ABSTRACT
Patupilone is a microtubule-targeted cytotoxic agent with clinical efficacy, but causes diarrhoea in more than 80% of patients. The efficacy and tolerability of patupilone delivered continuously by subcutaneous (s.c.) mini-pumps [(mini-pump dose (MPD)] or by intravenous bolus administration [intravenous bolus dose (IVBD)] were compared preclinically to determine whether the therapeutic index could be improved. The antiproliferative potency in vitro of patupilone was determined by measuring total cell protein. Tumours were grown s.c. in rats (A15) or nude mice (KB31, KB8511) or intracranially in nude mice (NCI-H460-Luc). Efficacy was monitored by measuring tumour volumes, bioluminescence or survival. Toxicity was monitored by body weight and/or diarrhoea. Total drug levels in blood, plasma, tissues or dialysates were quantified ex-vivo by liquid chromatography-mass spectroscopy/mass spectroscopy. Patupilone was potent in vitro with GI50s of 0.24-0.28 nmol/l and GI90s of 0.46-1.64 nmol/l. In rats, a single IVBD of patupilone dose dependently inhibited the growth of A15 tumours, but also caused dose-dependent body weight loss and diarrhoea, whereas MPD achieved similar efficacy, but no toxicity. In mice, MPD showed efficacy similar to that of IVBD against KB31 and KB8511 tumours, but with reduced toxicity. In a mouse intracranial tumour model, IVBD was more efficacious than MPD, consistent with patupilone concentrations in the brain. MPD provided constant plasma levels, whereas IVBD had very high C0/Cmin ratios of 70-280 (rat) or 8000 (mouse) over the dosing cycle. Overall, the correlation of plasma and tumour levels with response indicated that a Cave of at least GI90 led to tumour stasis. Continuous low concentrations of patupilone by MPD increased the therapeutic index in s.c. rodent tumour models compared with IVBD by maintaining efficacy, but reducing toxicity.
Subject(s)
Antineoplastic Agents/administration & dosage , Brain Neoplasms/drug therapy , Epothilones/administration & dosage , Infusion Pumps , Neoplasms, Experimental/drug therapy , Animals , Antineoplastic Agents/blood , Antineoplastic Agents/therapeutic use , Brain Neoplasms/blood , Brain Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Epothilones/blood , Epothilones/therapeutic use , Female , Humans , Infusions, Subcutaneous , Injections, Intravenous , Mice , Mice, Nude , Microdialysis , Neoplasms, Experimental/blood , Neoplasms, Experimental/pathology , Rats , Species Specificity , Xenograft Model Antitumor AssaysABSTRACT
Epothilones are relatively new tubulin-poison anticancer drugs. Iso-fludelone (KOS-1803) is a synthetic third generation epothilone drug discovered at Memorial Sloan Kettering Cancer Center, and currently in phase I clinical trials. We report an LC-MS/MS assay for the sensitive, accurate and precise quantitation of iso-fludelone in 0.2mL of human plasma. Validation was performed according to FDA guidance. The assay comprised of KOS-1724 as the internal standard and an MTBE liquid-liquid extraction with a water wash step. Separation was achieved with an YMC-Pack ODS-AQ column and an isocratic mobile phase of 0.1% formic acid in acetonitrile and water (70:30, v/v) at 0.3mL/min for 4min. Chromatographic separation was followed by electrospray, positive-mode ionization tandem mass spectrometric detection in the multiple reaction monitoring (MRM) mode. The assay was linear from 0.1 to 300ng/mL and was accurate (-9.41 to -7.07%) and precise (1.03-13.7%) which fulfilled FDA criteria for validation. Recovery from plasma was 73.9-79.7% and ion suppression was negligible (-22.8 to -31.3%). Plasma freeze-thaw stability (99.97-105.7%), stability for 11 months at -80°C (94.93-107.9%), and stability for 6h at room temperature (94.75-105.5%) were all acceptable. This assay is currently being applied to quantitate iso-fludelone in clinical samples.
Subject(s)
Antineoplastic Agents/blood , Chromatography, Liquid , Epothilones/blood , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry , Tubulin Modulators/blood , Calibration , Chromatography, Liquid/standards , Cold Temperature , Drug Stability , Humans , Liquid-Liquid Extraction , Reference Standards , Reproducibility of Results , Spectrometry, Mass, Electrospray Ionization/standards , Tandem Mass Spectrometry/standards , Time FactorsABSTRACT
A sensitive, accurate and rugged UHPLC-MS/MS method was developed and validated for the quantitation of Epothilone D (EpoD), a microtubule stabilizer in development for treatment of Alzeimer's disease, in rat plasma. The ester group in EpoD can be hydrolyzed by esterases in blood or plasma, which creates a stability concern for the bioanalysis of EpoD. Species differences in the stability of EpoD in plasma were observed. Carboxylesterases were identified as the likely esterases responsible for the hydrolysis of EpoD in plasma ex vivo, and the cause of the species different stability. Phenylmethanesulfonyl fluoride, a carboxylesterase inhibitor, was used to stabilize EpoD in rat blood during sample collection, processing, and storage. A systematic method screening and optimization strategy was used to improve the assay sensitivity and minimize potential bioanalytical risks. The stabilized plasma samples were extracted by liquid-liquid extraction. Chromatographic separation was achieved on an Acquity UPLC BEH Phenyl column with a gradient elution. EpoD and its stable isotope labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 0.100 to 100ng/mL was fitted to a 1/x(2) weighted linear regression model. The intra-assay precision was within ±3.6% CV and inter-assay precision was within ±4.2% CV. The assay accuracy was within ±8.3% of the nominal values. Assay recovery of EpoD was high (â¼90%) and matrix effect was minimal (1.02-1.05). EpoD was stable in stabilized rat plasma for at least 30h at room temperature, 180 days at -20°C, and following three freeze-thaw cycles. The validated method was successfully applied to sample analysis in toxicology studies.
Subject(s)
Chromatography, High Pressure Liquid/methods , Epothilones/blood , Epothilones/chemistry , Phenylmethylsulfonyl Fluoride/chemistry , Tandem Mass Spectrometry/methods , Animals , Carboxylic Ester Hydrolases/antagonists & inhibitors , Carboxylic Ester Hydrolases/drug effects , Drug Stability , Epothilones/pharmacokinetics , Female , Linear Models , Phenylmethylsulfonyl Fluoride/pharmacology , Rats , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
A phase 1, open-label, non-randomized, single center study was conducted to determine the pharmacokinetics, distribution, metabolism, elimination, and mass balance of patupilone in patients with advanced solid tumors. Five patients with advanced solid tumors received 10 mg/m(2) (1.1 MBq) of (14) C-radiolabeled patupilone at cycle 1 as a 20-minute intravenous infusion every 3 weeks until disease progression. Sequential samples of blood/plasma were taken for 3 weeks and urine and fecal samples were collected for seven days after the first dose of patupilone. Patupilone blood levels decreased rapidly after the infusion. The compound showed a large volume of distribution (Vss: 2242 L). The main radiolabeled component in blood was patupilone itself, accompanied by the lactone hydrolysis products that are unlikely to contribute to the pharmacological effect of patupilone. The blood clearance of patupilone was relatively low at 14 L/h. The administered radioactivity dose was excreted slowly (46 % of dose up to 168 h) but ultimately accounted for 91 % of the dose by extrapolation. The fecal excretion of radioactivity was 2-3 times higher than the urinary excretion consistent with hepato-biliary elimination. Three patients had progressive disease and two patients had stable disease as their best response. Patupilone was generally well tolerated in patients with advanced solid tumors with no newly occurring safety events compared to previous clinical studies. In adult solid tumor patients, intravenous radiolabeled patupilone undergoes extensive metabolism with fecal excretion of radioactive metabolites predominating over renal excretion.
Subject(s)
Antineoplastic Agents/pharmacokinetics , Epothilones/pharmacokinetics , Neoplasms/metabolism , Tubulin Modulators/pharmacokinetics , Antineoplastic Agents/blood , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/urine , Epothilones/blood , Epothilones/therapeutic use , Epothilones/urine , Feces/chemistry , Female , Humans , Male , Middle Aged , Neoplasms/drug therapy , Tubulin Modulators/blood , Tubulin Modulators/therapeutic use , Tubulin Modulators/urineABSTRACT
A simple LC-MS/MS method was developed for determination and pharmacokinetic study of Epothilone B in rat plasma.Plasma sample pretreatment involved a one-step liquid-liquid extraction of 100 µL plasma. The chromatographic separation was carried out on a Agilent Zobax SB C18 column with a mobile phase consisting of 10 mmol/L ammonium acetate-acetonitrile (35:65, v/v) at a flow rate of 0.2 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by SRM via electro spray ionization source with positive mode.The standard curve for Epothilone B was linear over the concentration range of 1-100 ng/mL with a lower limit of quantification of 0.5 ng/mL. The intra- and inter-day precision (relative standard deviation) values were not higher than 15% and the accuracy (relative error) was < 10% at 3 quality control levels. Pharmacokinetic parameters were as follows: t1/2, 3.56 (1.12) h; AUC0-24 h, 295.7 (65.3) ng · h/mL and AUC0-∞, 339.2 (87.4) ng · h/mL, CL, 5.77 (0.67) mL/h; MRT, 7.55 (2.41) h, respectively.This simple, fast and highly sensitive method was fully validated and successfully applied to a preclinical pharmacokinetic study of Epothilone B in rats after i. v. administration.
Subject(s)
Antineoplastic Agents/blood , Antineoplastic Agents/pharmacokinetics , Epothilones/blood , Epothilones/pharmacokinetics , Animals , Chromatography, High Pressure Liquid , Female , Indicators and Reagents , Liquid-Liquid Extraction , Male , Quality Control , Rats , Rats, Sprague-Dawley , Reference Standards , Reproducibility of Results , Spectrometry, Mass, Electrospray Ionization , Tandem Mass SpectrometryABSTRACT
BACKGROUND: Patients with glioblastoma (GBM) inevitably develop recurrent or progressive disease after initial multimodal treatment and have a median survival of 6-9 months from time of progression. To date, there is no accepted standard treatment for GBM relapse or progression. Patupilone (EPO906) is a novel natural microtubule-stabilizing cytotoxic agent that crosses the blood-brain barrier and has been found to have preclinical activity in glioma models. METHODS: This is a single-institution, early-phase I/II trial of GBM patients with tumor progression who qualified for second surgery with the goal of evaluating efficacy and safety of the single-agent patupilone (10 mg/m(2), every 3 weeks). Patients received patupilone 1 week prior to second surgery and every 3 weeks thereafter until tumor progression or toxicity. Primary end points were progression-free survival (PFS) and overall survival (OS) at 6 months as well as patupilone concentration in tumor tissue. Secondary end points were toxicity, patupilone concentration in plasma and translational analyses for predictive biomarkers. RESULTS: Nine patients with a mean age of 54.6 ± 8.6 years were recruited between June 2008 and April 2010. Median survival and 1-year OS after second surgery were 11 months (95% CI, 5-17 months) and 45% (95% CI, 14-76), respectively. Median PFS was 1.5 months (95% CI, 1.3-1.7 months) and PFS6 was 22% (95% CI, 0-46), with 2 patients remaining recurrence-free at 9.75 and 22 months. At the time of surgery, the concentration of patupilone in tumor tissue was 30 times higher than in the plasma. Tumor response was not predictable by the tested biomarkers. Treatment was generally well tolerated with no hematological, but cumulative, though reversible sensory neuropathy grade ≤3 was seen in 2 patients (22%) at 8 months and grade 4 diarrhea in the 2nd patient (11%). Non-patupilone-related peri-operative complications occurred in 2 patients resulting in discontinuation of patupilone therapy. There were no neurocognitive changes 3 months after surgery compared to baseline. CONCLUSIONS: In recurrent GBM, patupilone can be given safely pre- and postoperatively. The drug accumulates in the tumor tissue. The treatment results in long-term PFS in some patients. Patupilone represents a valuable novel compound which deserves further evaluation in combination with radiation therapy in patients with GBM.
Subject(s)
Antineoplastic Agents/therapeutic use , Central Nervous System Neoplasms/drug therapy , Epothilones/therapeutic use , Glioblastoma/drug therapy , Aged , Antineoplastic Agents/adverse effects , Antineoplastic Agents/blood , Apoptosis/drug effects , Brain Neoplasms/drug therapy , Brain Neoplasms/mortality , Brain Neoplasms/pathology , Brain Neoplasms/surgery , Central Nervous System Neoplasms/mortality , Central Nervous System Neoplasms/pathology , Central Nervous System Neoplasms/surgery , Combined Modality Therapy , Epothilones/adverse effects , Epothilones/blood , Glioblastoma/mortality , Glioblastoma/pathology , Glioblastoma/surgery , Humans , Ki-67 Antigen/analysis , Middle Aged , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/mortality , Treatment Outcome , Tubulin/analysisABSTRACT
BACKGROUND: Ixabepilone, which stabilizes microtubules, has low susceptibility to drug resistance mediated by P-glycoprotein or ßIII-tubulin. MATERIALS AND METHODS: This study was designed to determine the maximum tolerated dose (MTD) of oral ixabepilone when administered every 6 h for three doses, every 3 weeks, to patients with refractory advanced cancers. Eighteen patients were treated with escalating doses of ixabepilone: three at cohort 1 (30 mg/dose; 90 mg on Day 1), nine at cohort 2 (40 mg/dose; 120 mg on Day 1), and six at cohort 3 (50 mg/dose; 150 mg on Day 1). Serial plasma samples were collected during cycle 1 for pharmacokinetic (PK) measurements. RESULTS: Of the 18 treated patients, eight were male and ten were female. The median age was 59 years, and most had an excellent performance status (KPS 90-100; 61%). There were two dose limiting toxicities (DLT): Grade 4 febrile neutropenia at the 120 mg dose and Grade 4 neutropenic sepsis at the 150 mg dose. Because of the severity and duration of neutropenic sepsis at level 3, level 2 (120 mg) was defined as the MTD and this cohort was expanded to nine patients. High inter-individual variability in plasma drug concentrations was observed during the study, with particularly high levels in two patients with DLT. CONCLUSIONS: On the basis of this safety profile, the MTD of oral ixabepilone was defined as 120 mg given as three 40 mg doses each separated by 6 h on Day 1 of a 3-week cycle. However, the PK variability observed makes further development of this oral formulation unlikely.
Subject(s)
Epothilones/administration & dosage , Neoplasms/drug therapy , Tubulin Modulators/administration & dosage , Administration, Oral , Adult , Aged , Drug Administration Schedule , Epothilones/blood , Epothilones/pharmacokinetics , Female , Humans , Male , Maximum Tolerated Dose , Middle Aged , Neoplasms/metabolism , Treatment Outcome , Tubulin Modulators/blood , Tubulin Modulators/pharmacokineticsABSTRACT
PURPOSE: Sagopilone has recently been identified and preferentially used for the treatment of taxane-resistant cancer. The purpose of this dose-escalation study was to investigate the safety, tolerability, and pharmacokinetics (PK) of sagopilone in refractory solid tumors. METHODS: A total of 17 Japanese patients received sagopilone in this Phase I study. Sagopilone was given as a 30-min intravenous infusion once every 3 weeks (one course) with an initial dose of 12.4 mg/m(2) up to 22.0 mg/m(2) for a maximum of 6 courses. RESULTS: The maximum tolerated dose (MTD) was determined to be 16.5 mg/m(2). The major dose-limiting toxicity (DLT) was peripheral sensory neuropathy. The PK data demonstrated that sagopilone did not accumulate after repeated administration. Two patients had stable disease (SD) over a period of 12 weeks. CONCLUSIONS: Our study demonstrated clinically favorable safety, tolerability, and efficacy of sagopilone, which will help define the treatment of advanced tumors in more extensive clinical trials.
Subject(s)
Antineoplastic Agents/pharmacokinetics , Benzothiazoles/pharmacokinetics , Epothilones/pharmacokinetics , Neoplasms/metabolism , Adult , Aged , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/blood , Area Under Curve , Asian People , Benzothiazoles/administration & dosage , Benzothiazoles/blood , Drug Administration Schedule , Epothilones/administration & dosage , Epothilones/blood , Female , Humans , Male , Maximum Tolerated Dose , Middle Aged , Neoplasms/drug therapy , Treatment OutcomeABSTRACT
PURPOSE: To determine the maximum tolerated dose and safety of the epothilone, KOS-862, in patients with advanced solid tumors or lymphoma. PATIENTS AND METHODS: Patients were treated weekly for 3 out of 4 weeks (Schedule A) or 2 out of 3 weeks (Schedule B) with KOS-862 (16-120 mg/m(2)). Pharmacokinetic (PK) sampling was performed during cycles 1 and 2; pharmacodynamic (PD) assessment for microtubule bundle formation (MTBF) was performed after the 1st dose, only at or above 100 mg/m(2). RESULTS: Thirty-two patients were enrolled, and twenty-nine completed ≥1 cycle of therapy. Dose limiting toxicity [DLT] was observed at 120 mg/m(2). PK data were linear from 16 to 100 mg/m(2), with proportional increases in mean C(max) and AUC(tot) as a function of dose. Full PK analysis (mean ± SD) at 100 mg/m(2) revealed the following: half-life (t (½)) = 9.1 ± 2.2 h; volume of distribution (V(z)) = 119 ± 41 L/m(2); clearance (CL) = 9.3 ± 3.2 L/h/m(2). MTBF (n = 9) was seen in 40% of PBMCs within 1 h and in 15% of PBMC at 24-hours post infusion at 100 mg/m(2). Tumor shrinkage (n = 2, lymphoma), stable disease >3 months (n = 5, renal, prostate, oropharynx, cholangiocarcinoma, and Hodgkin lymphoma), and tumor marker reductions (n = 1, colorectal cancer/CEA) were observed. CONCLUSION: KOS-862 was well tolerated with manageable toxicity, favorable PK profile, and the suggestion of clinical activity. The maximum tolerated dose was determined to be 100 mg/m(2) weekly 3-on/1-off. MTBF can be demonstrated in PBMCs of patients exposed to KOS-862.
Subject(s)
Epothilones/administration & dosage , Tubulin Modulators/administration & dosage , Adult , Aged , Epothilones/blood , Epothilones/pharmacokinetics , Female , Humans , Leukocytes, Mononuclear/metabolism , Lymphoma/metabolism , Male , Microtubules/metabolism , Middle Aged , Neoplasms/metabolism , Tubulin Modulators/blood , Tubulin Modulators/pharmacokineticsABSTRACT
BACKGROUND: Sagopilone (ZK 219477), a lipophylic and synthetic analog of epothilone B, that crosses the blood-brain barrier has demonstrated preclinical activity in glioma models. PATIENTS AND METHODS: Patients with first recurrence/progression of glioblastoma were eligible for this early phase II and pharmacokinetic study exploring single-agent sagopilone (16 mg/m(2) over 3 h every 21 days). Primary end point was a composite of either tumor response or being alive and progression free at 6 months. Overall survival, toxicity and safety and pharmacokinetics were secondary end points. RESULTS: Thirty-eight (evaluable 37) patients were included. Treatment was well tolerated, and neuropathy occurred in 46% patients [mild (grade 1) : 32%]. No objective responses were seen. The progression-free survival (PFS) rate at 6 months was 6.7% [95% confidence interval (CI) 1.3-18.7], the median PFS was just over 6 weeks, and the median overall survival was 7.6 months (95% CI 5.3-12.3), with a 1-year survival rate of 31.6% (95% CI 17.7-46.4). Maximum plasma concentrations were reached at the end of the 3-h infusion, with rapid declines within 30 min after termination. CONCLUSIONS: No evidence of relevant clinical antitumor activity against recurrent glioblastoma could be detected. Sagopilone was well tolerated, and moderate-to-severe peripheral neuropathy was observed in despite prolonged administration.
Subject(s)
Antineoplastic Agents/therapeutic use , Benzothiazoles/therapeutic use , Brain Neoplasms/drug therapy , Epothilones/therapeutic use , Glioblastoma/drug therapy , Neoplasm Recurrence, Local/drug therapy , Adult , Aged , Antineoplastic Agents/adverse effects , Antineoplastic Agents/blood , Antineoplastic Agents/pharmacokinetics , Astrocytoma/blood , Astrocytoma/drug therapy , Benzothiazoles/adverse effects , Benzothiazoles/blood , Benzothiazoles/pharmacokinetics , Brain Neoplasms/blood , Disease Progression , Disease-Free Survival , Epothilones/adverse effects , Epothilones/blood , Epothilones/pharmacokinetics , Female , Glioblastoma/blood , Humans , Infusions, Intravenous , Male , Middle Aged , Neoplasm Recurrence, Local/blood , Young AdultABSTRACT
Patupilone is a novel microtubule-targeting cytotoxic agent, which exerts its antitumor effect through microtubule stabilization. Pharmacokinetics, pharmacodynamics, and safety of warfarin when administered concomitantly with patupilone were investigated, and antitumor activity was assessed. This was a phase I, two-center, drug-drug interaction study. In the core phase of the study, treatment consisted of warfarin 20 mg orally (days 1 and 29) and patupilone 10 mg/m(2) i.v. (days 8 and 29). Patients benefiting from patupilone treatment continued treatment every 3 weeks (extension phase) until progression of disease, death, or unacceptable toxicity. Seventeen patients were treated (core phase, 17; extension, 9). The geometric mean ratios (comedication/monotherapy) for C(max) and area under the curve(0-168) of warfarin were near unity and their 90% confidence intervals were within the equivalence limits of 0.80 and 1.25. The half-life, plasma clearance, and International Normalized Ratio (INR) of warfarin were not affected by patupilone coadministration. The most common adverse events were diarrhea, nausea, vomiting, abdominal pain, anorexia, dehydration, asthenia, and peripheral neuropathy. Five (29.4%) patients experienced grade 3 study drug-related adverse events (diarrhea, 17.6%; increased INR, 11.8%; dehydration, 5.9%; and neutropenia, 5.9%). One patient with triple-negative breast cancer (estrogen receptor, progesterone receptor, and HER2/neu negative) had a partial response (35% decrease in tumor measurements by Response Evaluation Criteria in Solid Tumors), and 11 had stable disease for 6 weeks or more (≥12 weeks, 6 patients). The pharmacokinetics and pharmacodynamics of warfarin were not affected by patupilone coadministration, suggesting that patupilone has no clinically relevant effect on CYP2C9 metabolism. Patupilone showed antitumor activity in triple-negative breast cancer.
Subject(s)
Antineoplastic Agents/administration & dosage , Epothilones/administration & dosage , Neoplasms/drug therapy , Neoplasms/metabolism , Warfarin/pharmacokinetics , Adult , Aged , Antineoplastic Agents/adverse effects , Dose-Response Relationship, Drug , Drug Interactions , Epothilones/adverse effects , Epothilones/blood , Female , Humans , Male , Middle Aged , Neoplasms/blood , Warfarin/administration & dosage , Warfarin/adverse effects , Warfarin/blood , Young AdultABSTRACT
A sensitive method was developed and validated for the measurement of ixabepilone (BMS-247550, Ixempra) using a demethylated analogue of ixabepilone (BMS-212188) as an internal standard. A 0.050 mL portion of each plasma sample was extracted with 0.450 mL of acetonitrile containing the internal standard via protein precipitation. The supernatant was analyzed on a LC-MS/MS system. Chromatography was carried out on a 2.0 mm x 100 mm YMC ODS-AQ 3 microm column using an isocractic mobile phase consisting of acetonitrile:10 mM ammonium acetate, pH 5.0 (70:30, v/v) at a flow rate of 0.30 mL/min. The mass spectrometer was fitted with a TurboIonSpray source and operated in negative ionization mode. Detection of ixabepilone and BMS-212188 were accomplished using multiple reaction monitoring (MRM) of precursor>product ion pairs of m/z 505.2>405.2, and 492.1>392.1, respectively. The assay range was 2.00-500 ng/mL and was fitted to a 1/x(2) weighted quadratic regression model. Replicate sample analysis indicated that intra- and inter-day accuracy and precision are within +/-15.0%. The recovery of ixabepilone from 0.050 mL of plasma containing 5.00 and 400 ng/mL was greater than 94%. The method was demonstrated to be sensitive, selective and robust, and was successfully used to support clinical studies. This paper also discussed approaches used for resolving a curve splitting issue observed during quantitative analysis of ixabepilone in biological matrices. Finally, to adapt the methodology to pharmacokinetics of ixabepilone after oral administration, the potential interference of chemical degradants on the determination of ixabepilone was evaluated.
Subject(s)
Chromatography, Liquid/methods , Epothilones/blood , Tandem Mass Spectrometry/methods , Tubulin Modulators/blood , Epothilones/administration & dosage , Humans , Sensitivity and Specificity , Tubulin Modulators/administration & dosageABSTRACT
A sensitive, simple and rapid high performance liquid chromatographic-tandem mass spectrometric (HPLC-MS/MS) method was developed for simultaneous quantitation of epothilone D and its major metabolite, the hydrolytic metabolite, epothilone C as internal standard in human plasma. Plasma samples were precipitated with acetonitrile, the analysis used a Venusil ASB C(18) analytical column. A tandem mass spectrometer equipped with electrospray ionization source was used as detector and operated in the positive-ion mode. Selected reaction monitoring (SRM) using the precursor to product ion pairs of m/z 492.3-->304.1 (epothilone D), m/z 510.3-->492.3 (metabolite), m/z 478.3-->290.1 (internal standard) was used for quantification. The analytical method was validated in terms of specificity, precision, accuracy, extraction recovery, stability, matrix effect and dilution effect. The linear calibration curves of epothilone D and metabolite were obtained over the concentration range of 0.2-1000 ng/ml and 5.0-1000 ng/ml, respectively. Lower limits of quantification (LLOQ) of epothilone D and metabolite were 0.2 ng/ml and 5.0 ng/ml, respectively. Due to its high sensitivity, specificity and simplicity, the method could be used for pharmacokinetic studies of both epothilone D and its hydrolytic metabolite.
Subject(s)
Chromatography, High Pressure Liquid/methods , Epothilones/blood , Epothilones/pharmacokinetics , Tandem Mass Spectrometry/methods , Humans , Reproducibility of Results , Spectrometry, Mass, Electrospray Ionization/methodsABSTRACT
A novel ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method has been established for the determination of a newly synthesized epothilone D analog (AV-EPO-106) in human plasma. The plasma samples were prepared by liquid-liquid extraction with cold tert-butyl methyl ether. The chromatographic separation was achieved within 5 min on a C(18) column with water-methanol (10:90, v/v) as mobile phase at a flow-rate of 0.8 mL/min. Mass transition of m/z 568.2 to 386.1 was measured for AV-EPO-106 in positive atmospheric pressure chemical ionization mode. A detailed validation of the method was performed as per the USFDA guidelines. For AV-EPO-106 at the concentrations of 1.0, 5.0 and 10.0 microg/mL in human plasma, the absolute extraction recoveries were 86.17, 85.24 and 85.69%, respectively. The linear quantification range of the method was 0.10-20.0 microg/mL in human plasma with linear correlation coefficients greater than 0.999. The intra-day and inter-day accuracy for AV-EPO-106 at the levels of 1.0, 5.0 and 10.0 microg/mL in human plasma fell in the ranges of 98.25-100.47 and 94.19-97.25%, and the intra- and inter-day precision were in the ranges of 4.75-6.30% and 8.89-10.45%, respectively. The method was successfully applied to quantify AV-EPO-106 in human plasma to determine the half-life of this compound in human plasma.
Subject(s)
Epothilones/blood , Tubulin Modulators/blood , Chromatography, High Pressure Liquid , Drug Stability , Half-Life , Humans , Linear Models , Reproducibility of Results , Sensitivity and Specificity , Tandem Mass SpectrometryABSTRACT
Ixabepilone (BMS-247550) is a semi-synthetic, microtubule stabilizing epothilone B analogue which is more potent than taxanes and has displayed activity in taxane-resistant patients. The human plasma pharmacokinetics of ixabepilone have been described. However, the excretory pathways and contribution of metabolism to ixabepilone elimination have not been determined. To investigate the elimination pathways of ixabepilone we initiated a mass balance study in cancer patients. Due to autoradiolysis, ixabepilone proved to be very unstable when labeled with conventional [14C]-levels (100 microCi in a typical human radio-tracer study). This necessitated the use of much lower levels of [14C]-labeling and an ultra-sensitive detection method, Accelerator Mass Spectrometry (AMS). Eight patients with advanced cancer (3 males, 5 females; median age 54.5 y; performance status 0-2) received an intravenous dose of 70 mg, 80 nCi of [14C]ixabepilone over 3 h. Plasma, urine and faeces were collected up to 7 days after administration and total radioactivity (TRA) was determined using AMS. Ixabepilone in plasma and urine was quantitated using a validated LC-MS/MS method. Mean recovery of ixabepilone-derived radioactivity was 77.3% of dose. Fecal excretion was 52.2% and urinary excretion was 25.1%. Only a minor part of TRA is accounted for by unchanged ixabepilone in both plasma and urine, which indicates that metabolism is a major elimination mechanism for this drug. Future studies should focus on structural elucidation of ixabepilone metabolites and characterization of their activities.
Subject(s)
Antineoplastic Agents/pharmacology , Epothilones/pharmacokinetics , Neoplasms/metabolism , Adenocarcinoma/metabolism , Antineoplastic Agents/blood , Antineoplastic Agents/urine , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Squamous Cell/metabolism , Chromatography, Liquid/methods , Colonic Neoplasms/metabolism , Epothilones/blood , Epothilones/urine , Feces/chemistry , Female , Humans , Lung Neoplasms/metabolism , Male , Middle Aged , Ovarian Neoplasms/metabolism , Pancreatic Neoplasms/metabolism , Sigmoid Neoplasms/metabolism , Tandem Mass Spectrometry/methodsABSTRACT
A novel reversed-phase high-performance liquid chromatographic (HPLC) method has been established for the determination of a newly developed anti-cancer agent desoxyepothilone B (dEpo B) in nude mice plasma. The sample preparation involved deproteination of 200 microl of plasma sample first, followed by liquid-liquid extraction of the resultant supernatant with chloroform. The compound taxol was used as the internal standard. Chromatographic separations were carried out on a 250 mm x 4.6 mm Zorbax SB-phenyl column with acetonitrile-0.25% orthophosphoric acid (50/50, v/v) as mobile phase and UV detection at 250 nm. For dEpo B and taxol at the concentration level of 10 microg/ml in nude mice plasma, the absolute extraction recoveries were 85.3 and 87.2%, respectively. The linear quantification range of the method was 0.1-100 microg/ml in nude mice plasma with linear correlation coefficients greater than 0.999. The within-day and between-day relative standard deviations (R.S.D.s) for dEpo B at 0.5, 2.5 and 10 microg/ml levels in nude mice plasma fell in the range of 2.8-4.8 and 1.5-4.6%, and the within-day and between-day recoveries were in the range of 96.5-101.7 and 97.7-101.2%, respectively.
Subject(s)
Antineoplastic Agents/blood , Chromatography, High Pressure Liquid/methods , Epothilones/blood , Animals , Antineoplastic Agents/pharmacokinetics , Epothilones/pharmacokinetics , Mice , Mice, Nude , Reference Standards , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
PURPOSE: To determine, for various species, the pharmacological and biochemical properties of epothilone D (EpoD) that are relevant in establishing an appropriate animal model for further evaluation of this promising antitumor agent. METHODS: A method involving high-performance liquid chromatography (HPLC) was developed and used to assess the stability and protein binding of EpoD in plasma from various species, its metabolism by various S9 fractions, and its pharmacokinetics in mice. RESULTS: EpoD was stable in dog and human plasma. In plasma from other species, stability decreased in the order: hamster > mouse > guinea pig > rat. EpoD was highly bound to proteins in dog and human plasma. In an evaluation of S9 fractions from mouse, rat, guinea pig, dog, and human, mouse S9 was most efficient in metabolizing EpoD. Following administration to CD2F1 mice, the initial half-lives for plasma elimination of EpoD were <5 min for an intravenous dose and <20 min for an intraperitoneal dose. CONCLUSIONS: The species differences in EpoD biostability and metabolism may have implications in assessing its antitumor activity and pharmacologic and toxicologic profiles in humans. Relative to humans, the mouse is not a good model for disposition of EpoD; the dog would be more appropriate.
Subject(s)
Antineoplastic Agents/pharmacokinetics , Epothilones/pharmacokinetics , Animals , Antineoplastic Agents/blood , Dogs , Drug Evaluation, Preclinical , Drug Stability , Epothilones/blood , Guinea Pigs , Humans , Male , Mice , Mice, Inbred Strains , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Protein Binding , Rats , Species SpecificityABSTRACT
The total synthesis of a family of (E)-9,10-dehydro derivatives of epothilone D (i.e., 12,13-desoxyepothilone B) is described. The route is particularly concise and amenable to production of new congeners. Furthermore, the chemistry described herein constitutes a major simplification in the total synthesis of EpoD, which is in human clinical trials. This new family of epothilones shows major advantages in terms of their potency and pharmacostability relative to the wild-type saturated analogues in the D series. From the perspective of compound availability through synthesis, potency, and pharmacokinetic properties, these compounds could well warrant advancement to clinical evaluation in humans.