 are relevant to the pharmacokinetics of losartan and valsartan, respectively, but not the pharmacokinetics of candesartan or telmisartan.

Imatinib assay by high-performance liquid chromatography in tandem mass spectrometry with solid-phase extraction in human plasma.

We have developed a method of liquid chromatography in tandem with mass spectrometry to monitor therapeutic levels of imatinib in plasma, a selective inhibitor of protein tyrosine kinase. After solid-phase extraction of plasma samples, imatinib and its internal standard, imatinib-D8, were eluted with Zorbax SB-C18 at 60 °C, under isocratic conditions through a mobile phase consisting of 4 mm ammonium formate, pH: 3.2 (solution A) and acetonitrile solution B. The flow rate was 0.8 mL/min with 55% solution A + 45% solution B. Imatinib was detected and quantified by mass spectrometry with electrospray ionization operating in selected-reaction monitoring mode. The calibration curve was linear in the range 10-5000 ng/mL, the lower limit of quantitation being 10 ng/mL. The method was validated according to the recommendations of the Food and Drug Administration, including tests of matrix effect (bias < 10%) and recovery efficiency (>80 and <120%). The method is precise (coefficient of variance intra-day <2% and inter-day <7%), accurate (95-108%), sensitive and specific. It is a simple method with very fast recording time (1.2 min) that is applicable to clinical practice. This will permit improvement of the pharmacological treatment of patients.

High-Fat Breakfast Increases Bioavailability of Albendazole Compared to Low-Fat Breakfast: Single-Dose Study in Healthy Subjects.

Purpose: Albendazole is a benzimidazole carbamate drug with anthelmintic and antiprotozoal activity against intestinal and tissue parasites. It has been described that the administration with meals increases albendazole absorption. Our aim was to compare the systemic exposure in healthy volunteers of two albendazole formulations after a single oral dose under fed conditions and to evaluate the effect of breakfast composition on albendazole and albendazole sulfoxide bioavailability. Methods: 12 healthy volunteers were included in a 4-period, 4-sequence, crossover, open, randomized, bioequivalence clinical trial, including two stages to compare two formulations of albendazole. Single oral doses of 400 mg albendazole were administered under fed conditions (a low-fat breakfast in first stage and a high-fat breakfast in the second) separated by 7-day washout periods. Plasma albendazole and albendazole sulfoxide concentrations were measured by HPLC-MS/MS. Findings: Albendazole absorption was clearly influenced by the meal composition. A high-fat breakfast increased albendazole and albendazole sulfoxide area under the concentration-time curve (AUC) and maximum concentration (Cmax) by double, compared to a low-fat breakfast. The bioavailability of the two formulations was very similar, although the sample size was not sufficient to demonstrate bioequivalence because the intraindividual variability of albendazole was approximately 60%. Implications: The higher albendazole and albendazole sulfoxide levels when administered with a high-fat meal could be of importance in clinical practice. Since albendazole labeling recommends its administration with meals, it is necessary to insist on taking it with a fatty meal so that the effectiveness of albendazole is not compromised.

Effective quantification of 11 tyrosine kinase inhibitors and caffeine in human plasma by validated LC-MS/MS method with potent phospholipids clean-up procedure. Application to therapeutic drug monitoring.

Therapeutic drug monitoring (TDM) help to improve treatment efficacy and safety. Therefore, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous monitoring of 11 tyrosine kinase inhibitors (TKIs) in human plasma. TKIs included in the assay are used in the treatment of chronic myeloid leukemia (CML: imatinib, dasatinib, nilotinib, bosutinib, ponatinib), polycythemia vera (ruxolitinib), chronic lymphocytic leukemia (ibrutinib) and rheumatoid arthritis (filgotinib, tofacitinib, baricitinib, peficitinib). Caffeine was also included in the method. Caffeine increases the acidity of the stomach and decreases its pH as well as is a competitive inhibitor of cytochrome P450 isoenzymes. Thus, it may influence absorption and metabolism of some TKIs, by modifying their plasma levels. The analytes of interest and their stable isotope-labeled internal standards were extracted from 200 µL of human plasma. Microelution-solid phase extraction (µ-SPE) was optimized for method validation and compared to simple protein precipitation (PPT). A gradient elution on a Poroshell 120 EC-C18 column at 60 °C and a flow rate of 0.5 mL/min was applied for analyte separation. The analytical run lasted 8 min and it was followed by a re-equilibration time of 4 min. Dynamic multiple reaction monitoring scan in the positive ionization mode was applied to improve method sensitivity. Endogenous plasma phospholipids can strongly affect MS analysis. Hence, the monitoring of endogenous phospholipids was included in the assay. Full validation of the method was achieved, including tests of precision, accuracy, trueness, linearity, extraction recovery, matrix effect, process efficiency, stability, sensitivity (with excellent LLOQs), selectivity, identity confirmation and carry-over effect. Regarding sample cleanup, more than 91% of early eluting and more than 96% of late eluting endogenous phospholipids were eliminated by µ-SPE when compared to PPT. This method enables the simultaneous plasma monitoring of 11 TKIs and caffeine and ensures high effectiveness in phospholipids elimination. The present approach is currently used in our clinical practice, being applied to TDM of dasatinib, imatinib, nilotinib and ponatinib. TKIs plasma monitoring helps to individualize dose adjustment and manage adverse effects in CML patients.

Simultaneous determination of six antipsychotics, two of their metabolites and caffeine in human plasma by LC-MS/MS using a phospholipid-removal microelution-solid phase extraction method for sample preparation.

A simple and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated in human plasma for the simultaneous determination of aripiprazole (ARI) and its metabolite dehydro-aripiprazole (DARI); olanzapine (OLA), risperidone (RIS), paliperidone (PAL), quetiapine (QUE), clozapine (CLO) and caffeine (CAF). CAF is included to the method because it can have an influence on drug metabolism due to competitive inhibition. The above mentioned compounds and their isotope-labeled internal standards were extracted from 200 µL human plasma samples by both, effective phospholipids-eliminating three-step microelution-solid-phase extraction (µ-SPE) and protein precipitation (PPT) for comparison. A combination of formic acid (0.2%)-acetonitrile (pH 3.0; 65:35, v/v) was used as mobile phase and the chromatogram was run under gradient conditions at a flow rate of 0.6 mL/min. Run time lasted 6 min, followed by a re-equilibration time of 3 min. All analytes were monitored by mass spectrometric detection operating in multiple reaction monitoring mode and the method was validated covering the corresponding therapeutic ranges: 0.18-120 ng/mL for ARI, 0.25-80 ng/mL for DARI, 1.00-100 ng/mL for OLA, 0.70-60 ng/mL for RIS, 0.20-30 ng/mL for PAL, 0.50-160 ng/mL for QUE, 0.50-1000 ng/mL for CLO, and finally 1200-3700 ng/mL for CAF. The method was validated based on the recommendations of regulatory agencies through tests of precision, accuracy, extraction recovery, identity confirmation, trueness, matrix effect, process efficiency, stability, selectivity, linearity and carry-over effect fulfilling the guideline requirements. Our µ-SPE method results in the elimination of more than 99% of early eluting and more than 92% of late-eluting phospholipids compared to PPT. Additionally, the method was successfully applied for quantifying ARI and OLA plasma concentrations from healthy volunteers.

Utility of Therapeutic Drug Monitoring of Imatinib, Nilotinib, and Dasatinib in Chronic Myeloid Leukemia: A Systematic Review and Meta-analysis.

PURPOSE: This study examined the utility of therapeutic drug monitoring (TDM) of imatinib, nilotinib, and dasatinib in adult patients with chronic-phase chronic myeloid leukemia (CML). TDM in CML entails the measurement of plasma tyrosine kinase inhibitor (TKI) concentration to predict efficacy and tolerability outcomes and to aid in clinical decision making. TDM was to be deemed useful if it could be used for predicting the effectiveness of a drug and/or the occurrence of adverse reactions. It was expected that the findings from the present study would allow for the definition of a therapeutic range of each TKI. METHODS: A systematic review of studies reporting trough TKI levels (Cmin) and clinical outcomes was performed. We included randomized clinical trials, nonrandomized controlled studies, interrupted time series studies, and case series studies that provided information about plasma levels of imatinib, nilotinib, or dasatinib and relevant clinical end points in adult patients with chronic-phase CML treated with the corresponding TKI as the single antiproliferative therapy. Meta-analyses, Student t tests, and receiver operating characteristic analyses were performed to detect mean differences between groups of patients with or without: (1) the achievement of major molecular response and (2) adverse reactions. FINDINGS: A total of 38 studies (28 for imatinib, 7 for nilotinib, and 3 for dasatinib) were included in the systematic review. TDM was found useful in predicting the efficacy of imatinib, with a Cmin cutoff value of 1000 ng/mL, consistent with guideline recommendations. We suggest a therapeutic range of imatinib at a Cmin of 1000-1500 ng/mL because higher concentrations did not increase efficacy. The findings from the rest of the comparisons were inconclusive. IMPLICATIONS: TDM is useful in predicting the efficacy of imatinib in CML. Further research is needed to determine its validity with nilotinib and dasatinib.

Human brain gene expression profiles of the cathepsin V and cathepsin L cysteine proteases, with the PC1/3 and PC2 serine proteases, involved in neuropeptide production.

Proteases are required to generate active peptide neurotransmitters, known as neuropeptides, from pro-neuropeptides. Model animal systems have recently illustrated roles for the cathepsin V (CTSV) and cathepsin L (CTSL) cysteine proteases, combined with the serine proteases PC1/3 (PCSK1) and PC2 (PCSK2), and exopeptidases in the production of neuropeptides. There is notable interest in the human-specific cathepsin V gene which is not present in rodent and other animal models used in prior studies of neuropeptide production. A gap in the field is knowledge of the human brain gene expression patterns of these neuropeptide-producing protease systems. Therefore, the goal of this study was to characterize the expression profiles of these pro-neuropeptide processing proteases in human brain. Quantitative gene expression microarray data for 169 human brain regions was obtained from the Allen Institute Human Brain Atlas resource, analyzed as log2 of gene expression intensity normalized to the mean of human genes (21,245 genes) expressed in human brain. These proteases had log2 values of 2-12, indicating expression levels above the average of all genes in the human brain, with varying expression levels among the 169 brain regions. CTSV and CTSL displayed moderate to high expression values of 1.9-8.6 and 7.1-10.6, respectively. Interestingly, CTSV and CTSL showed high expression in white matter composed of myelinated axons, consistent with the knowledge that neuropeptide production occurs in axons within transported neuropeptide secretory vesicles to nerve terminals. PCSK1 had a broad range of moderate to very high expression with log2 of 2-12. PCSK2 had somewhat lower expression levels than PCSK1. The exopeptidase genes RNPEP, CTSH, and CPE each showed fairly even levels of expression throughout the brain, with CPE displaying high expression. The prevalence of these processing proteases throughout human brain regions, including areas rich in neuropeptides such as hypothalamus, is consistent with their roles for neuropeptide production. Further, proenkephalin and NPY precursors, substrates of CTSV and CTSL shown in prior model animal studies, were co-expressed with CTSV and CTSL. These data demonstrate that the human brain expresses the neuropeptide-producing cysteine and serine proteases, with exopeptidases, throughout a multitude of brain regions.

How to make P-glycoprotein (ABCB1, MDR1) harbor mutations and measure its expression and activity in cell cultures?

Several polymorphisms have been identified in ABCB1, the gene encoding for the P-glycoprotein. This transporter alters the pharmacokinetics or effectiveness of drugs by excreting them from cells where it is expressed (e.g., blood-brain barrier, intestine or tumors). No consensus has been reached regarding the functional consequences of these polymorphisms in the transporter's function. The aim of this review was to describe a methodology that allows the assessment of P-gp function when harboring polymorphisms. We describe how to obtain cell lines with high expression levels of the transporter with polymorphisms and several tactics to measure its expression and activity. This methodology may help elucidate the contribution of polymorphisms in ABCB1 to drug pharmacokinetics, effectiveness and safety or to cancer chemotherapy failure.

Effective phospholipids removing microelution-solid phase extraction LC-MS/MS method for simultaneous plasma quantification of aripiprazole and dehydro-aripiprazole: Application to human pharmacokinetic studies.

A simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for simultaneous quantification of aripiprazole and its active metabolite, dehydro-aripiprazole, in human plasma. Stable isotopically labeled aripiprazole, aripiprazole-D8, has been used as the internal standard (IS) for both analytes. Only 200 µl of human plasma was needed for analyte extraction, using effective phospholipids-eliminating three-step microelution-solid-phase extraction (SPE, Oasis PRiME HLB 96-well µElution Plate). An ACE C18-PFP column was applied for chromatographic separation at 25 °C, protected by a 0.2-µm on-line filter. A combination of ammonium formate (5 mM)-acetonitrile (pH 4.0; 65:35, v/v) was used as mobile phase and the chromatogram was run under gradient conditions at a flow rate of 0.6 ml/min. Run time lasted 5 min, followed by a re-equilibration time of 3 min, to give a total run time of 8 min. Five µl of the sample was injected into the chromatographic system. Aripiprazole, dehydro-aripiprazole and IS were detected using the mode multiple reaction monitoring in the positive ionization mode. The method was linear in the concentration range of 0.18-110 ng/ml and 0.35-100 ng/ml for aripiprazole and dehydro-aripiprazole, respectively. Our method has been validated according to the recommendations of regulatory agencies through tests of precision, accuracy, recovery, matrix effect, stability, sensitivity, selectivity and carry-over. Our microelution-SPE method removes more than 99% of main plasma phospholipids compared to protein precipitation and was successfully applied to several bioequivalence studies.

Effects of aripiprazole on pupillometric parameters related to pharmacokinetics and pharmacogenetics after single oral administration to healthy subjects.

BACKGROUND: Pupillometry is used for the detection of autonomic dysfunction related to numerous diseases and drug administration. Genetic variants in cytochrome P450 ( CYP2D6, CYP3A4), dopamine receptor ( DRD2, DRD3), serotonin receptor ( HTR2A, HTR2C) and ATP-binding cassette subfamily B ( ABCB1) genes were previously associated with aripiprazole response. AIMS: Our aim was to evaluate if aripiprazole affects pupil contraction and its relationship with pharmacokinetics and pharmacogenetics. METHODS: Thirty-two healthy volunteers receiving a 10 mg single oral dose of aripiprazole were genotyped for 15 polymorphisms in ABCB1, CYP2D6, DRD2, DRD3, HTR2A and HTR2C genes by reverse transcription polymerase chain reaction. Aripiprazole and dehydro-aripiprazole plasma concentrations were measured by high-performance liquid chromatography tandem mass spectrometry. Pupil examination was performed by automated pupillometry. RESULTS: Aripiprazole caused pupil constriction and reached the peak value at Cmax. HTR2A rs6313 T allele carriers and HTR2C rs3813929 C/T subjects showed higher maximum constriction velocity and maximum pupil diameter. Besides, Gly/Gly homozygotes for DRD3 rs6280 showed significantly lower maximum constriction velocity values. A/G heterozygotes for DRD2 rs6277 showed higher total time taken by the pupil to recover 75% of the initial resting size values. CYP2D6 intermediate metabolisers showed higher area under the curve, Cmax and T1/2 than extensive metabolisers. ABCB1 G2677T/A A/A homozygotes had greater T1/2 in comparison with C/C homozygotes. ABCB1 C3435T T allele carriers and C1236T C/T subjects showed greater area under the curve than C/C homozygotes. CONCLUSIONS: Aripiprazole affects pupil contraction, which could be a secondary effect through dopamine and serotonin receptors. Pupillometry could be a useful tool to assess autonomic nervous system activity during antipsychotic treatment.

Influence of CYP2D6, CYP3A4, CYP3A5 and ABCB1 Polymorphisms on Pharmacokinetics and Safety of Aripiprazole in Healthy Volunteers.

The aim of this study was to investigate the effect of polymorphisms in cytochrome P450 (CYP) 2D6, CYP3A4 and CYP3A5 enzymes and in P-glycoprotein (P-gp) on the pharmacokinetics and safety of aripiprazole and, its active metabolite, dehydro-aripiprazole, in 148 healthy volunteers from six bioequivalence trials receiving a single oral dose of aripiprazole. The plasma concentrations of both analytes were measured by LC-MS/MS. CYP2D6 (*3,*4,*5,*6,*7,*9 and copy number variations), CYP3A4 (*20 and *22), CYP3A5*3 and C3435T, C1236T and G2677T/A in ABCB1 gene were determined. As the number of active CYP2D6 alleles decreased, AUC0-t , Cmax and t1/2 of aripiprazole were higher and clearance of aripiprazole, AUC0-t of dehydro-aripiprazole and ratio dehydro-aripiprazole/aripiprazole were lower. AUC0-t of aripiprazole of poor metabolizer (PM) subjects was increased by 50% compared to extensive metabolizers (EM), and AUC0-t of dehydro-aripiprazole was decreased by 33%. ABCB1 1236TT subjects had a lower clearance of aripiprazole (p = 0.023) and AUC0-t (p = 0.039) and Cmax of dehydro-aripiprazole (p = 0.036) compared to C/C. CYP3A5*3/*3 subjects had a 10% lower ratio dehydro-aripiprazole/aripiprazole than *1/*3 (p = 0.019). Adverse drug reactions (ADRs) had a directly proportional relationship with AUC0-t of aripiprazole (p = 0.001), especially nausea/vomiting, which were more common in women (p = 0.005). Women and CYP3A5*1/*1 subjects showed more often dizziness (p = 0.034; p = 0.009). Pharmacokinetics of aripiprazole is affected by CYP2D6 phenotype but also by sex and C1236T (ABCB1 gene), while dehydro-aripiprazole pharmacokinetics is affected by CYP2D6 and C1236T. The ratio dehydro-aripiprazole/aripiprazole was influenced by CYP2D6 phenotype and CYP3A5*3. Concentrations of aripiprazole, sex, CYP3A5*3 and CYP2D6 were involved in the development of ADRs.

Effect of ABCB1 C3435T Polymorphism on Pharmacokinetics of Antipsychotics and Antidepressants.

P-glycoprotein, encoded by ABCB1, is an ATP-dependent drug efflux pump which exports substances outside the cell. Some studies described connections between C3435T polymorphism T allele and lower P-glycoprotein expression; therefore, homozygous T/T could show higher plasma levels. Our aim was to evaluate the effect of C3435T on pharmacokinetics of 4 antipsychotics (olanzapine, quetiapine, risperidone and aripiprazole) and 4 antidepressants (trazodone, sertraline, agomelatine and citalopram). The study included 473 healthy volunteers receiving a single oral dose of one of these drugs, genotyped by real-time PCR. Multivariate analysis was performed to adjust the effect of sex and genotype of the main cytochrome P450 enzymes. C3435T polymorphism had an effect on olanzapine pharmacokinetics, as T/T individuals showed lower clearance and volume of distribution. T/T individuals showed lower T1/2 of 9-OH-risperidone, but this difference disappeared after multivariate correction. T/T homozygous individuals showed lower dehydro-aripiprazole and trazodone area under the concentration-time curve, along with lower half-life and higher clearance of trazodone. C/T genotype was associated to higher citalopram maximum concentration. C3435T had no effect on quetiapine, sertraline or agomelatine pharmacokinetics. C3435T can affect the elimination of some drugs in different ways. Regarding risperidone, trazodone and dehydro-aripiprazole, we observed enhanced elimination while it was reduced in olanzapine and citalopram. However, in quetiapine, aripiprazole, sertraline and agomelatine, no changes were detected. These results suggest that P-glycoprotein polymorphisms could affect CNS drugs disposition, but the genetic factor that alters its activity is still unknown. This fact leads to consider the analysis of ABCB1 haplotypes instead of individual variants.

The Neuroprotector Benzothiazepine CGP37157 Extends Lifespan in C. elegans Worms.

The benzothiazepine CGP37157 has shown neuroprotective effects in several in vitro models of excitotoxicity involving dysregulation of intracellular Ca2+ homeostasis. Although its mechanism of neuroprotection is unclear, it is probably related with some of its effects on Ca2+ homeostasis. CGP37157 is a well-known inhibitor of the mitochondrial Na+/Ca2+ exchanger (mNCX). However, it is not very specific and also blocks several other Ca2+ channels and transporters, including voltage-gated Ca2+ channels, plasma membrane Na+/Ca2+ exchanger and the Ca2+ homeostasis modulator 1 channel (CALHM1). In the present work, we have studied if CGP37157 could also induce changes in life expectancy. We now report that CGP37157 extends C. elegans lifespan by 10%-15% with a bell-shaped concentration-response, with high concentrations producing no effect. The effect was even larger (25% increase in life expectancy) in worms fed with heat-inactivated bacteria. The worm CGP37157 concentration producing maximum effect was measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and was close to the IC50 for inhibition of the Na+/Ca2+ exchanger. CGP37157 also extended the lifespan in eat-2 mutants (a model for caloric restriction), suggesting that caloric restriction is not involved in the mechanism of lifespan extension. Actually, CGP37157 produced no effect in mutants of the TOR pathway (daf15/unc24) or the insulin/insulin-like growth factor-1 (IGF-1) pathway (daf-2), indicating that the effect involves these pathways. Moreover, CGP37157 was also ineffective in nuo-6 mutants, which have a defect in the mitochondrial respiratory chain complex I. Since it has been described that neuroprotection by this compound in cell cultures is abolished by mitochondrial inhibitors, this suggests that life extension in C. elegans and neuroprotection in cell cultures may share a similar mechanism involving mitochondria.

Inclusion complex of ITH12674 with 2-hydroxypropyl-ß-cyclodextrin: Preparation, physical characterization and pharmacological effect.

ITH12674 is a multitarget drug, designed to exert a dual "drug-prodrug" mechanism of action, able to induce the phase II antioxidant and anti-inflammatory response for the treatment of brain ischemia. However, its physicochemical properties limit its potential preclinical development due to its low water solubility and instability towards heat and pH variations. In order to improve its properties, we prepared the inclusion complex of ITH12674 with 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) by the freeze-drying method. The formation of the inclusion complex was confirmed by FT-IR spectroscopy, PXRD, DSC, 1H NMR and SEM techniques. Experimental results showed that the inclusion complex enhanced its water solubility and stability against heat, acidic and basic conditions. Furthermore, the inclusion complex, prepared in water solution, exerted the same potency to induce the phase II antioxidant response as the pure ITH12674. Thus the formation of the inclusion complex with HP-ß-CD is a very effective method to stabilize and solubilize the active compound for its future preclinical development.