Research article Received: 4 February 2015,
Revised: 3 March 2015,
Accepted: 13 March 2015
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bmc.3476
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 Anitha Police, Sandip Gurav, Vinay Dhiman, Mohd Zainuddin, Ravi kanth Bhamidipati, Sriram Rajagopal and Ramesh Mullangi* ABSTRACT: 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 (r2 = 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. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: odanacatib; HPLC; method validation; rat plasma; human plasma; pharmacokinetics
Introduction Osteoporosis is a disease that reduces bone density and strength and results in an increased risk of bone fractures. Antiosteoporotic treatment options include bisphosphonates, estrogens, selective estrogen receptor modulators, anti-receptor activator of nuclear factor-kB-ligand monoclonal antibody and parathyroid hormone. Cathepsin-K is an enzyme that is richly expressed in osteoclasts (principal bone resorbing cells) and is instrumental in bone matrix degradation during bone remodeling (Gauthier et al., 2008; Stoch and Wagner, 2008). Several studies have shown that cathepsin-K deficiency leads to an increase in bone mineral density (Saftig et al., 1998). Inhibition of cathepsin-K is a novel approach to the treatment of osteoporosis. Odanacatib (Fig. 1) is a novel, oral selective cathepsin-K inhibitor that selectively inhibits the cathepsin-K enzyme. In a phase II study done in post-menopausal women, odanacatib resulted in a dose-dependent increase in bone mineral density both at lumbar spine and total hip (Bone et al., 2010; Eisman et al., 2011). Currently it is being developed for the treatment of osteoporosis and is undergoing large-scale evaluation in phase III clinical trials to assess vertebral, hip and nonvertebral fracture risk reduction. The pharmacokinetics and metabolism of odanacatib were studied extensively in rats, dogs and monkeys following oral and intravenous administration of 14C-odanacatib (Kassahun et al., 2011). Odanacatib exhibited low clearance in rats and monkeys and extremely low clearance in dogs. Consistent with low systemic clearance, the plasma elimination half-life is long in dogs. Owing to its poor low aqueous solubility, odanacatib oral bioavailability is
Biomed. Chromatogr. 2015
highly dependent on the vehicle used. With suspension formulation the oral bioavailability in these three pre-clinical species was 10 mg as a result of solubilitylimited absorption (Stoch et al., 2013). Only one LC-MS/MS bioanalytical method was reported in the literature for quantification of odanacatib in human analysis to support plasma samples analysis in clinical trials (Sun et al., 2012). LC-MS/MS is an expensive technique that requires a high level of investment in both equipment purchase and maintenance and it may be not available in all analytical laboratories. To date there is
* Correspondence to: R. Mullangi, Drug Metabolism and Pharmacokinetics, Jubilant Biosys Ltd, Industrial Suburb, Yeshwanthpur, Bangalore-560 022, India. Email: [email protected] Drug Metabolism and Pharmacokinetics, Jubilant Biosys Ltd, Industrial Suburb, Yeshwanthpur, Bangalore-560 022, India
Copyright © 2015 John Wiley & Sons, Ltd.
A. Police et al. prepared by spiking 180 μL of blank rat plasma with the appropriate working solution of odanacatib (20 μL) on the day of analysis. Samples for the determination of precision and accuracy were prepared by spiking blank rat and human plasma in bulk with odanacatib at appropriate concentrations (51.1, 153, 1019 and 1698 ng/mL) and 200 μL aliquots were distributed into different tubes. All the samples were stored together at 80 ± 10°C until analysis.
Figure 1. Structural representation of odanacatib.
no HPLC method reported for quantification of odanacatib in any pre-clinical species and human plasma. Hence, we felt that there is a great need to develop and validate an HPLC method for quantification of odanacatib in plasma, which will be a valuable tool to support pre-clinical and clinical pharmacokinetic studies. The objective of the present study was to develop and validate a simple, specific, sensitive and reproducible HPLC method for quantitation of odanacatib in rat and human plasma. The method was successfully applied to quantitate levels of odanacatib in a rat pharmacokinetic study.
Liquid–liquid extraction method was followed for extraction of odanacatib from rat/human plasma. To an aliquot of 200 μL plasma sample, IS solution (20 μL of 2000 ng/mL) was added and mixed for 15 s on a cyclomixer (Remi Instruments, Mumbai, India). After the addition of 1.5 mL of ter-butyl methyl ether, the mixture was vortexed for 3 min, followed by centrifugation for 10 min at 14,000 rpm on a Centrifuge 5430R (Eppendorff, Germany) at 4°C. The organic layer (1.3 mL) was separated and evaporated to dryness at 50°C using a gentle stream of nitrogen (Turbovap®, Zymark®, Kopkinton, MA, USA). The residue was reconstituted in 200 μL of the 80% acetonitrile in Milli Q water and 25 μL was injected onto HPLC system.
Validation procedures A full validation according to the US Food and Drug Administration guidelines (US DHHS et al., 2001) was performed for the assay in rat and human plasma.
Experimental Chemicals and reagents Odanacatib (purity >98%) was procured from Shanghai Arbor Chemical Co Ltd, China. Itraconazole was purchased from Sigma-Aldrich (St Louis, MO, USA). HPLC-grade acetonitrile and methanol were purchased from Rankem, Ranbaxy Fine Chemicals Limited, New Delhi, India. Potassium dihydrogen orthophosphate and ethylenediaminetetra-acetic acid disodium salt . (Na2EDTA) were purchased from Qualigens, Mumbai, India. Blank rat . plasma (with Na2EDTA as an anti-coagulant) was procured from Jubilant . Biosys Animal House, Bangalore, India. Blank human plasma (with Na2EDTA as an anti-coagulant) was procured Red Cross Society, Bangalore, India. All other chemicals/reagents were of research grade and used without further purification.
HPLC operating conditions Odanacatib was assayed using a Waters 2695 Alliance HPLC system (Waters, Milford, MA, USA) equipped with performance PLUS inline degasser along with an autosampler and UV detector set at 268 nm for odanacatib and internal standard (IS). The chromatographic separation of odanacatib and IS in processed samples was achieved on a Symmetry Shield RP18 column (250 × 4.6 mm, 5 μm; Waters Corporation, Milford, MA, USA) maintained at 35 ± 1°C and using an isocratic mobile phase [10 mM KH2PO4 (pH 3.0)–acetonitrile (30:70, v/v)] at 0.7 mL/min flow-rate.
Preparation of stock and standard solutions Odanacatib and IS were accurately weighed into volumetric flasks using an analytical microbalance. They were then diluted with methanol. Odanacatib stock solutions for spiking the calibration standards and quality control (QC) samples were prepared from a separate weighing at 194 μg/mL. The IS stock solution of 200 μg/mL was prepared in methanol. The stock solutions of odanacatib and IS were stored at 4°C, and were found to be stable for 1 month (data not shown) and successively diluted with methanol–water (50:50, v/v) to prepare working solutions to prepare the calibration curve (CC). Another set of working stock solutions of odanacatib were made in methanol–water (50:50, v/v) (from different stock) for preparation of QC samples. Appropriate dilutions of odanacatib stock solution were made in methanol–water (50:50, v/v) to produce working stock solutions for CC and QC spiking. A working IS solution (2000 ng/mL) was prepared in methanol–water (50:50, v/v). Calibration samples were
wileyonlinelibrary.com/journal/bmc
Sample preparation
Specificity and selectivity. The specificity of the method was evaluated by analyzing rat and human plasma samples from 10 different lots (six normal lots, two hemolyzed lots and two lipemic lots) to investigate the potential interferences at the LC peak region for analyte and IS. These samples were processed using the proposed extraction protocol and analyzed with the set chromatographic conditions at the lower limit of quantitation (LLOQ) level. The peak area of the co-eluting components or interferences in blank samples should be
Revised: 3 March 2015,
Accepted: 13 March 2015
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bmc.3476
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 Anitha Police, Sandip Gurav, Vinay Dhiman, Mohd Zainuddin, Ravi kanth Bhamidipati, Sriram Rajagopal and Ramesh Mullangi* ABSTRACT: 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 (r2 = 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. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: odanacatib; HPLC; method validation; rat plasma; human plasma; pharmacokinetics
Introduction Osteoporosis is a disease that reduces bone density and strength and results in an increased risk of bone fractures. Antiosteoporotic treatment options include bisphosphonates, estrogens, selective estrogen receptor modulators, anti-receptor activator of nuclear factor-kB-ligand monoclonal antibody and parathyroid hormone. Cathepsin-K is an enzyme that is richly expressed in osteoclasts (principal bone resorbing cells) and is instrumental in bone matrix degradation during bone remodeling (Gauthier et al., 2008; Stoch and Wagner, 2008). Several studies have shown that cathepsin-K deficiency leads to an increase in bone mineral density (Saftig et al., 1998). Inhibition of cathepsin-K is a novel approach to the treatment of osteoporosis. Odanacatib (Fig. 1) is a novel, oral selective cathepsin-K inhibitor that selectively inhibits the cathepsin-K enzyme. In a phase II study done in post-menopausal women, odanacatib resulted in a dose-dependent increase in bone mineral density both at lumbar spine and total hip (Bone et al., 2010; Eisman et al., 2011). Currently it is being developed for the treatment of osteoporosis and is undergoing large-scale evaluation in phase III clinical trials to assess vertebral, hip and nonvertebral fracture risk reduction. The pharmacokinetics and metabolism of odanacatib were studied extensively in rats, dogs and monkeys following oral and intravenous administration of 14C-odanacatib (Kassahun et al., 2011). Odanacatib exhibited low clearance in rats and monkeys and extremely low clearance in dogs. Consistent with low systemic clearance, the plasma elimination half-life is long in dogs. Owing to its poor low aqueous solubility, odanacatib oral bioavailability is
Biomed. Chromatogr. 2015
highly dependent on the vehicle used. With suspension formulation the oral bioavailability in these three pre-clinical species was 10 mg as a result of solubilitylimited absorption (Stoch et al., 2013). Only one LC-MS/MS bioanalytical method was reported in the literature for quantification of odanacatib in human analysis to support plasma samples analysis in clinical trials (Sun et al., 2012). LC-MS/MS is an expensive technique that requires a high level of investment in both equipment purchase and maintenance and it may be not available in all analytical laboratories. To date there is
* Correspondence to: R. Mullangi, Drug Metabolism and Pharmacokinetics, Jubilant Biosys Ltd, Industrial Suburb, Yeshwanthpur, Bangalore-560 022, India. Email: [email protected] Drug Metabolism and Pharmacokinetics, Jubilant Biosys Ltd, Industrial Suburb, Yeshwanthpur, Bangalore-560 022, India
Copyright © 2015 John Wiley & Sons, Ltd.
A. Police et al. prepared by spiking 180 μL of blank rat plasma with the appropriate working solution of odanacatib (20 μL) on the day of analysis. Samples for the determination of precision and accuracy were prepared by spiking blank rat and human plasma in bulk with odanacatib at appropriate concentrations (51.1, 153, 1019 and 1698 ng/mL) and 200 μL aliquots were distributed into different tubes. All the samples were stored together at 80 ± 10°C until analysis.
Figure 1. Structural representation of odanacatib.
no HPLC method reported for quantification of odanacatib in any pre-clinical species and human plasma. Hence, we felt that there is a great need to develop and validate an HPLC method for quantification of odanacatib in plasma, which will be a valuable tool to support pre-clinical and clinical pharmacokinetic studies. The objective of the present study was to develop and validate a simple, specific, sensitive and reproducible HPLC method for quantitation of odanacatib in rat and human plasma. The method was successfully applied to quantitate levels of odanacatib in a rat pharmacokinetic study.
Liquid–liquid extraction method was followed for extraction of odanacatib from rat/human plasma. To an aliquot of 200 μL plasma sample, IS solution (20 μL of 2000 ng/mL) was added and mixed for 15 s on a cyclomixer (Remi Instruments, Mumbai, India). After the addition of 1.5 mL of ter-butyl methyl ether, the mixture was vortexed for 3 min, followed by centrifugation for 10 min at 14,000 rpm on a Centrifuge 5430R (Eppendorff, Germany) at 4°C. The organic layer (1.3 mL) was separated and evaporated to dryness at 50°C using a gentle stream of nitrogen (Turbovap®, Zymark®, Kopkinton, MA, USA). The residue was reconstituted in 200 μL of the 80% acetonitrile in Milli Q water and 25 μL was injected onto HPLC system.
Validation procedures A full validation according to the US Food and Drug Administration guidelines (US DHHS et al., 2001) was performed for the assay in rat and human plasma.
Experimental Chemicals and reagents Odanacatib (purity >98%) was procured from Shanghai Arbor Chemical Co Ltd, China. Itraconazole was purchased from Sigma-Aldrich (St Louis, MO, USA). HPLC-grade acetonitrile and methanol were purchased from Rankem, Ranbaxy Fine Chemicals Limited, New Delhi, India. Potassium dihydrogen orthophosphate and ethylenediaminetetra-acetic acid disodium salt . (Na2EDTA) were purchased from Qualigens, Mumbai, India. Blank rat . plasma (with Na2EDTA as an anti-coagulant) was procured from Jubilant . Biosys Animal House, Bangalore, India. Blank human plasma (with Na2EDTA as an anti-coagulant) was procured Red Cross Society, Bangalore, India. All other chemicals/reagents were of research grade and used without further purification.
HPLC operating conditions Odanacatib was assayed using a Waters 2695 Alliance HPLC system (Waters, Milford, MA, USA) equipped with performance PLUS inline degasser along with an autosampler and UV detector set at 268 nm for odanacatib and internal standard (IS). The chromatographic separation of odanacatib and IS in processed samples was achieved on a Symmetry Shield RP18 column (250 × 4.6 mm, 5 μm; Waters Corporation, Milford, MA, USA) maintained at 35 ± 1°C and using an isocratic mobile phase [10 mM KH2PO4 (pH 3.0)–acetonitrile (30:70, v/v)] at 0.7 mL/min flow-rate.
Preparation of stock and standard solutions Odanacatib and IS were accurately weighed into volumetric flasks using an analytical microbalance. They were then diluted with methanol. Odanacatib stock solutions for spiking the calibration standards and quality control (QC) samples were prepared from a separate weighing at 194 μg/mL. The IS stock solution of 200 μg/mL was prepared in methanol. The stock solutions of odanacatib and IS were stored at 4°C, and were found to be stable for 1 month (data not shown) and successively diluted with methanol–water (50:50, v/v) to prepare working solutions to prepare the calibration curve (CC). Another set of working stock solutions of odanacatib were made in methanol–water (50:50, v/v) (from different stock) for preparation of QC samples. Appropriate dilutions of odanacatib stock solution were made in methanol–water (50:50, v/v) to produce working stock solutions for CC and QC spiking. A working IS solution (2000 ng/mL) was prepared in methanol–water (50:50, v/v). Calibration samples were
wileyonlinelibrary.com/journal/bmc
Sample preparation
Specificity and selectivity. The specificity of the method was evaluated by analyzing rat and human plasma samples from 10 different lots (six normal lots, two hemolyzed lots and two lipemic lots) to investigate the potential interferences at the LC peak region for analyte and IS. These samples were processed using the proposed extraction protocol and analyzed with the set chromatographic conditions at the lower limit of quantitation (LLOQ) level. The peak area of the co-eluting components or interferences in blank samples should be
