High-throughput screening assay for the quantification of Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, and d18:1/22:0 in HepG2 cells using RapidFire mass spectrometry.

Ceramides are known to be involved in various biological processes with their physiological levels elevated in various disease conditions such as diabetes, Alzheimer's, atherosclerosis. To facilitate the rapid screening of Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, and d18:1/22:0 inhibition in HepG2 cells, a RapidFire coupled to tandem mass spectrometry (RF-MS/MS) method has been developed. The RF platform provides an automated solid-phase extraction system that gave a throughput of 12.6 s per sample to an MS/MS system using electrospray ionization under the positive ion mode. Chromatographic separation of Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, and d18:1/22:0 was achieved using a ternary gradient on C8 type E cartridge. The MS/MS ion transitions monitored were 538.2 → 264.2, 650.7 → 264.2, 648.6 → 264.2, 566.4 → 264.2, 510.4 → 264.2, 594.4 → 264.2, 622.5 → 264.2, and 552.3 → 250.2 for Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, d18:1/22:0, and the internal standard (Cer d17:1/18:0), respectively. The RF-MS/MS methodology showed an excellent performance with an average Z' value of 0.5-0.7. This is the first report of an RF-MS/MS assay for screening of ceramides which is amenable for high-throughput screening.

Prediction of Human Pharmacokinetics of Fomepizole from Preclinical Species Pharmacokinetics Based on Normalizing Time Course Profiles.

Fomepizole is used as an antidote to treat methanol poisoning due to its selectivity towards alcohol dehydrogenase. In the present study, the goal is to develop a method to predict the fomepizole human plasma concentration versus time profile based on the preclinical pharmacokinetics using the assumption of superimposability on simulated time course profiles of animals and humans. Standard allometric equations with/without correction factors were also assimilated in the prediction. The volume of distribution at steady state (Vss) predicted by simple allometry (57.55 L) was very close to the reported value (42.17 L). However, clearance (CL) prediction by simple allometry was at least 3-fold higher to the reported value (33.86 mL/min); hence, multiple correction factors were used to predict the clearance. Both brain weight and maximum life span potential could predict the CL with 1.22- and 1.01-fold difference. Specifically, the predicted Vss and CL values via interspecies scaling were used in the prediction of series of human intravenous pharmacokinetic parameters, while the simulation of human oral profile was done by the use of absorption rate constant (Ka) from dog following the applicability of human bioavailability value scaled from dog data. In summary, the findings indicate that the utility of diverse allometry approaches to derive the human pharmacokinetics of fomepizole after intravenous/oral dosing.

Prediction of Human Pharmacokinetics of Bendamustine from Preclinical Species Pharmacokinetics Based on Normalizing Time Course Profiles.

Bendamustine, an alkylating anticancer agent, is used to treat chronic lymphocytic leukemia by intravenous infusion alone or in combination. The work aimed to develop a method to predict time vs. concentration profile for humans based on preclinical pharmacokinetics using the assumption of superimposability of normalized time course profiles of animals and humans. Standard allometric equations with/without correction factors (CF) were also used in prediction. The Vss was predicted by simple allometry of 0.312W0.871 (r2=0.987), where W is body weight; predicted Vss (19.71 L) was similar to the reported value (20.10 L). However, CL prediction involved both simple and CF allometry. Best proximity CL (543 vs. 598 mL/min) was obtained with maximum life span correction (MLP) [2.46W1.215 (r2=0.988)]. Normalized curves were obtained by normalizing the time (with mean residence time) vs. concentration (with dose/Vss) in animal species. The concentration vs. time profile in humans after intravenous infusion was then simulated using normalized curve for each animal species and the values of CL and Vss were predicted for humans. In summary the findings indicate that normalized time course approach could predict the bendamustine human pharmacokinetics and such an approach could be prospectively applied for analog drugs of this class.

Prediction of Human Pharmacokinetics of Ulixertinib, a Novel ERK1/2 Inhibitor from Mice, Rats, and Dogs Pharmacokinetics.

BACKGROUND AND OBJECTIVES: Ulixertinib (BVD-523) is a novel and selective reversible inhibitor of ERK1/ERK2. The primary objectives of the study were to evaluate the pharmacokinetics of ulixertinib in mice, rats, and dogs followed by prediction of human pharmacokinetic profile by allometric equations with/without correction factors. METHODS: Oral and intravenous pharmacokinetic profiles of ulixertinib were generated in mice, rats, and dogs. The human intravenous pharmacokinetics profiles [volume of distribution (Vss) and clearance (CL)] were predicted employing simple allometry and using correction factors [maximum life span potential (MLP) and brain weight (BW)]. Pharmacokinetic data obtained from dogs were used to simulate human oral profile [area under the curve (AUC) and maximum plasma concentrations (Cmax)]. RESULTS: Post-intravenous administration the CL was moderate in dogs (15.5 mL/min/kg) and low in mice (6.24 mL/min/kg) and rats (1.67 mL/min/kg). Vss was 0.56, 0.36, and 1.61 L/kg in mice, rats, and dogs, respectively. The half-life (t½) of ulixertinib ranged between 1.0 and 2.5 h across the animal species. Following oral administration ulixertinib attained maximum concentration in plasma (Tmax) within 0.50-0.75 h in mice and rats, indicating that absorption was rapid; however, in dogs, Tmax attained at 2 h. Absolute oral bioavailability in mice and rats was > 92%; however, in dogs, it was 34%. By different allometric approaches, simple method and brain weight correction factor shown clear improvement in the prediction efficiency of allometric scaling for Vss (1.34-1.70 L/kg) and CL (4.18-6.09 mL/min/kg), respectively, comparing with the MLP method and simple method for CL. Similarly, simulation of oral human profile was attained from scaled values and dog data to predict reported human profile (AUC and Cmax). CONCLUSIONS: The derived pharmacokinetic parameters (AUC and Cmax at 600 mg dose) and simulated plasma concentration-time profiles of ulixertinib in humans were predicted with good confidence by allometric approach.

A small molecule inhibitor of Nicotinamide N-methyltransferase for the treatment of metabolic disorders.

Nicotinamide N-methyltransferase (NNMT) is a cytosolic enzyme that catalyzes the transfer of a methyl group from the co-factor S-adenosyl-L-methionine (SAM) onto the substrate, nicotinamide (NA) to form 1-methyl-nicotinamide (MNA). Higher NNMT expression and MNA concentrations have been associated with obesity and type-2 diabetes. Here we report a small molecule analog of NA, JBSNF-000088, that inhibits NNMT activity, reduces MNA levels and drives insulin sensitization, glucose modulation and body weight reduction in animal models of metabolic disease. In mice with high fat diet (HFD)-induced obesity, JBSNF-000088 treatment caused a reduction in body weight, improved insulin sensitivity and normalized glucose tolerance to the level of lean control mice. These effects were not seen in NNMT knockout mice on HFD, confirming specificity of JBSNF-000088. The compound also improved glucose handling in ob/ob and db/db mice albeit to a lesser extent and in the absence of weight loss. Co-crystal structure analysis revealed the presence of the N-methylated product of JBSNF-000088 bound to the NNMT protein. The N-methylated product was also detected in the plasma of mice treated with JBSNF-000088. Hence, JBSNF-000088 may act as a slow-turnover substrate analog, driving the observed metabolic benefits.

Novel nicotinamide analog as inhibitor of nicotinamide N-methyltransferase.

Nicotinamide N-methyltransferase (NNMT) has been linked to obesity and diabetes. We have identified a novel nicotinamide (NA) analog, compound 12 that inhibited NNMT enzymatic activity and reduced the formation of 1-methyl-nicotinamide (MNA), the primary metabolite of NA by ∼80% at 2 h when dosed in mice orally at 50 mg/kg.

LC-ESI-MS/MS determination of 4-methylpyrazole in dog plasma and its application to a pharmacokinetic study in dogs.

A simple, specific, sensitive and rapid LC-ESI-MS/MS method has been developed and validated for the quantification of 4-methylpyrazole in dog plasma using N-methylnicotinamide-d4 as an internal standard (IS) as per regulatory guidelines. Sample preparation was accomplished through a simple protein precipitation. Chromatographic separation of 4-methylpyrazole and the IS was performed on a monolithic (Chromolith RP18e ) column using an isocratic mobile phase comprising 0.2% formic acid in water and acetonitrile (20:80, v/v) at a flow rate of 1.0 mL/min. Elution of 4-methylpyrazole and the IS occurred at ~1.60 and 1.56 min, respectively. The total chromatographic run time was 3.2 min. A linear response function was established in the concentration range of 4.96-4955 ng/mL. The intra- and inter-day accuracy and precision were in the ranges 1.81-12.9 and 3.80-11.1%, respectively. This novel method has been applied to a pharmacokinetic study in dogs.

LC-MS/MS determination of tideglusib, a novel GSK-3ß inhibitor in mice plasma and its application to a pharmacokinetic study in mice.

A sensitive, specific and rapid LC-ESI-MS/MS method has been developed and validated for the quantification of tideglusib in mice plasma using warfarin as an internal standard (I.S.) as per regulatory guidelines. Sample preparation was accomplished through liquid-liquid extraction process. Chromatographic separation was performed on Atlantis dC18 column using mobile phase A (acetonitrile) and B (5mM ammonium acetate in water) in a flow-gradient mode. Elution of tideglusib and the I.S. occurred at ∼2.06 and 1.29min, respectively. The total chromatographic run time was 3.2min. A linear response function was established in the concentration range of 20.2-1008ng/mL. The intra- and inter-day accuracy and precision were in the range of 4.61-12.6 and 6.04-11.8%, respectively. This novel method has been applied to a pharmacokinetic study in mice.

Area under the curve predictions of dalbavancin, a new lipoglycopeptide agent, using the end of intravenous infusion concentration data point by regression analyses such as linear, log-linear and power models.

1. Dalbavancin, a lipoglycopeptide, is approved for treating gram-positive bacterial infections. Area under plasma concentration versus time curve (AUCinf) of dalbavancin is a key parameter and AUCinf/MIC ratio is a critical pharmacodynamic marker. 2. Using end of intravenous infusion concentration (i.e. Cmax) Cmax versus AUCinf relationship for dalbavancin was established by regression analyses (i.e. linear, log-log, log-linear and power models) using 21 pairs of subject data. 3. The predictions of the AUCinf were performed using published Cmax data by application of regression equations. The quotient of observed/predicted values rendered fold difference. The mean absolute error (MAE)/root mean square error (RMSE) and correlation coefficient (r) were used in the assessment. 4. MAE and RMSE values for the various models were comparable. The Cmax versus AUCinf exhibited excellent correlation (r > 0.9488). The internal data evaluation showed narrow confinement (0.84-1.14-fold difference) with a RMSE < 10.3%. The external data evaluation showed that the models predicted AUCinf with a RMSE of 3.02-27.46% with fold difference largely contained within 0.64-1.48. 5. Regardless of the regression models, a single time point strategy of using Cmax (i.e. end of 30-min infusion) is amenable as a prospective tool for predicting AUCinf of dalbavancin in patients.

Sensitive LC-MS/MS Method for the Simultaneous Determination of Bendamustine and its Active Metabolite, γ-Hydroxybendamustine in Small Volume Mice and Dog Plasma and its Application to a Pharmacokinetic Study in Mice and Dogs.

A highly sensitive, specific and rapid LC-ESI-MS/MS method has been developed and validated for the simultaneous quantification of bendamustine (BM) and γ-hydroxybendamustine (HBM) in small volume (20 µL) mice and dog plasma using phenacetin as an internal standard (IS) as per regulatory guidelines. Both the analytes and IS were extracted from mice and dog plasma using a liquid-liquid extraction method. Chromatography was achieved on Atlantis dC18 column using an isocratic mobile phase (0.2% formic acid:acetonitrile, 25:75) at a flow rate of 0.40 mL/min. The total chromatographic run time was 3.0 min and the elution of BM, HBM and IS occurred at ~1.2, 1.2 and 2.0 min, respectively. A linear response function was established 0.11-518 ng/mL for both the analytes in mice and dog plasma. The intra- and inter-day accuracy and precisions were in the range of 3.46-12.9 and 3.63-8.23%; 1.15-9.00 and 7.86-9.49% for BM and HBM, respectively in mice plasma and 2.15-6.49 and 1.73-13.1%; 4.35-13.9 and 4.33-10.5% for BM and HBM, respectively in dog plasma. This novel method has been applied to a pharmacokinetic study in mice and dogs.

Interspecies scaling of urinary excretory amounts of nine drugs belonging to different therapeutic areas with diverse chemical structures - accurate prediction of the human urinary excretory amounts.

1. The human urinary excretory amounts of total drug (parent + metabolites) were predicted for nine drugs with diverse chemical structures using simple allometry. The drugs used for scaling were cephapirin, olanzapine, labetolol, carisbamate, voriconazole, tofacitinib, nevirapine, ropinirole, and cyclindole. 2. The traditional allometric scaling was attempted using Y = aWb relationship. The corresponding predicted urinary amounts were converted into % recovery by using appropriate human dose. Appropriate statistical tests comprising of fold-difference (predicted/observed values) and error calculations (MAE and RMSE) were performed. 3. The interspecies scaling of all nine drugs tested showed excellent correlation (r > 0.9672). The predictions for eight out of nine drugs (exception was cephaphirin) were contained within 0.80-1.25 fold-differences. The MAE and RMSE were within ± 18% and 14.64%, respectively. 4. The present work supported the potential application of prospective allometry scaling to predict the urinary excretory amounts of the total drug and gauge any issues for the renal handling of the total drug.

Validation of an LC-MS/MS method for simultaneous detection of four HDAC inhibitors - belinostat, panobinostat, rocilinostat and vorinostat in mouse plasma and its application to a mouse pharmacokinetic study.

A sensitive and rapid LC-MS/MS method was developed and validated for the simultaneous quantitation of four HDAC inhibitors, namely belinostat (BST), panobinostat (PST), rocilinostat (RST) and vorinostat (VST), in mouse plasma as per regulatory guidelines. The analytes and internal standard were extracted from 50 µL mouse plasma by protein precipitation, followed by chromatographic separation using an Atlantis C18 column with an isocratic mobile phase comprising 0.1% formic acid-acetonitrile (25:75, v/v) at a flow rate of 0.5 mL/min within 2.5 min. Detection and quantitation were done by multiple reaction monitoring on a triple quadrupole mass spectrometer following the transitions: m/z 319 → 93, 350 → 158, 434 → 274 and 265 → 232 for BST, PST, RST and VST, respectively, in the positive ionization mode. The calibration curves were linear from 2.92 to 2921 ng/mL for BST and PST and from 1.01 to 1008 ng/mL for RST and VST with r2 ≥ 0.99 for all of the analytes. The intra- and inter-batch accuracy and precision (CV) across quality controls varied from 85.5 to 112% and from 2.30 to 12.5, respectively, for all of the analytes. Analytes were found to be stable under different stability conditions. The method was applied to an i.v. pharmacokinetic study in mice.

Sensitive method for the determination of rocilinostat in small volume mouse plasma by LC-MS/MS and its application to a pharmacokinetic study in mice.

A highly sensitive, specific and rapid LC-ESI-MS/MS method has been developed and validated for the quantification of rocilinostat in small volume mouse plasma (20 µL) using vorinostat as an internal standard (IS) as per regulatory guidelines. Sample preparation was accomplished through a protein precipitation procedure with acetonitrile. Chromatography was achieved on Prodigy ODS-2 column using a binary gradient using mobile phase A (0.2% formic acid in water) and B (acetonitrile) at a flow rate of 0.38 mL/min. The total chromatographic run time was 4.1 min and the elution of rocilinostat and IS occurred at ~3.2 and 2.9 min, respectively. A linear response function was established in the concentration range of 0.28-1193 ng/mL in mouse plasma. The intra- and inter-day accuracy and precisions were in the ranges of 3.12-8.93 and 6.41-11.6%, respectively. This novel method has been applied to a pharmacokinetic study in mice. Copyright © 2016 John Wiley & Sons, Ltd.

Retrospective and Prospective Human Intravenous and Oral Pharmacokinetic Projection of Dipeptidyl peptidase-IV Inhibitors Using Simple Allometric Principles - Case Studies of ABT-279, ABT-341, Alogliptin, Carmegliptin, Sitagliptin and Vildagliptin.

PURPOSE: The purpose of this exercise was to explore the utility of allometric scaling approach for the prediction of intravenous and oral pharmacokinetics of six dipeptidy peptidase-IV (DPP-IV) inhibitors viz. ABT-279, ABT-341, alogliptin, carmegliptin, sitagliptin and vildagliptin. METHODS: The availability of intravenous and oral pharmacokinetic data in animals enabled the allometry scaling of 6 DPP-IV inhibitors. The relationship between the main pharmacokinetic parameters [viz. volume of distribution (Vd) and clearance (CL)] and body weight was studied across three or four mammalian species, using double logarithmic plots to predict the human pharmacokinetic parameters of CL and Vd using simple allometry. RESULTS: A simply allometry relationship: Y = aWb was found to be adequate for the prediction of intravenous and oral human clearance/volume of distribution for DPP-IV inhibitors. The allometric equations for alogliptin, carmegliptin, sitagliptin, vildagliptin, ABT-279 and ABT-341 were 1.867W0.780, 1.170W0.756, 2.020W0.529, 1.959 W0.847, 0.672 W1.016, 1.077W 0.649, respectively, to predict intravenous clearance (CL) and the corresponding equations to predict intravenous volume of distribution (Vd) were: 3.313W0.987, 6.096W0.992, 7.140W0.805, 2.742W0.941, 1.299W0.695 and 5.370W0.803. With the exception of a few discordant values the exponent rule appeared to hold for CL (0.75) and Vd (1.0) for the predictions of various DPP-IV inhibitors. Regardless of the routes, the predicted values were within 2-3 fold of observed values and intravenous allometry was better than oral allometry. CONCLUSION: Simple allometry retrospectively predicted with reasonable accuracy the human reported values of gliptins and could be used as a prospective tool for this class of drugs.

Development and validation of an RP-HPLC method for the quantitation of odanacatib in rat and human plasma and its application to a pharmacokinetic study.

A simple, specific, sensitive and reproducible high-performance liquid chromatography (HPLC) assay method has been developed and validated for the estimation of odanacatib in rat and human plasma. The bioanalytical procedure involves extraction of odanacatib and itraconazole (internal standard, IS) from a 200 µL plasma aliquot with simple liquid-liquid extraction process. Chromatographic separation was achieved on a Symmetry Shield RP18 using an isocratic mobile phase at a flow rate of 0.7 mL/min. The UV detection wave length was 268 nm. Odanacatib and IS eluted at 5.5 and 8.6 min, respectively with a total run time of 10 min. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 50.9-2037 ng/mL (r(2) = 0.994). The intra- and inter-day precisions were in the range of 2.06-5.11 and 5.84-13.1%, respectively, in rat plasma and 2.38-7.90 and 6.39-10.2%, respectively, in human plasma. The validated HPLC method was successfully applied to a pharmacokinetic study in rats.

Validation of a simple bioanalytical method for the determination of DRF-6196, a novel oxazolidinone, in mouse plasma: application to a single-dose pharmacokinetic study.

An isocratic simple, specific, sensitive and reproducible high performance liquid chromatography (HPLC) method was developed and validated for the estimation of DRF-6196, a novel oxazolidinone in mouse plasma. This method involves a simple liquid/liquid extraction of DRF-6196 and the internal standard (IS; chlorzoxazone, CAS 95-25-0) from plasma into dichloromethane/ethyl acetate mixture that was evaporated under nitrogen. The HPLC analysis was carried out on an Inertsil ODS 2 column using 0.01 mol/L potassium dihydorgen ortho phosphate (pH 3.2) and acetonitrile (65:35, v/v) as mobile phase. The eluate was monitored using an UV detector set at 266 nm. Ratio of peak area of analyte to IS was used for quantification of plasma samples. The retention time of DRF-6196 and IS were 8.2 and 11.1 min, respectively. The assay was linear (r2 > 0.999) in the concentration range 0.1-50 microg/ml. Absolute recovery for analyte and IS was > 94 % from mouse plasma. The lower limit of quantification (LLOQ) of DRF-6196 was 0.1 microg/ml. The inter- and intra-day precision in the measurement of quality control (QC) samples, 0.1, 0.3, 15.0 and 40.0 microg/ml, were in the range 3.64 to 9.51 % relative standard deviation (RSD) and 0.92 to 6.23 % RSD, respectively. Accuracy in the measurement of QC samples was in the range 88.15 to 106.05 % of the nominal values. The analyte and IS were stable in the stability studies viz., benchtop, autosampler and freeze/thaw cycles. The stability of DRF-6196 was established for 1 month at -80 degrees C. The assay method was successfully applied to a pharmacokinetic study of DRF-6196 in mice.

HPLC method for determination of DRF-4367 in rat plasma: validation and its application to pharmacokinetics in Wistar rats.

A specific, accurate, precise and reproducible high-performance liquid chromatography (HPLC) method was developed for the estimation of DRF-4367, a novel cyclooxygenase-2 inhibitor in rat plasma. The assay procedure involved simple liquid/liquid extraction of DRF-4367 and internal standard (IS, celecoxib) from plasma into dichloromethane. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto a Kromasil KR 100-5C(18) column (4.6 x 250 mm, 5 microm). The mobile phase consisting of 0.01 M potassium dihydrogen ortho-phosphate (pH 3.2) and acetonitrile (40:60, v/v) was used at a flow rate of 1.0 mL/min. The eluate was monitored using an UV detector set at 247 nm. The ratio of peak area of analyte to IS was used for quantification of plasma samples. Nominal retention times of DRF-4367 and IS were 6.6 and 11.2 min, respectively. The standard curve for DRF-4367 was linear (r(2) > 0.999) in the concentration range 0.1-20 micro g/mL. Absolute recovery was >86% from rat plasma for both analyte and IS. The lower limit of quantification of DRF-4367 was 0.1 micro g/mL. The inter- and intra-day precisions in the measurement of quality control samples, 0.1, 0.3, 8.0 and 15.0 microg/mL, were in the range 6.93-9.34% relative standard deviation (RSD) and 0.48-6.59% RSD, respectively. Accuracy in the measurement of QC samples was in the range 91.24-109.36% of the nominal values. Analyte and IS were stable in the battery of stability studies, viz. benchtop, autosampler and freeze-thaw cycles. Stability of DRF-4367 was established for 1 month at -80 degrees C. The application of the assay to a pharmacokinetic study in rats is described.