ABSTRACT
Lysine specific demethylase 1 (LSD1) and HDAC6 are epigenetic proteins associated with several diseases, including cancer and combined inhibition of these proteins could be highly beneficial in treating some cancers such as AML, MM and solid tumors. Multiple myeloma (MM) is a challenging cancer with fast relapse rate where novel treatment options are the need of the hour. We have designed and developed novel, LSD1 and HDAC6 selective dual inhibitors to target MM. Our dual inhibitor compound 1 shows superior potency in multiple MM cell lines. In MM.1S xenograft model compound 1 shows superior efficacy compared to single agent LSD1 and HDAC6 inhibitors by oral administration and is well tolerated. Further evaluation of the molecule in other cancers is in progress.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Histone Deacetylase 6/antagonists & inhibitors , Histone Demethylases/antagonists & inhibitors , Multiple Myeloma/drug therapy , Administration, Oral , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/chemistry , Histone Deacetylase 6/metabolism , Histone Demethylases/metabolism , Humans , Mice , Molecular Structure , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Structure-Activity RelationshipABSTRACT
AMG 510 is the first-in-class KRASG12C inhibitor, currently in phase 2 clinical trials as an orphan drug to treat non-small cell lung cancer patients. We developed and validated a sensitive, selective, and high-throughput HPLC-MS/MS method for the quantitation of AMG 510 in mouse plasma per the regulatory guideline of the US Food and Drug and Administration. AMG 510 and the IS (MRTX-1257) were extracted from mouse plasma using tert-butyl methyl ether and chromatographed using an isocratic mobile phase (0.2% formic acid:acetonitrile; 25:75, v/v) at a flow rate of 0.65 mL/min on an Atlantis dC18 column. AMG 510 and the IS eluted at ~0.95 and 0.73 min, respectively. AMG 510 and the IS were detected by positive electrospray ionization in multiple reaction monitoring using transition pair (Q1 â Q3) m/z 561.1 â 134.1 and m/z 566.5 â 98.2, respectively. Excellent linearity was achieved in the concentration range of 1.08-5040 ng/mL (r > 0.0996). No matrix effect and carryover were observed. Intra- and inter-day accuracies and precisions were within the acceptance range. AMG 510 was demonstrated to be stable under the tested storage conditions. This novel method has been applied to a pharmacokinetic study in mice.
Subject(s)
Chromatography, High Pressure Liquid/methods , Piperazines/blood , Piperazines/pharmacokinetics , Pyridines/blood , Pyridines/pharmacokinetics , Pyrimidines/blood , Pyrimidines/pharmacokinetics , Tandem Mass Spectrometry/methods , Animals , Linear Models , Male , Mice , Mice, Inbred BALB C , Piperazines/chemistry , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Pyridines/chemistry , Pyrimidines/chemistry , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Phosphatidylinositol 3-kinase (PI3K) inhibitors are a novel class of anticancer drugs that are approved to treat various malignancies. We report the development and validation of a HPLC method for the simultaneous quantitation of three PI3K inhibitors, namely copanlisib, duvelisib and idelalisib, in rat plasma as per the regulatory guidelines of the United States Food and Drug Administration. The method involves extraction of copanlisib, duvelisib and idelalisib along with an internal standard (IS; filgotinib) from rat plasma (100 µL) using a liquid-liquid extraction process. The chromatographic separation of the analytes was achieved using step-wise gradient elution on a Hypersil Gold C18 column. The UV detection wavelength was set at λmax = 280 nm. Copanlisib, duvelisib, idelalisib and the IS eluted at 7.16, 12.6, 11.9 and 9.86 min, respectively, with a total run time of 15 min. The calibration curve ranged from 50 to 5000 ng/mL for all the analytes. Inter- and intra-day precision and accuracy, stability studies, dilution integrity and incurred sample reanalysis were investigated for all three analytes, and the results met the acceptance criteria. The validated HPLC method was successfully applied to a pharmacokinetic study in rats.
Subject(s)
Chromatography, High Pressure Liquid/methods , Liquid-Liquid Extraction/methods , Phosphoinositide-3 Kinase Inhibitors/blood , Phosphoinositide-3 Kinase Inhibitors/pharmacokinetics , Animals , Antineoplastic Agents/blood , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Isoquinolines/blood , Isoquinolines/chemistry , Isoquinolines/pharmacokinetics , Linear Models , Male , Phosphoinositide-3 Kinase Inhibitors/chemistry , Purines/blood , Purines/chemistry , Purines/pharmacokinetics , Pyrimidines/blood , Pyrimidines/chemistry , Pyrimidines/pharmacokinetics , Quinazolines/blood , Quinazolines/chemistry , Quinazolines/pharmacokinetics , Quinazolinones/blood , Quinazolinones/chemistry , Quinazolinones/pharmacokinetics , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Levodropropizine (LDP) is a non-opioid anti-tussive. The stereoselective pharmacokinetics and tissue distribution (TD) of LDP vs. dextrodropropizine (DDP) have been characterized after oral and intravenous (IV) administration of LDP and rac-dropropozine in rats.Oral/IV doses of 50/5.0 mg/kg and 25/2.5 rac-dropropizine and LDP were employed. TD study focused on tissues such as liver, lung and kidney. Blood samples were collected for pharmacokinetic and TD evaluation. Validated methods were used to quantitate LDP, DDP and rac-dropropizine.No stereoselectivity in pharmacokinetics was observed between LDP vs. DDP following rac-dropropizine. However, LDP pharmacokinetics after LDP administration (oral/IV) appeared to be different compared to LDP derived from rac-dropropizine.TD data were similar between the two enantiomers regardless of oral/IV rac-dropropizine administration. When LDP alone was administered, levels were comparable to those derived for LDP from rac-dropropizine after oral/IV. However, in the lung and kidney tissues, the exposure after oral dosing was higher for LDP alone as compared to LDP from rac-dropropizine.In summary, complete characterization of stereoselective pharmacokinetics and TD of rac-dropropizine has been reported after oral/IV routes. It was evident that the presence of DDP, increased the plasma/tissue exposure of LDP which was evident after oral rac-dropropizine dosing.
Subject(s)
Antitussive Agents/pharmacokinetics , Propylene Glycols/pharmacokinetics , Administration, Intravenous , Administration, Oral , Animals , Male , Rats , Rats, Sprague-Dawley , StereoisomerismABSTRACT
Isocitrate dehydrogenase (IDH) inhibitors comprise a novel class of anticancer drugs, which are approved to treat acute myeloid leukemia patients having mutations on IDH1/2. We report the development and validation of a high-performance liquid chromatography (HPLC) method for the simultaneous quantitation of IDH inhibitors, namely enasidenib (EDB), ivosidenib (IDB) and vorasidenib (VDB), in mouse plasma as per the US Food and Drug Administration regulatory guidelines. The method involves extraction of EDB, IDB and VDB along with internal standard (IS; phenacetin) from mouse plasma (100 µl) using a simple protein precipitation process. The chromatographic analysis was performed on an HPLC system using a gradient mobile phase (comprising 10 mm ammonium acetate and acetonitrile in a flow-gradient) and an X-Terra Phenyl column. The UV detection wave length was set at λmax 265 nm. EDB, IDB, VDB and the IS eluted at 7.36, 8.60, 9.50 and 5.12 min, respectively, with a total run time of 10 min. The calibration curve was linear over a concentration range of 0.20-12.5 µg/ml for EDB and 0.50-12.5 µg/ml for IDB and VDB (r2 = ≥0.998 for all of the analytes). Validation results met the acceptance criteria. The validated HPLC method was successfully applied to a pharmacokinetic study in mice.
Subject(s)
Aminopyridines/blood , Antineoplastic Agents/blood , Chromatography, High Pressure Liquid/methods , Diamines/blood , Glycine/analogs & derivatives , Isocitrate Dehydrogenase/antagonists & inhibitors , Pyridines/blood , Triazines/blood , Aminopyridines/chemistry , Aminopyridines/pharmacokinetics , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Diamines/chemistry , Diamines/pharmacokinetics , Drug Stability , Glycine/blood , Glycine/chemistry , Glycine/pharmacokinetics , Limit of Detection , Linear Models , Male , Mice , Pyridines/chemistry , Pyridines/pharmacokinetics , Reproducibility of Results , Triazines/chemistry , Triazines/pharmacokineticsABSTRACT
A simple, sensitive and rapid assay method has been developed and validated for the estimation of apalutamide on mouse dried blood spots (DBS) using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode. The method utilizes liquid extraction of apalutamide from 3 mm punched disks from DBS cards (spiked or study samples). The extracted sample was chromatographed on an Atlantis dC18 column using gradient elution with 0.2% formic acid and acetonitrile at a flow rate of 1.00 mL/min. The total run time was 3.0 min. The MS/MS ion transitions monitored were m/z 478 â 450 for apalutamide and m/z 481 â 453 for the IS (apalutamide-d3 ). Method validation was performed as per regulatory guidelines. The assay was linear in the range of 0.95-2030 ng/mL. The intra- and inter-day precisions were in the ranges of 2.37-8.53 and 6.76-11.5%, respectively. Stability studies showed that apalutamide was stable on DBS cards for one month. This novel method has been applied to analyze the DBS samples of apalutamide obtained from a pharmacokinetic study in mice.
Subject(s)
Chromatography, Liquid/methods , Dried Blood Spot Testing/methods , Tandem Mass Spectrometry/methods , Thiohydantoins/blood , Thiohydantoins/pharmacokinetics , Animals , Linear Models , Male , Mice , Mice, Inbred BALB C , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
A sensitive, specific and rapid LC-ESI-MS/MS method has been developed and validated for the quantification of epacadostat in mouse plasma using tolbutamide as an internal standard (IS) as per regulatory guidelines. Sample preparation was accomplished through a protein precipitation. Chromatographic separation was performed on an Atlantis dC18 column using a binary gradient using mobile phase A (acetonitrile) and B (0.2% formic acid in water) at a flow rate of 0.90 mL/min. Elution of epacadostat and IS occurred at ~2.41 and 2.51 min, respectively. The total chromatographic run time was 3.2 min. A linear response function was established in the concentration range of 1.07-533 ng/mL. The intra- and inter-day accuracy and precision were in the ranges of 1.81-12.9 and 3.80-11.1%, respectively. This novel method has been applied to a pharmacokinetic study in mice.
Subject(s)
Chromatography, High Pressure Liquid/methods , Enzyme Inhibitors/blood , Indoleamine-Pyrrole 2,3,-Dioxygenase/antagonists & inhibitors , Oximes/blood , Sulfonamides/blood , Tandem Mass Spectrometry/methods , Animals , Limit of Detection , Male , Mice, Inbred BALB C , Spectrometry, Mass, Electrospray Ionization/methodsABSTRACT
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 â 93, 350 â 158, 434 â 274 and 265 â 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.
Subject(s)
Chromatography, Liquid/methods , Histone Deacetylase Inhibitors/blood , Tandem Mass Spectrometry/methods , Animals , Calibration , Histone Deacetylase Inhibitors/pharmacokinetics , Limit of Detection , Male , Mice , Mice, Inbred BALB C , Reference Standards , Reproducibility of ResultsABSTRACT
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.
Subject(s)
Chromatography, Liquid/methods , Hydroxamic Acids/blood , Pyrimidines/blood , Tandem Mass Spectrometry/methods , Animals , Limit of Detection , Male , Mice , Mice, Inbred BALB CABSTRACT
A highly sensitive, specific and rapid LC-ESI-MS/MS method has been developed and validated for simultaneous quantification of methotrexate (MTX) and tofacitinib (TFB) in rat plasma (50 µL) using phenacetin as an internal standard (IS), as per the US Food and Drug Administration guidelines. After a solid-phase extraction procedure, the separation of the analytes and IS was performed on a Chromolith RP18e column using an isocratic mobile phase of 5 m m ammonium acetate (pH 5.0) and acetonitrile at a ratio of 25:75 (v/v) using flow-gradient with a total run time of 3.5 min. The detection was performed in multiple reaction monitoring mode, using the transitions of m/z 455.2 â 308.3, m/z 313.2 â 149.2 and m/z 180.3 â 110.2 for MTX, TFB and IS, respectively. The calibration curves were linear over the range of 0.49-91.0 and 0.40-74.4 ng/mL for MTX and TFB, respectively. The intra- and interday accuracy and precision values for MTX and TFB were <15% at low quality control (QC), medium QC and high QC and <20% at lower limit of quantification. The validated assay was applied to derive the pharmacokinetic parameters for MTX and TFB post-dosing of MTX and TFB orally and intravenously to rats.
Subject(s)
Chromatography, High Pressure Liquid/methods , Methotrexate/blood , Piperidines/blood , Pyrimidines/blood , Pyrroles/blood , Tandem Mass Spectrometry/methods , Animals , Male , Methotrexate/pharmacokinetics , Piperidines/pharmacokinetics , Pyrimidines/pharmacokinetics , Pyrroles/pharmacokinetics , Rats , Rats, Sprague-DawleyABSTRACT
A novel, simple, specific, sensitive and reproducible high-performance liquid chromatography (HPLC) assay method has been developed and validated for the estimation of tofacitinib in rat plasma. The bioanalytical procedure involves extraction of tofacitinib and itraconazole (internal standard, IS) 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 1.0 mL/min and C18 column maintained at 40 ± 1 °C. The eluate was monitored using an UV detector set at 287 nm. Tofacitinib and IS eluted at 6.5 and 8.3 min, respectively and the total run time was 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 182-5035 ng/mL (r(2) = 0.995). The intra- and inter-day precisions were in the range of 1.41-11.2 and 3.66-8.81%, respectively, in rat plasma. The validated HPLC method was successfully applied to a pharmacokinetic study in rats.
Subject(s)
Chromatography, High Pressure Liquid/methods , Chromatography, Reverse-Phase/methods , Piperidines/blood , Pyrimidines/blood , Pyrroles/blood , Animals , Drug Stability , Male , Piperidines/pharmacokinetics , Protein Kinase Inhibitors/blood , Protein Kinase Inhibitors/pharmacokinetics , Pyrimidines/pharmacokinetics , Pyrroles/pharmacokinetics , Rats , Rats, Sprague-DawleyABSTRACT
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.
Subject(s)
Biphenyl Compounds/pharmacokinetics , Chromatography, High Pressure Liquid/methods , Chromatography, Reverse-Phase/methods , Animals , Area Under Curve , Biphenyl Compounds/blood , Half-Life , Humans , Rats , Reproducibility of Results , Spectrophotometry, UltravioletABSTRACT
Sarcopenia is associated with numerous adverse health outcomes, including frailty, disability, and mortality. Since the Asian Working Group for Sarcopenia 2019 guidelines, which were published in 2020, are relatively new, studies on the association between sarcopenia as defined by these guidelines and mortality are limited in the Asian region. Accordingly, this study aimed to examine the all-cause mortality risk associated with sarcopenia among community-dwelling older adults in rural Malaysia. This cohort study included 2404 older adults residing in Kuala Pilah District, Negeri Sembilan, Malaysia who were followed up for 83 months. The prevalence rates of sarcopenia and severe sarcopenia were 5.0% and 3.60%, respectively. Older adults with sarcopenia and severe sarcopenia had a 114% (hazard ratio [HR]: 2.14) and 146% (HR: 2.46) increased mortality risk compared with those without sarcopenia (HR: 2.14). Our findings indicate that early intervention is recommended to prevent sarcopenia in older adults.
Subject(s)
Frailty , Mortality , Sarcopenia , Aged , Humans , Cohort Studies , Frailty/epidemiology , Independent Living , Prevalence , Sarcopenia/epidemiologyABSTRACT
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 â 95.0 for TAK-700 and m/z 180.2 â 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.
Subject(s)
Chromatography, High Pressure Liquid/methods , Imidazoles/blood , Imidazoles/chemistry , Naphthalenes/blood , Naphthalenes/chemistry , Tandem Mass Spectrometry/methods , Animals , Imidazoles/pharmacokinetics , Linear Models , Male , Naphthalenes/pharmacokinetics , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Sensitivity and Specificity , StereoisomerismABSTRACT
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.
Subject(s)
Androstenols/blood , Chromatography, Reverse-Phase/methods , Androstenes , Androstenols/chemistry , Androstenols/pharmacokinetics , Animals , Chromatography, High Pressure Liquid/methods , Drug Stability , Linear Models , Liquid-Liquid Extraction , Male , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Sensitivity and Specificity , Spectrophotometry, UltravioletABSTRACT
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.
Subject(s)
Chromatography, High Pressure Liquid/methods , Imidazoles/blood , Naphthalenes/blood , Animals , Chromatography, Reverse-Phase , Drug Stability , Imidazoles/chemistry , Imidazoles/pharmacokinetics , Linear Models , Male , Naphthalenes/chemistry , Naphthalenes/pharmacokinetics , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Sensitivity and Specificity , Steroid 17-alpha-Hydroxylase/antagonists & inhibitorsABSTRACT
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 â 156.0 for ART and 180.2 â 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.
Subject(s)
Androstenols/blood , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Androstenes , Androstenols/chemistry , Androstenols/pharmacokinetics , Animals , Drug Stability , Humans , Male , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Sensitivity and Specificity , Spectrometry, Mass, Electrospray Ionization/methodsABSTRACT
Neutrophils are closely involved in the regulation of tumor progression and formation of premetastatic niches. However, the mechanisms of their involvement and therapeutic regulation of these processes remain elusive. Here, we report a critical role of neutrophil peptidylarginine deiminase 4 (PAD4) in neutrophil migration in cancer. In several transplantable and genetically engineered mouse models, tumor growth was accompanied by significantly elevated enzymatic activity of neutrophil PAD4. Targeted deletion of PAD4 in neutrophils markedly decreased the intratumoral abundance of neutrophils and led to delayed growth of primary tumors and dramatically reduced lung metastases. PAD4-mediated neutrophil accumulation by regulating the expression of the major chemokine receptor CXCR2. PAD4 expression and activity as well as CXCR2 expression were significantly upregulated in neutrophils from patients with lung and colon cancers compared with healthy donors, and PAD4 and CXCR2 expression were positively correlated in neutrophils from patients with cancer. In tumor-bearing mice, pharmacologic inhibition of PAD4 with the novel PAD4 isoform-selective small molecule inhibitor JBI-589 resulted in reduced CXCR2 expression and blocked neutrophil chemotaxis. In mouse tumor models, targeted deletion of PAD4 in neutrophils or pharmacologic inhibition of PAD4 with JBI-589 reduced both primary tumor growth and lung metastases and substantially enhanced the effect of immune checkpoint inhibitors. Taken together, these results suggest a therapeutic potential of targeting PAD4 in cancer. SIGNIFICANCE: PAD4 regulates tumor progression by promoting neutrophil migration and can be targeted with a small molecule inhibitor to suppress tumor growth and metastasis and increase efficacy of immune checkpoint blockade therapy.
Subject(s)
Extracellular Traps , Lung Neoplasms , Animals , Disease Models, Animal , Extracellular Traps/metabolism , Immune Checkpoint Inhibitors , Lung Neoplasms/pathology , Mice , Neutrophils , Protein-Arginine Deiminase Type 4 , Receptors, Chemokine/metabolismABSTRACT
BACKGROUND: Darolutamide is recently approved for the treatment of non-metastatic castrate resistance prostate cancer. Hitherto, no stereoselective pharmacokinetic data have been published pertaining to darolutamide and its diastereomers in animals or humans. The key aims of the experiment were to examine darolutamide, S,S-darolutamide and S,R-darolutamide with respect to (a) assessment of in vitro metabolic stability and protein binding and (b) characterization of in vivo oral and intravenous pharmacokinetics in mice. METHODS: In vitro (liver microsomes stability and protein binding) and in vivo experiments (oral/intravenous dosing to mice) were carried out using darolutamide, S,S-darolutamide and S,Rdarolutamide. Besides, tissue levels of darolutamide, S,S-darolutamide and S,R-darolutamide were measured following oral and intravenous dosing. Appropriate plasma/tissue samples served to determine the pharmacokinetics of various analytes in mice. Liquid chromatography in tandem with mass spectrometry procedures enabled the delineation of the plasma pharmacokinetics, in vitro and tissue uptake data of the various analytes. RESULTS: Chiral inversion was absent in the metabolic stability study. However, darolutamide showed profound stereoselectivity (S,S-darolutamide greater than S,R-darolutamide) after either intravenous or oral dosing. S,R-darolutamide but not S,S-darolutamide showed conversion to its antipode post oral and intravenous dosing to mice. Regardless of oral or intravenous dosing, active keto darolutamide formation was evident after administration of darolutamide, S,S-darolutamide or S,R- darolutamide. Tissue data supported the observations in plasma; however, tissue exposure of darolutamide, S,Sdarolutamide and S,R-darolutamide was much lower as compared to plasma. CONCLUSION: In lieu of the human pharmacokinetic data, although the administration of diastereomeric darolutamide was justified, it is proposed to delineate the clinical pharmacokinetics of S,Rdarolutamide and S,S-darolutamide relative to darolutamide in future clinical pharmacology studies.
Subject(s)
Prostatic Neoplasms , Pyrazoles , Administration, Oral , Animals , Chromatography, Liquid , Humans , Male , Mice , Microsomes, Liver , Prostatic Neoplasms/drug therapy , StereoisomerismABSTRACT
Copanlisib is a pan phosphatidylinositol 3-kinase (PI3K) inhibitor approved for follicular lymphoma. In this paper, we present the data of development and validation of a high-performance liquid chromatography (HPLC) method for the quantitation of copanlisib in mice plasma as per the FDA regulatory guideline. The method involves the extraction of copanlisib along with internal standard (IS, enasidenib) from mice plasma (100 µL) using ethyl acetate as an extraction solvent. The chromatographic resolution of copanlisib and the IS was achieved on a Hypersil Gold C18 column maintained at 40 °C using a binary gradient mobile phase [10 mM ammonium formate (pH 4.0) and acetonitrile]. The flow-rate was 0.8 mL/min. For the detection of copanlisib and the IS, the photo-diode array detector was set at λmax 310 nm. Copanlisib and the IS eluted at 6.60 and 7.80 min, respectively with a total run time of 10 min. The calibration curve was linear over a concentration range of 50 to 5000 ng/mL for copanlisib (r2≥ 0.998). The results of intra- and inter-day accuracy and precision studies were within the acceptable limits. Copanlisib was stable on bench-top, in auto-sampler, up to three freeze/thaw cycle and long-term storage at -80 °C. The application of the validated method was shown in a mice pharmacokinetic study.