Evaluation of OCT2-mediated drug-drug interactions between ulotaront and metformin in subjects with schizophrenia.

Ulotaront (SEP-363856) is a TAAR1 agonist, with 5-HT1A agonist activity, currently in clinical development for the treatment of schizophrenia. In vitro studies indicate ulotaront is an OCT2-specific inhibitor with IC50 of 1.27 µM. The primary objective of this study is to determine if a single dose of ulotaront affects the PK of metformin, an index substrate of OCT2, in subjects with schizophrenia. In a randomized, single-blind, 2-period crossover study, 25 adults with schizophrenia received a single dose of metformin-HCl 850 mg (approximately 663 mg metformin) with and without coadministration of 100 mg ulotaront. The plasma samples were analyzed by fully validated LC-MS/MS methods. The primary PK endpoints for metformin were AUCinf, AUClast, Cmax, and tmax. The highest-anticipated clinical dose of ulotaront (100 mg) had no statistically significant effect on the PK of a single dose of metformin based on Cmax and AUCinf. Geometric least squares mean ratios were 89.98% and 110.63%, respectively, with the 90% confidential interval (CI) for each parameter contained within 80%-125%. Median tmax was comparable across the treatments. Ulotaront does not act as a perpetrator of OCT2-mediated DDI against metformin. Co-administration of ulotaront is not expected to require dose adjustment of metformin or other drugs cleared by OCT2.

A Phase I, Open-Label, Fixed Sequence Study to Investigate the Effect of Cytochrome P450 2D6 Inhibition on the Pharmacokinetics of Ulotaront in Healthy Subjects.

BACKGROUND: Ulotaront is a novel psychotropic agent with agonist activity at trace amine-associated receptor 1 (TAAR1) and 5-hydroxytryptamine type 1A (5-HT1A) receptors in phase III clinical development for the treatment of schizophrenia. OBJECTIVE: This study aimed to investigate the effect of paroxetine, a strong cytochrome P450 (CYP) 2D6 inhibitor, on ulotaront pharmacokinetics (PK) in healthy volunteers. METHODS: Subjects received a single oral dose of 25 mg ulotaront on Day 1 and an oral dose of 20 mg paroxetine once daily from Days 5 to 10 to achieve steady-state plasma paroxetine levels. On Day 11, subjects received another single oral dose of 25 mg ulotaront, with continued daily oral dosing of 20 mg paroxetine from Days 11 to 14. All 24 subjects were CYP2D6 normal metabolizers. RESULTS: Coadministration of paroxetine increased ulotaront maximum observed plasma concentration (Cmax) and area under the plasma concentration-time curve from time zero to infinity (AUC∞) by 31% and 72%, respectively, and decreased ulotaront apparent clearance (CL/F) by approximately 42%. While coadministration of paroxetine increased AUC∞ of active but minor metabolite SEP-363854 by 32%, it had no effect on SEP-363854 Cmax, or on SEP-363854 to the ulotaront AUC from time zero to the last quantifiable concentration (AUClast) ratio. Based on the acceptable adverse event profile of ulotaront across previous phase II studies, the increase in ulotaront exposure is unlikely to be clinically meaningful. CONCLUSIONS: Weak drug-drug interactions were observed between ulotaront and the strong CYP2D6 inhibitor paroxetine; however, dose adjustment as a precondition when ulotaront is coadministered with strong CYP2D6 inhibitors or administered to CYP2D6 poor metabolizers should not be necessary.

Comparative Bioequivalence of Tablet and Capsule Formulations of Ulotaront and the Effect of Food on the Pharmacokinetics of the Tablet Form in Humans.

INTRODUCTION: Ulotaront (SEP-363856), a dual trace animeassociated receptor 1 (TAAR1) and 5-HT1A receptor agonist, is in phase 3 clinical development for the treatment of schizophrenia. This study evaluated the comparative bioequivalence (BE) between tablet and capsule formulations of ulotaront and the food effect (FE) on pharmacokinetics (PK) of tablet form in healthy adult human subjects. METHODS: The BE study applied an open-label two-period crossover design in 24 healthy volunteers. Subjects were randomly assigned (1:1) to dosing sequence AB or BA (A, 25 mg ulotaront tablet; B, 25 mg ulotaront capsule). The FE study also used an open-label randomized two-period crossover design in 20 healthy volunteers. Subjects were fasted overnight then randomly assigned (1:1) to dosing sequence AB or BA (A, fasted condition; B, fed condition). Dosing periods were separated by 1 week for both studies. Serial plasma samples from each period were collected and analyzed by LC-MS/MS. PK parameters were calculated using Phoenix WinNonlin® software. RESULTS: For the BE study, geometric mean ulotaront Cmax values were 93.28 and 86.98 ng/mL for tablet and capsule, respectively. Cmax ratio was 107.25% (90% CI 101.84-112.94%). Geometric mean ulotaront area under the plasma concentration-time curve from time 0 to infinity (AUC0-∞) values were 868.8 and 829.3 ng·h/mL for tablet and capsule, respectively. AUC0-∞ ratio was 104.76% (90% CI 100.68109.01%). For the FE study, geometric mean ulotaront Cmax was 157.89 and 157.95 ng/mL under fed and fasted conditions, respectively. Geometric mean ratio of Cmax was 99.96% (90% CI 94.48-105.77%). Geometric mean ulotaront AUC0-∞ was 1584.2 ng·h/mL fed and 1589.2 ng·h/mL fasted. Geometric mean ratio for AUC0-∞ was 99.69% (90% CI 95.02-104.58%). There was a delay in tmax (median difference 1.47 h) in the fed condition. CONCLUSIONS: The results showed geometric mean ratios and 90% CIs for both Cmax and AUC0-∞ for ulotaront were well within typical bioequivalence criteria of 80-125% for both the BE and FE studies, thereby confirming the bioequivalence of the two dosage forms and no significant food effect.

In Vitro ADME and Preclinical Pharmacokinetics of Ulotaront, a TAAR1/5-HT1A Receptor Agonist for the Treatment of Schizophrenia.

PURPOSE: Ulotaront (SEP-363856) is a TAAR1 agonist with 5-HT1A agonist activity currently in clinical development for the treatment of schizophrenia. The objectives of the current study were to characterize the in vitro ADME properties, preclinical PK, and to evaluate the DDI potential of ulotaront and its major metabolite SEP-383103. METHODS: Solubility, permeability, plasma protein binding, CYP inhibition and induction, transporter inhibition and uptake studies were conducted in vitro. Phenotyping studies were conducted using recombinant human CYPs and FMOs, human liver microsomes and human liver homogenates. Preclinical plasma and brain pharmacokinetics were determined after a single intraperitoneal, intravenous, and oral administration of ulotaront. RESULTS: Ulotaront is a compound of high solubility, high permeability, and low binding to plasma proteins. Ulotaront metabolism is mediated via both NADPH-dependent and NADPH-independent pathways, with CYP2D6 as the major metabolizing enzyme. Ulotaront is an inducer of CYP2B6, and an inhibitor of CYP2D6, OCT1 and OCT2, while SEP-383103 is neither a CYP inducer nor a potent inhibitor of CYPs and human transporters. Ulotaront exhibits rapid absorption, greater than 70% bioavailability, approximately 3.5 L/kg volume of distribution, 1.5-4 h half-life, 12-43 ml/min/kg clearance, and good penetration across the blood-brain barrier in preclinical species. CONCLUSIONS: Ulotaront has been designated as a BCS1 compound by US FDA. The ability of ulotaront to penetrate the blood-brain barrier for CNS targeting has been demonstrated in mice and rats. The potential for ulotaront and SEP-383103 to act as perpetrators of CYP and transporter-mediated DDIs is predicted to be remote.

Investigation of Human in vivo Metabolism of SEP-227900 Using the Samples from First-in-Human Study by LC-HRMS/UV and NMR.

OBJECTIVE: The study aims to explore the human in vivo metabolism of SEP-227900 (4H-furo[3, 2-b] pyrrole-carboxylic acid, m.w 151.03), a D-amino-acid oxidase (DAAO) inhibitor, by using plasma and urine samples from first-in-human study. METHODS: The human plasma and urine samples were from a single dose cohort that consisted of 9 healthy male volunteers each received an 80- mg dose of SEP-227900 orally. The pooled pre-dose urine and the pooled 0-24 h urine sample were created across 9 subjects by equal volume. Plasma samples were pooled by equal volume across 9 subjects to obtain 0-12 h plasma for metabolite searching, and also pooled by timepoints across 9 subjects to obtain 0.5, 5, and 12-h plasma for semi-quantitation. The plasma was de-proteinized by acetonitrile (1:3 v/v plasma-acetonitrile), then the supernatant was dried down, reconstituted, and injected for LC-HRMS/UV analysis. The urine sample was just simply centrifuged before analysis. LC-HRMS/UV was utilized to search predictable and unknown metabolites and estimate their relative abundances. Accurate mass measurement by Orbitrap-MS and MS/MS was used for metabolite identification. Chromatographic separation was achieved on a MACMOD AQ C8 column (250 × 4.6 mm, 5-µm) with a gradient mobile phase (A: 10 mM NH4Ac; B: acetonitrile; flowrate: 0.700 ml/min) for a total run-time of 65 min. The definite position in the molecule for the glucuronidation metabolism was characterized by the detected migration phenomenon, methylation with diazomethane (CH2N2), and NMR. RESULTS: Unchanged parent drug and four metabolite peaks were detected in humans: M1 was a mono-oxidative metabolite of SEP-227900; M2 was a glucuronide conjugate of SEP-227900; M3 was a glycine conjugate of SEP-227900; M4 was a glycine conjugate of M1. The specific position of the oxidation in M1 solely based on the mass spectral (MS and MS/MS) data was not identified. However, for the major metabolite M2, the acyl glucuronidation was unambiguously determined through multiple pieces of experimental evidence such as the observation of a migration pattern, mono-methylation by diazomethane, and NMR measurement. This determination is of significance related to the safety evaluation of investigational new drug development. The glycine conjugate of SEP-227900, i.e., M3, was found to be the most abundant metabolite in human urine (approximately 3-fold higher level than the glucuronide level). All together (mainly glycine-conjugate and glucuronide), it resulted in greater than 80% of the dosed amount in urine excretion (a separate measurement showed 23% of the dosed amount in urine excretion as the glucuronide). CONCLUSION: Four metabolites were found in humans: SEP-227900-glycine conjugate, SEP- 227900-glucuronide, mono-oxidative metabolite, and its consequent glycine conjugate. The glucuronide metabolite was identified as acyl glucuronide. Greater than 80% of the dosed amount of SEP-227900 was excreted in the urine, mainly in the forms of glycine- and glucuronide- conjugates.

A sensitive LC-MS/MS method for simultaneous quantification of ulotaront and its N-desmethyl metabolite in human plasma and application to a clinical study.

Ulotaront (SEP-363856) is a novel non-D2-receptor-binding agent under development for the treatment of patients with schizophrenia. A highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with lower limit of quantitation of 0.0200 ng/mL (i.e. 20.0 pg/mL) was successfully developed and validated for the simultaneous quantitation of ulotaront and its N-desmethyl metabolite (M11A) in human plasma. Plasma samples were extracted by solid phase extraction with Oasis MCX 96-well plate, followed by a reversed phase LC separation coupled with MS/MS detection in positive mode (m/z 184.1 → 135.0 for ulotaront and 170.1 → 135.0 for M11A). Stable isotope-labeled compounds SEP-363856-d3 and M11A-d4 were used as internal standards (IS) for corresponding analytes. The validated calibration curve range was 0.0200-20.0 ng/mL for both analytes using a 0.200 mL plasma. Extraction recoveries were found to be 75.7% and 75.1% for ulotaront and IS1, and 82.7% and 83.9% for M11A and IS2, respectively. Frozen plasma samples were confirmed to be stable for up to 730 days at both -20 °C and -70 °C. The validated method has been successfully used to evaluate the pharmacokinetics (PK) of ulotaront and M11A in clinical studies. The application to the first-in-human PK study (single ascending dose) presented in this work demonstrated that ulotaront exhibited near dose proportionality for both Cmax (maximum concentration) and AUC (area under the curve) over the dose range from 5 to 125 mg. M11A was found as a minor metabolite with an exposure of about 2-3% of the parent compound.

A phase I study of the WT2725 dosing emulsion in patients with advanced malignancies.

WT2725 is a Wilms' tumor gene 1 (WT1)-derived-oligopeptide vaccine designed to induce WT1-specific cytotoxic T-lymphocytes against WT1+ tumors in human leukocyte antigen (HLA)-A*0201+ and/or HLA-A*0206+ patients. Here, we report the results of a phase I study of WT2725. In this phase I, open-label, dose-escalation and expansion two-part study, the WT2725 dosing emulsion was administered as a monotherapy to patients with advanced malignancies known to overexpress WT1, including glioblastoma. In part 1, 44 patients were sequentially allocated to four doses: 0.3 mg (n = 5), 0.9 mg (n = 5), 3 mg (n = 6), and 9 mg (n = 28). In part 2, 18 patients were allocated to two doses: 18 mg (n = 9) and 27 mg (n = 9). No dose-limiting toxicities were observed, so the maximum tolerated dose was not reached. Median progression-free survival was 58 (95% confidence interval [CI] 56-81) days (~ 2 months) across all patients with solid tumors; median overall survival was 394 days (13.0 months) (95% CI 309-648). Overall immune-related response rate in solid tumor patients was 7.5% (95% CI 2.6-19.9); response was most prominent in the glioblastoma subgroup. Overall, 62.3% of patients were considered cytotoxic T-lymphocyte responders; the proportion increased with increasing WT2725 dosing emulsion dose. WT2725 dosing emulsion was well tolerated. Preliminary tumor response and biological marker data suggest that WT2725 dosing emulsion may exert antitumor activity in malignancies known to overexpress the WT1 protein, particularly glioblastoma, and provide a rationale for future clinical development.Trial registration: NCT01621542.

An ultrasensitive LC-MS/MS method and application to clinical dose-range finding for glycopyrrolate via an e-Nebulizer.

Aim: Glycopyrrolate (GLY) has been widely used to treat chronic obstructive pulmonary disease (COPD). Recent technical advancement significantly improves the drug delivery efficiency. In a clinical dose-range determination study via electronic nebulizer, the starting dose was as low as 3-µg, thus, a bioanalytical method capable of measuring down to sub-pg/ml or fg/ml level was required to characterize pharmacokinetics of GLY. Methods: A straightforward solid-phase extraction coupled with reversed-phase LC-MS/MS method was successfully developed to quantify plasma GLY concentrations of 0.500-250 pg/mL. MS/MS detection was at ESI+ by monitoring m/z 318.3→116.1 for GLY and 321.3→119.1 for the internal standard GLY-d3. Results and Conclusion: The method was validated with adequate selectivity, intra- and inter-assay precision and accuracy, dilution linearity, and sufficient stability for measuring GLY in human plasma. This method has been used for sample analysis of a clinical dose-range finding study (3-50 µg twice daily doses via an electronic nebulizer). The assay exhibited good precision, accuracy, and robustness. Pharmacokinetic analysis showed good dose proportionality over the studied dose range, where Cmax was found to be Y = 1.354*X + 1.968 (r2 = 0.9984), and AUC0-12 h was Y = 4.661*X + 23.32 (r2 = 0.9976). The study clearly demonstrated the importance of an ultrasensitive (500 fg/ml LLOQ) method.

A sensitive liquid chromatography-tandem mass spectrometry method for quantitative determination of dasotraline in human plasma and its clinical application.

This manuscript describes a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of dasotraline in human plasma. Dasotraline and the internal standard (IS) d4-13C4-dasotraline were extracted from the 0.500-mL plasma pre-mixed with 0.20-mL of 0.5 M sodium bicarbonate solution by a 3-mL of hexane containing 0.7 % sec-butyl alcohol. The organic extract, after dried down, was reconstituted in 150 µL acetonitrile containing 0.1 % formic acid. Forty (40) µL of the resulted sample was injected into LC-MS/MS for analysis. Chromatographic separation was on a Betasil Silica column. MS/MS detection was by monitoring m/z 275→159 and 283→160 for dasotraline and IS, respectively. Peak area ratio of analyte/IS was used for constructing calibration curve and calculating sample concentration. The retention time was ∼3.1 min for both dasotraline and IS. The validated linear range was 5-5000 pg/mL with correlation coefficient r ≥ 0.999. Intra-run precision and accuracy were ≤ 7.3 % CV (n = 6) and 94.4-101.0 % of nominals. Inter-run precision and accuracy were ≤ 4.7 % CV (n = 18) and 96.1-99.8 % of nominals. Plasma sample was confirmed stable for 8 cycles of freeze/thaw, 29 h on bench-top, and up to 977 days of storage at both -20 °C and -70 °C. This method was successfully applied to analyze pharmacokinetic (PK) samples from a single ascending dose (SAD) clinical study with healthy subjects. PK results indicated that dasotraline was slowly absorbed (tmax: 10-12 h) and slowly eliminated (terminal elimination half-life, i.e. t1/2: 47-77 h) with dose proportional Cmax but slightly greater than dose proportional AUC with increase of dosed amount.

LC-MS/MS simultaneous quantification of apomorphine and its major metabolites in human plasma: Application to clinical comparative bioavailability evaluation for the apomorphine sublingual film and a subcutaneous product.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous quantification of apomorphine and its metabolites apomorphine sulfate and norapomorphine in human plasma for supporting clinical development of a novel apomorphine sublingual thin film (APL) for the treatment of Parkinson's disease. Analytes and internal standards (IS) were extracted from human plasma by Oasis HLB SPE cartridge, followed by a reversed phase LC-MS/MS analysis using multiple reaction monitoring (MRM) in positive mode (m/z 268 → 237 for apomorphine, 348 → 237 for apomorphine sulfate, and 348 → 237 for norapomorphine). Stable isotope-labeled compounds were used as IS for respective analytes. The validated curve ranges were 0.02-20 ng/mL, 10-1000 ng/mL, and 0.5-20 ng/mL for apomorphine, apomorphine sulfate and norapomorphine, respectively. Extraction recoveries were found to be 73.4 % (apomorphine), 81.1 % (apomorphine sulfate), and 58.6 % (norapomorphine). Established long-term plasma frozen storage stabilities were 504 days at -20 °C and276 days at -60 °C, respectively. The method has been successfully used for analyzing pharmacokinetics (PK) samples collected from a comparative bioavailability study of APL and the marketed apomorphine subcutaneous (s.c.) product Apo-go®. The results demonstrated that the 15-mg APL film administrated via sublingual produced comparable PK characteristics of apomorphine when compared to the commercial product Apo-go (2-mg) via s.c. administration, hence establishing the dose regimen for this sublingual formulation. It was also noticed that the sublingual 15-mg APL film produced a significantly higher apomorphine sulfate metabolite level than the 2-mg s.c. Apo-go, and both treatments yielded a negligible level of norapomorphine metabolite in humans.

Validation of a sensitive and specific LC-MS/MS method and application to evaluate the systemic exposure and bioavailability of glycopyrrolate via different drug delivery approaches/devices.

A specific and sensitive LC-MS/MS method using d3 -glycopyrrolate as the internal standard (IS) was developed for the quantitative determination of glycopyrrolate (GLY) in human plasma over a concentration range of 4.00-2000 pg/ml. The GLY and IS were extracted using a solid-phase extraction cartridge, then eluted with 0.5% formic acid in 70:30 acetonitrile-water. The eluate was directly injected into an Agilent Pursuit 5PFP column for analysis using an isocratic mobile phase of 50:50 A:B at a flow-rate of 0.500 ml/min (A, 10 mm ammonium acetate in 1% formic acid; B, methanol). The MS detection was in positive mode by monitoring m/z 318.3 → 116.1 (GLY) and 321.3 → 119.1 (IS). The method validation showed the linearity of r2 ≥ 0.9960, intra-/inter-run precisions of ≤11.1% coefficient of variation and accuracies ranging from -2.5 to 12.8% relative error for all levels of quality control samples. This method was successfully employed to support a clinical study to compare absorption and bioavailability of GLY administered by a Magnair® eFlow nebulizer to a Seebri® Breezhaler® with/without a charcoal blockade of gastric absorption. By comparison with intravenous administration, respective bioavailabilities of ~15% for GLY/Magnair and ~22% for the Seebri Breezhaler were found. The bioanalytical reliability was also demonstrated by satisfactory incurred sample reanalysis performance.

An Ultrasensitive LC-APPI-MS/MS Method for Simultaneous Determination of Ciclesonide and Active Metabolite Desisobutyryl-Ciclesonide in Human Serum and Its Application to a Clinical Study.

BACKGROUND: The development of more efficient drug delivery devices for ciclesonide inhalation products requires an ultrasensitive bioanalytical method to measure systematic exposure of ciclesonide (CIC) and its active metabolite desisobutyryl-ciclesonide (des-CIC) in humans. METHOD: Serum sample was extracted with 1-chlorobutane. A reversed-phase liquid chromatography coupled with atmospheric pressure photoionization-tandem mass spectrometry (LC-APPI-MS/MS) method was used for quantification of 1-500 pg/mL for both analytes in a 0.500-mL serum. The analysis time was 4.7 min/injection. CIC-d11 and des-CIC-d11 were used as the internal standards. RESULTS: Calibration curves showed good linearity (r2 > 0.99) for both analytes. This novel method was precise and accurate with interassay precision and accuracy of all within 9.6% CV and ± 4.0% bias for regular QC samples. Extraction recovery was approximately 85% for both analytes. Serum samples are stable for 3 freeze-thaw cycles, 24 h at bench top, and up to 706 days at both -20 °C and -70 °C. This method was successfully used to support a pharmacokinetic (PK) comparison between the inhalation suspensions and an inhalation aerosol of ciclesonide in healthy participants. The method robustness was also supported by the good incurred sample reanalysis reproducibility. CONCLUSION: APPI, a highly selective and sensitive ionization source, made possible for quantifying CIC and des-CIC with a lower limit of quantification (LLOQ) of 1 pg/mL in human serum by LC-MS/MS. A 10-fold sensitivity improvement from the most sensitive reported method (LLOQ, 10 pg/mL) is essential to fully characterize the PK profiles of CIC and des-CIC in support of the clinical development of the ciclesonide-related medications for patients.

Absorption, distribution, metabolism, and excretion of [14C]-dasotraline in humans.

Dasotraline is a dopamine and norepinephrine reuptake inhibitor, and the early clinical trials show a slow absorption and long elimination half-life. To investigate the absorption, distribution, metabolism, and excretion of dasotraline in humans, a single dose of [14C]-dasotraline was administered to eight healthy male adult volunteers. At 35 days, 90.7% of the dosed radioactivity was recovered in the urine (68.3%) and feces (22.4%). The major metabolic pathways involved were: (1) amine oxidation to form oxime M41 and sequential sulfation to form M42 or glucuronidation to form M43; (2) N-hydroxylation and sequential glucuronidation to form M35; (3) oxidative deamination to form (S)-tetralone; (4) mono-oxidation of (S)-tetralone and sequential glucuronidation to form M31A and M32; and (5) N-acetylation to form (1R,4S)-acetamide M102. A total of 8 metabolites were detected and structurally elucidated with 4 in plasma (M41, M42, M43, and M35), 7 in urine (M41, M42, M43, M31A, M32, M35, and (S)-tetralone), and 3 in feces (M41, (S)-tetralone, and (1R,4S)-acetamide). The 2 most abundant circulating metabolites were sulfate (M42) and glucuronide (M43) conjugates of the oxime of dasotraline, accounting for 60.1% and 15.0% of the total plasma radioactivity, respectively; unchanged dasotraline accounted for 8.59%. The oxime M41 accounted for only 0.62% of the total plasma radioactivity and was detected only at early time points. M35 was a minor glucuronide metabolite, undetectable by radioactivity but identified by mass spectrometry. The results demonstrate that dasotraline was slowly absorbed, and extensively metabolized by oxidation and subsequent phase II conjugations. The findings from this study also demonstrated that metabolism of dasotraline by humans did not produce metabolites that may cause a safety concern.

Exploratory Biomarker Study of the Triple Reuptake Inhibitor SEP-432 Compared to the Dual Reuptake Inhibitor Duloxetine in Healthy Normal Subjects.

INTRODUCTION: SEP-432 is a triple monoamine reuptake inhibitor of norepinephrine (NE), serotonin (5-HT), and dopamine (DA), based on in vitro binding studies. We sought evidence that SEP-432 engages these monoamine systems by measuring concentrations of monoamines and/or their main metabolites in cerebrospinal fluid (CSF) and plasma and comparing results to duloxetine, a dual reuptake inhibitor of NE and 5-HT. METHODS: Eighteen healthy normal subjects received either SEP-432 (300 mg/day), duloxetine (60 mg/day), or placebo for 14 days in-clinic (double blind) with CSF and plasma collections at baseline (single lumbar puncture) and Day 14 (24-h CSF and plasma collection). Concentrations of monoamines and their metabolites, as well as pharmacokinetic concentrations of SEP-432 and metabolite, were quantified by liquid chromatography-tandem mass spectrometry. RESULTS: Compared to placebo in the Day 14 area under the curve 24-h (AUC0-24 h ) analysis, SEP-432 significantly (P < 0.05) decreased the NE metabolite dihydroxyphenylglycol (DHPG) in CSF and plasma, decreased 5-HT in plasma, and did not affect DA metabolites, while duloxetine had significant effects on DHPG and 5-HT. Time-matched baseline to Day 14 biomarker comparisons confirmed these findings. CONCLUSION: CSF monoamine biomarkers confirmed central NET activity for SEP-432 and duloxetine's dual reuptake inhibition.

Automatic Supported Liquid Extraction (SLE) Coupled with HILIC-MS/MS: An Application to Method Development and Validation of Erlotinib in Human Plasma.

A novel bioanalytical method was developed and validated for the quantitative determination of erlotinib in human plasma by using the supported liquid extraction (SLE) sample cleanup coupled with hydrophilic interaction liquid chromatography and tandem mass spectrometric detection (HILIC-MS/MS). The SLE extract could be directly injected into the HILIC-MS/MS system for analysis without the solvent evaporation and reconstitution steps. Therefore, the method is simple and rapid. In the present method, erlotinib-d6 was used as the internal standard. The SLE extraction recovery was 101.3%. The validated linear curve range was 2 to 2,000 ng/mL based on a sample volume of 0.100-mL, with a linear correlation coefficient of > 0.999. The validation results demonstrated that the present method gave a satisfactory precision and accuracy: intra-day CV < 5.9% (.

Simultaneous determination of three isomeric metabolites of tacrolimus (FK506) in human whole blood and plasma using high performance liquid chromatography-tandem mass spectrometry.

An ammonium-adduct based liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous determination of three isomeric metabolites of tacrolimus (FK506), 13-O-demethylated (M1), 31-O-demethylated (M2) and 15-O-demethylated (M3) tacrolimus in human whole blood and plasma. These metabolites and the internal standards were extracted from biological matrix by methylbutyl ether (MTBE). Separation was achieved on a Genesis C(18) column with a gradient mobile phase elution. Ammonium-adduct ions formed by a Turbo Ionspray in positive ion mode were used to detect each analyte and internal standard. The MS/MS detection was by monitoring the fragmentation of 807.5-->772.4 (m/z) for M1, 807.5-->754.5 (m/z) for both M2 and M3, 795.5-->760.5 (m/z) for IS1 (FR298701) and 961.5-->908.5 (m/z) for IS2 (FR290198) on a triple quadrupole mass spectrometer (Sciex API 3000). The retention times were approximately 4.1 min for M1, 6.8 min for M2, 6.0 min for M3, and 3.9 min for IS1 and 6.4 min for IS2, respectively. The validated dynamic range was 0.2-20 ng/ml for all three metabolites based on a sample volume of 0.25-ml. The linearity of calibration curves for M1, M2, and M3 in both matrices had a correlation coefficient of >/=0.9984. In whole blood, validation data showed intra-batch (n=6) CVs of

In vivo metabolism for the hydroxylation of FK778 to the metabolite M3 in humans studied by enantioselective liquid chromatography/tandem mass spectrometry.

A major active metabolite of malononitrilamide FK778 (an immunosuppressant under development) is labeled M3. Due to a chiral center created during in vivo metabolism, the exploration of enantiomer profiles in clinical samples is critical to the characterization of the immunosuppressive activity of M3. An enantioselective liquid chromatography method with detection by tandem mass spectrometry (LC/MS/MS) was developed for the resolution of M3 enantiomers. It was experimentally confirmed that no interconversion between the two enantiomers occurred during sample preparation. This new approach was applied to measure the enantioselectivity of the M3 metabolite in human plasma samples from kidney transplanted patients. The assay results of 91 in vivo human samples from three subjects showed a ratio of 57:43 for the (-)-enantiomer (the 2nd eluter) vs. the (+)-enantiomer (1st eluter), indicating that the enantiometabolism of FK778 through human enzymes is essentially non-specific.

Effects of St. John's wort (Hypericum perforatum) on tacrolimus pharmacokinetics in healthy volunteers.

Tacrolimus is an immunosuppressant approved for the prevention of rejection following transplantation and is a substrate for CYP3A and P-glycoprotein. A pharmacokinetic interaction between St. John's wort (antidepressant herbal product and inducer of CYP3A and P-glycoprotein) and tacrolimus was evaluated in 10 healthy volunteers. The pharmacokinetics of tacrolimus were obtained from serial blood samples collected following single oral doses (0.1 mg/kg) prior to and during an 18-day concomitant St. John's wort dosing phase (300 mg orally three times daily). Coadministration of St. John's wort significantly decreased tacrolimus AUC (306.9 microg.h/L +/- 175.8 microg.h/L vs. 198.7 microg.h/L +/- 139.6 microg.h/L; p=0.004) and increased apparent oral clearance (349.0 mL/h/kg +/- 126.0 mL/h/kg vs. 586.4 mL/h/kg +/- 274.9 mL/h/kg; p=0.01) and apparent oral volume of distribution at steady state (11.5 L/kg +/- 4.3 L/kg vs. 17.6 L/kg +/- 9.6 L/kg; p=0.04). St. John's wort appears to induce tacrolimus metabolism, most likely through induction of CYP3A and P-glycoprotein.

Quantification of 6-deoxy-6-demethyl-4-dedimethylaminotetracycline (COL-3) in human plasma using liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

An accurate and reliable liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method has been developed and validated for the determination of 6-deoxy-6-demethyl-4-dedimethylaminotetracycline (COL-3) in human plasma. The assay used chrysin as an internal standard (I.S.). The analyte and the I.S. were extracted from acidified plasma by methyl-t-butyl ether. Separation was achieved on a YMCbasic column using acetonitrile-water-formic acid mobile phase. The MS-MS detection was by monitoring fragmentation 372.1-->326.2 (m/z) for COL-3 and 255.1-->153.1 (m/z) for the I.S. on a Sciex API 365 using a Turbo Ionspray in positive ion mode. The retention times were approximately 1.7 min for COL-3 and 1.8 min for the I.S. The validated dynamic range was 0.03-10.0 microg/ml using 0.25-ml plasma with correlation coefficients of >or=0.9985. The precision and accuracy for the calibration standards (n=3) were RSD

Conformations of gas-phase lysozyme ions produced from two different solution conformations.

Near pH 2.0, lysozyme in water is in its native conformation, and in water/methanol (2/8) it adopts a helical denatured conformation (Kamatari et al. Protein Sci. 1998, 7, 681-688). Hydrogen/deuterium (H/D) exchange of lysozyme in solution confirms that it is partially unfolded at pH 2.0 in water/methanol (v/v = 2/8). With electrospray ionization (ESI) mass spectrometry (MS), lysozyme in water produces ions with charges +7 to +12, with the greatest intensity at +10, whereas lysozyme in water/methanol (2/8) produces ions with charges +6 to +12 with the greatest intensity at +7. Thus, lysozyme is an exception to the rule that a protein denatured in solution forms higher charge states than the same protein in its folded native conformations in solution. Because the same charge states are produced from these two solution conformations, a direct comparison of the properties of the gas-phase ions produced from two very different solution conformations is possible. The conformations of lysozyme ions in the gas phase were studied using cross section measurements and gas-phase H/D exchange. Similar cross sections and H/D exchange levels were observed for same-charge states of lysozyme ions formed from the native and helical denatured conformations in solution. Cross sections show that the ions have compact structures. Thus, disulfide-intact gaseous lysozyme ions generated from the denatured state in water/methanol (2/8) refold into compact structures in the gas phase on a time scale of milliseconds or less.