Preclinical assessment of Orteronel(®), a CYP17A1 enzyme inhibitor in rats.

Orteronel (TAK-700) is a novel and selective inhibitor of CYP17A1, which is expressed in testicular, adrenal and prostate tumor tissues. Orteronel is currently in Phase-III clinical development for metastatic castration-resistant prostate patients. The objective of the study is to assess the permeability, metabolic stability (in various preclinical and human liver microsomes), identify the major CYPs involved in the metabolism of Orteronel. We have also studied the pharmacokinetics and excretion of Orteronel in Sprague-Dawley rats. Orteronel was found to be stable in various liver microsomes tested. The half-life (t ½) of Orteronel with intravenous (i.v.) route was found to be 1.65 ± 0.22 h. The clearance and volume of distribution by i.v. route for Orteronel were found to be 27.5 ± 3.09 mL/min/kg and 3.94 ± 0.85 L/kg, respectively. The absorption of Orteronel was rapid, with maximum concentrations of drug in plasma of 614 ± 76.4, 1,764 ± 166, 4,652 ± 300 and 17,518 ± 3,178 ng/mL attained at 0.38, 0.75, 0.50 and 0.83 h, respectively, after oral administration of Orteronel at 5, 10, 30 and 100 mg/kg as a suspension. In the dose proportional oral pharmacokinetic study, the mean t ½ by oral route was found to be ~3.5 h and bioavailability ranged between 69 and 89 %. The primary route of elimination for Orteronel is urine.

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.

Development and validation of an RP-HPLC method for the quantitation of Orteronel (TAK-700), a CYP17A1 enzyme inhibitor, in rat plasma and its application to a pharmacokinetic study.

A novel, simple, specific, sensitive and reproducible high-performance liquid chromatography assay method has been developed and validated for the estimation of Orteronel in rat plasma. The bioanalytical procedure involves extraction of Orteronel and phenacetin (internal standard) from rat plasma with a simple liquid-liquid extraction process. The chromatographic analysis was performed on a Waters Alliance system using a gradient mobile phase conditions at a flow rate of 1 mL/min and a C18 column maintained at ambient room temperature. The eluate was monitored using a photodiode array detector set at 242. Orteronel and internal standard eluted at 4.8 and 6.2 min, respectively and the total run time was 9 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 100-3149 ng/mL (r(2) = 0.995). The intra- and inter-day precisions were in the ranges of 0.31-7.87 and 3.97-6.35, respectively, in rat plasma. The validated HPLC method was successfully applied to a pharmacokinetic study of Orteronel in rats.

Validated RP-HPLC/UV method for the quantitation of abiraterone in rat plasma and its application to a pharmacokinetic study in rats.

A novel, simple, specific, sensitive and reproducible high-performance liquid chromatography (HPLC) assay method has been developed and validated for the estimation of abiraterone (ART) in rat plasma. The analytical procedure involves extraction of ART and diclofenac (internal standard, IS) from rat plasma with a simple liquid-liquid extraction process. The chromatographic analysis was performed on a Waters Alliance system with a Betasil C(18) column maintained at ambient room temperature and an isocratic mobile phase [acetonitrile-water-10 mm potassium dihydrogen phosphate (pH 3.0), 55:5:40, v/v/v] at a flow rate of 1.00 mL/min with a total run time of 10 min. The eluate was monitored using an UV detector set at 255 nm. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 93.4-3251 ng/mL (r(2) = 0.997). The intra- and inter-day precisions were 0.56-4.98 and 3.03-7.18, respectively, in rat plasma. The validated HPLC method was successfully applied to a pharmacokinetic study of ART in rats.

Development and validation of an enantioselective LC-MS/MS method to quantify enantiomers of (±)-TAK-700 in rat plasma: lack of in vivo inversion of (+)-TAK-700 (Orteronel) to its antipode.

A highly sensitive, specific and enantioselective assay has been developed and validated for the estimation of TAK-700 enantiomers [(+)-TAK-700 and (-)-TAK-700] in rat plasma on LC-MS/MS-ESI in the positive-ion mode. Liquid-liquid extraction was used to extract (±)-TAK-700 enantiomers and IS (phenacetin) from rat plasma. TAK-700 enantiomers were separated using methanol and 5 mm ammonium acetate (80:20, v/v) at a flow rate of 0.7 mL/min on a Chiralcel OJ-RH column. The total run time was 7.0 min and the elution of (+)-TAK-700, (-)-TAK-700 and IS occurred at 3.71, 4.45 and 4.33 min, respectively. The MS/MS ion transitions monitored were m/z 308.2 → 95.0 for TAK-700 and m/z 180.2 → 110.1 for IS. The standard curves for TAK-700 enantiomers were linear (r(2) > 0.998) in the concentration range 2.01-2015 ng/mL for each enantiomer. The inter- and intra-day precisions were in the ranges 3.74-7.61 and 2.06-8.71% and 3.59-9.00 and 2.32-11.0% for (+)-TAK-700 and (-)-TAK-700, respectively. Both the enantiomers were found to be stable in a battery of stability studies. This novel method was applied to the study of stereoselective oral pharmacokinetics of (+)-TAK-700 and it was unequivocally demonstrated that (+)-TAK-700 does not undergo chiral inversion to its antipode in vivo.

Development and validation of a highly sensitive LC-ESI-MS/MS method for the determination of Orteronel® (TAK-700) in rat plasma: application to a pharmacokinetic study.

BACKGROUND: A highly sensitive, specific and high-throughput LC-ESI-MS/MS method in the positive mode has been developed and validated for the quantitation of Orteronel® (TAK-700) in rat plasma using YM-55208 as an internal standard. RESULTS: The assay procedure involves extraction of Orteronel and internal standard from rat plasma with liquid-liquid extraction method. Chromatographic separation was achieved using an isocratic mobile phase at a flow rate of 0.6 ml/min on an Atlantis dC18 column with a total run time of 2.5 min. The MS/MS ion transitions monitored were m/z 308.4→95.0 for Orteronel and m/z 262.3→194.2 for IS. Method validation was performed as per US FDA guidelines. The LLOQ achieved was 0.42 ng/ml and the linearity range extended from 0.42 to 814 ng/ml. CONCLUSION: The results met the acceptance criteria. The validated method was successfully applied to characterize the pharmacokinetic parameters of Orteronel in rat plasma.

Development and validation of a highly sensitive method for the determination of abiraterone in rat and human plasma by LC-MS/MS-ESI: application to a pharmacokinetic study.

A highly sensitive, rapid assay method has been developed and validated for the estimation of abiraterone (ART) in rat and human plasma with liquid chromatography coupled to tandem mass spectrometry and electrospray ionization in the positive-ion mode. The assay procedure involves extraction of ART and phenacetin (internal standard, IS) from rat and human plasma with a simple protein precipitation extraction process. Chromatographic separation was achieved using an isocratic mobile (10 mm ammonium acetate:acetonitrile, 10:90, v/v) at a flow-rate of 0.70 mL/min on an Atlantis dC(18) column maintained at 40 °C with a total run time of 3.5 min. The MS/MS ion transitions monitored were 350.3 → 156.0 for ART and 180.2 → 110.1 for IS. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.20 ng/mL and the linearity range extended from 0.20 to 201 ng/mL. The intra- and inter-day precisions were in the ranges 2.39-10.4 and 4.84-9.53% in rat plasma and 3.82-10.8 and 6.97-8.94% in human plasma.

A strategy for extending the applicability of a validated plasma calibration curve to quantitative measurements in multiple tissue homogenate samples: a case study from a rat tissue distribution study of JI-101, a triple kinase inhibitor.

A general practice in bioanalysis is that, whatever the biological matrix the analyte is being quantified in, the validation is performed in the same matrix as per regulatory guidelines. In this paper, we are presenting the applicability of a validated LC-MS/MS method in rat plasma for JI-101, to estimate the concentrations of JI-101 in various tissues that were harvested in a rat tissue distribution study. A simple protein precipitation technique was used to extract JI-101 and internal standard from the tissue homogenates. The recovery of JI-101 in all the matrices was found to be >70%. Chromatographic separation was achieved using a binary gradient using mobile phase A (acetonitrile) and B (0.2% formic acid in water) at a flow rate of 0.30 mL/min on a Prodigy ODS column with a total run time of 4.0 min. The MS/MS ion transitions monitored were 466.1 → 265 for JI-101 and 180.1 → 110.1 for internal standard. The linearity range was 5.02-4017 ng/mL. The JI-101 levels were quantifiable in the various tissue samples harvested in this study. Therefore, the use of a previously validated JI-101 assay in plasma circumvented the tedious process of method development/validation in various tissue matrices.

Development and validation of a highly sensitive LC-MS/MS method for simultaneous quantitation of ethionamide and ethionamide sulfoxide in human plasma: application to a human pharmacokinetic study.

A highly sensitive and specific LC-MS/MS method has been developed for simultaneous quantification of ethionamide and ethionamide sulfoxide in human plasma (300 µL) using prothionamide as an internal standard (IS). Solid-phase extraction was used to extract ethionamide, ethionamide sulfoxide and IS from human plasma. The chromatographic separation of ethionamide, ethionamide sulfoxide and IS was achieved with a mobile phase consisting of 0.1% acetic acid : acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a Peerless Basic C(18) column. The total run time was 3.5 min and the elution of ethionamide, ethionamide sulfoxide and IS occurred at 2.50, 2.18 and 2.68 min, respectively. A linear response function was established for the range of concentrations 25.7-6120 ng/mL (r > 0.998) for ethionamide and 50.5-3030 ng/mL (r > 0.998) for ethionamide sulfoxide. The intra- and inter-day precision values for ethionamide and ethionamide sulfoxide met the acceptance as per FDA guidelines. Ethionamide and ethionamide sulfoxide were stable in battery of stability studies, viz. bench-top, autosampler and freeze-thaw cycles. The developed assay was applied to a pharmacokinetic study in humans.

Highly sensitive method for the determination of a novel triple kinase inhibitor with anti-cancer activity, JI-101, in rat plasma by liquid chromatography-electrospray ionization tandem mass spectrometry: application to a pharmacokinetic study.

A highly sensitive, rapid assay method has been developed and validated for the estimation of JI-101 in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode. The assay procedure involves extraction of JI-101 and phenacetin (internal standard, IS) from rat plasma with a solid-phase extraction process. Chromatographic separation was achieved using a binary gradient using mobile phase A (acetonitrile) and B (0.2% formic acid in water) at a flow rate of 0.30 mL/min on a Prodigy ODS column with a total run time of 4.0 min. The MS/MS ion transitions monitored were 466.1 → 265 for JI-101 and 180.1 → 110.1 for IS. Method validation and sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 5.03 ng/mL and the linearity range extended from 5.03 to 2014 ng/mL. The intra-day and inter-day precisions were in the ranges of 1.17-19.6 and 3.09-10.4%, respectively. This method has been applied to a pharmacokinetic study of JI-101 in rats.