Generic UPLC-MS/MS and Gyrolab assays with blood microsampling for pharmacokinetic assessments of therapeutic antibodies in mice.

Serial blood sampling from one animal is useful to understand relationship between pharmacokinetics (PK) and pharmacological or toxicological events in individual animals. To assess its feasibility in mice, two therapeutic antibodies were used to evaluate impacts by different blood sampling methods, sampling sites, and assay platforms on PK. Denosumab and Panitumumab were intravenously administered to mice and only 0.05 mL of blood sample per point was collected from jugular vein or tail vein. Blood samples were collected serially from a mouse or collected by traditional composite sampling from each mouse. Plasma concentrations of the two drugs were assayed by a generic ligand binding assay using Gyrolab or by a generic ultra-performance liquid chromatography with tandem mass spectrometry. The two assay platforms showed acceptable accuracy and precision and gave comparable PK parameters of the drugs, suggesting that both assays were successfully applied to the PK assessments. Comparable results in the PK profiles were noted between serial and composite blood samplings and differences in the two sampling sites did not impact PK. These findings suggest that microsampling combined with generic assays is useful to assess PK profiles of therapeutic antibodies in mice.

A sensitive assay for the determination of E7090, a novel selective inhibitor of fibroblast growth factor receptors, and its metabolite in human plasma by UPLC-MS/MS with at-column dilution.

E7090, a novel fibroblast growth factor receptors inhibitor, is currently under clinical development for the treatment of patients with solid tumors. The previous assay was insufficient in detection sensitivity for E7090 and high exposure of a dealkylated metabolite, M2, was noted in a clinical trial at low doses. Thus, a sensitive assay for the simultaneous determination of E7090 and M2 in human plasma has been developed using ultra-performance liquid chromatography with tandem mass spectrometer (UPLC-MS/MS). E7090 and M2 were extracted from 0.1 mL of plasma by protein precipitation and chromatographed on a reverse phase column utilizing at-column dilution which enables larger volume sample injection to the UPLC-MS/MS. E7090 and M2 were quantifiable from 0.025 ng/mL, which was 40-fold higher sensitivity than the previous assay. Accuracy as relative error and precision as relative standard deviation were within ± 15% and 15%, respectively, ensuring the reproducibility of the assay. The developed assay method was applied to a clinical trial of E7090, and plasma concentrations of E7090 and M2 were quantifiable up to 144 h postdose. These results indicated that the developed more sensitive assay was reproducible and was successfully applied to a clinical trial of E7090.

A Simple UPLC-MS/MS Assay of Rifampin in a Small Volume of Human Plasma.

Rifampin (RIF) is a typical cytochrome P450 (CYP) 3A inducer and inhibitor of organic anion transporting polypeptide (OATP) 1B1 to assess drug-drug interaction (DDI) via CYP3A or OATP1B1 in clinical settings. To ensure sufficient exposure of RIF in DDI studies, it is important to determine plasma RIF concentrations. In this study, we developed a simple RIF assay in a small volume of human plasma by ultraperformance liquid chromatography with tandem mass spectrometry. RIF in 0.02 mL of plasma was extracted using protein precipitation and separated on a reverse phase column under gradient elution of three mobile phases, where the mobile phase C containing 1% formic acid was exclusively used to reduce the carryover of RIF. RIF and the internal standard were detected by multiple reaction monitoring in positive-ion electrospray ionization. RIF was quantifiable at 0.025-10 µg/mL without the carryover issue. The intra- and inter-run assays confirmed the reproducibility of the assay. Stability assessments ensured that RIF in human plasma was stable for 6 h at room temperature and for 409 days at -15 °C or below. The assay was successfully applied to a pharmacokinetic study with successful incurred sample reanalysis.

A validated UPLC-MS/MS assay of E7090, a novel selective inhibitor of fibroblast growth factor receptors, in human plasma and urine.

E7090, a novel fibroblast growth factor receptors inhibitor, is currently under clinical development for the treatment of patients with solid tumors. Assays for the determination of E7090 concentrations in human plasma and urine have been developed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to evaluate pharmacokinetic profiles of E7090. E7090 and a deuterated labeled internal standard (IS) were extracted from 50 µL of plasma by protein precipitation. In quantification of E7090 in urine, 50 µL of urine samples fortified with 15 µL of ethanol (10:3, v/v) to minimize nonspecific binding of E7090 to urine containers were subjected to the assay without extraction. E7090 and the IS were separated by chromatography on a reverse phase column and were detected by selected reaction monitoring in the positive ion mode. The lower limit of quantification was set at 1 ng/mL and E7090 was quantifiable from 1 to 3000 ng/mL in plasma and urine. Accuracy and precision were measured during the reproducibility assessments and were within ± 7.0% and 9.1%, respectively, in plasma and within ± 7.0% and 5.8%, respectively, in urine, indicating sufficient reproducibility. The validated methods were successfully applied to the quantification of E7090 in human plasma and urine to support a Phase-1 clinical trial.

Liposome-Encapsulated Eribulin Shows Enhanced Antitumor Activity over Eribulin for Combination Therapy with Anti-PD-1 Antibody.

Eribulin is a microtubule dynamics inhibitor with tumor microenvironment modulation activity such as vascular remodeling activity. Here, we investigated antitumor and immunomodulatory activities of eribulin and its liposomal formulation (eribulin-LF) as monotherapies or in combination with anti-programmed death 1 (PD-1) Ab. The antitumor activity of eribulin or eribulin-LF as monotherapy or in combination with anti-PD-1 Ab was examined in a P-glycoprotein-knockout 4T1 model. Eribulin and eribulin-LF showed stronger antitumor activity in immunocompetent mice compared with immunodeficient mice, indicating that they have immunomodulatory activity that underlies its antitumor activity. Combination therapy of eribulin and eribulin-LF with anti-PD-1 Ab showed antitumor activity, and the combination activity of eribulin-LF with anti-PD-1 Ab was observed at a lower dose and longer interval of administration compared with that using eribulin. To examine the immunomodulatory activity of eribulin and eribulin-LF and its underlying mechanisms, we performed flow cytometry, IHC, and gene expression profiling. IHC and flow cytometry revealed that eribulin-LF increased microvessel density and intratumoral populations of cytotoxic T cells and natural killer cells rather than eribulin. Gene expression profiling demonstrated that eribulin-LF induces IFNγ signaling. Furthermore, IHC also showed that eribulin-LF increased infiltration of CD8-positive cells together with increased CD31-positive cells. Eribulin-LF also increased ICAM-1 expression, which is essential for lymphocyte adhesion to vascular endothelial cells. In conclusion, eribulin showed combination antitumor activity with anti-PD-1 Ab via immunomodulation due to its vascular remodeling activity, and the liposomal formulation showed improved antitumor activity over the standard formulation.

Quantitative and qualitative application of a novel capillary microsampling device, Microsampling Wing™ (MSW), using antiepileptic drugs in rats.

A novel microsampling device, namely, the Microsampling Wing™ (MSW), was evaluated using three anti-epileptic drugs (AEDs): carbamazepine, lamotrigine, and phenytoin. A simultaneous assay method of the three AEDs was developed and qualified via liquid chromatography with tandem mass spectrometry. Using 2.8 µL plasma, the three AEDs were quantifiable from 1 or 2 ng/mL. According to the intra-assay reproducibility assessment and additional validation parameters, the established method is reproducible. To apply the device to a pharmacokinetic (PK) study in rats, a cocktail of the three AEDs was orally administered to rats. Whole blood samples were serially collected using the MSW device and a glass capillary from the tail vein, and plasma samples (each 2.8 µL) from each device were assayed to compare PK parameters. The PK parameters of the three AEDs were similar between the two devices. A metabolite identification study was also conducted after oral administration of carbamazepine to rats. At least seven metabolites were detected in plasma, and the major metabolite was carbamazepine 10,11-epoxide, which is in accordance with the reported results. These findings suggest that the MSW device is a useful microsampling device for PK and metabolite identification studies.

A sensitive bioanalytical assay for methylcobalamin, an endogenous and light-labile substance, in human plasma by liquid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study.

Methylcobalamin (mecobalamin) is a vitamin B12 coenzyme and is effective for the treatment of peripheral neuropathies. As mecobalamin is an endogenous substance in human plasma at low levels and is light-labile, a sensitive assay method has been developed by using liquid chromatography with tandem mass spectrometry with particular care taken for light exposure. Stability tests performed under light showed that mecobalamin was stable in plasma in amber tubes only when exposed to <10 lx light, and thus, all the assays used for the method validation and the in-study sample assays should be performed under red light with minimal light exposure. Mecobalamin and its stable isotope, which was used as an internal standard, were extracted from 0.1 mL plasma by protein precipitation with methanol and were quantifiable in a range from 0.05 to 20 ng/mL. The reproducibility assessment showed acceptable accuracy and precision (≤15 %). Mecobalamin was stable in plasma at room temperature for 21 h when exposed to <10 lx light in amber tubes and for 205 days when stored frozen. The validated method was successfully applied to an in-study sample assay by performing a clinical pharmacokinetic study of mecobalamin, in which a successful incurred sample reanalysis was performed.

Establishment of a simultaneous assay for lemborexant, a novel dual orexin receptor antagonist, and its three metabolites, and its application to a clinical protein binding study.

A simultaneous assay for the determination of lemborexant and three metabolites (M4, M9, and M10) in human plasma and phosphate buffered saline (PBS) was developed and validated using liquid chromatography with tandem mass spectrometry, in support of plasma protein binding (PPB) studies. The analytes were extracted from plasma and PBS by solid phase extraction and then chromatographed on a reversed phase C18 column to ensure peak separation of three metabolites with the same mass transition. The analytes and the corresponding deuterated substances used as an IS were detected in the positive ion mode by multiple reaction monitoring. Lemborexant and three metabolites were quantifiable from 4 and 300 pg/mL in PBS and plasma, respectively, without any carryover. Extraction recovery was almost complete without matrix effects. The accuracy and precision in the intra- and inter-assay reproducibility were within the criteria as well as in the dilution integrity. Stability of the four analytes was ensured to cover duration of equilibrium dialysis and storage of samples. The established method was applied to an ex vivo PPB study in humans.

Relative Activity Factor (RAF)-Based Scaling of Uptake Clearance Mediated by Organic Anion Transporting Polypeptide (OATP) 1B1 and OATP1B3 in Human Hepatocytes.

In vitro-in vivo extrapolation based on uptake clearance determined in human hepatocytes has been used to predict in vivo hepatic clearance of organic anion transporting polypeptide (OATP) substrates. This study evaluated the relative activity factor (RAF) approach to extrapolate active uptake clearance in transporter-transfected cell systems (CLuptake) to that in human hepatocyte suspensions (PSinf,act). RAF values for OATP1B1 and OATP1B3 were determined in two batches of cryopreserved human hepatocytes using estrone-3-sulfate and cholecystokinin octapeptide as reference substrates, respectively. Fourteen OATP1B substrate drugs selected (atorvastatin, bosentan, cerivastatin, fexofenadine, fluvastatin, glibenclamide, irbesartan, nateglinide, pitavastatin, pravastatin, rosuvastatin, telmisartan, torasemide, and valsartan) showed temperature-dependent uptake in human hepatocytes. In transporter-transfected cells, OATP1B1- and OATP1B3-mediated uptake was observed in all compounds except for telmisartan. RAF-based net CLuptake was mainly accounted for by OATP1B1 (72.3-99.7%) and fell within the 3-fold of PSinf,act observed in human hepatocytes in 11 out of 13 compounds (excluding telmisartan). This study demonstrated that the RAF approach provides a quantitative index of OATP1B1- and OATP1B3-mediated PSinf,act in human hepatocytes, which will facilitate the optimization of the pharmacokinetic properties of OATP1B substrates at nonclinical stages of drug development.

A dried blood spot assay with UPLC-MS/MS for the simultaneous determination of E6005, a phosphodiesterase 4 inhibitor, and its metabolite in human blood.

E6005, a novel phosphodiesterase 4 inhibitor, is currently under clinical development for the treatment of atopic dermatitis. To support pediatric clinical trials, the dried blood spot assay for simultaneous determination of E6005 and its main metabolite, ER-392710 (M11), has been developed using ultra-performance liquid chromatography with tandem mass spectrometry. E6005 and M11, in 25 µL blood spotted onto FTA™ DMPK-C cards, were extracted by water/acetonitrile (1:1, v/v), and then chromatographed on a reversed phase column under gradient elution. The mass transitions, m/z 473.1 → 163.0 for E6005 and m/z 459.1 → 149.0 for M11, with corresponding stable isotope internal standard, m/z 477.2 → 167.0, and m/z 463.2 → 153.0, were monitored. E6005 and M11 were quantifiable from 1 to 200 ng/mL as free base. Accuracy and precision of the two analytes in the intra- and inter-batch reproducibility were within ±8.0% and 15.7%, respectively. Extraction recoveries of the analytes were 73% or more and hematocrit ranging from 26.9% to 51.8% did not impact the analytes' accuracy. Various stability assessments, including possible conversion of E6005 to M11, were thoroughly performed, and bench-top stability was ensured up to 160 days. The DBS method was applied to determine E6005 and M11 concentrations in blood samples supporting a pediatric clinical trial.

Simultaneous Determination of Donepezil and Its Three Metabolites in Human Plasma Using LC-MS-MS.

A simple and selective assay for the determination of donepezil (Aricept(®)) and its three metabolites including 6-O-desmethyl (M1), 5-O-desmethyl (M2) and N-oxide (M6) metabolites in human plasma was developed and validated using liquid chromatography with tandem mass spectrometry. An analog of donepezil was used as an internal standard (IS) for all the analytes. The analytes and the IS were extracted from plasma by solid-phase extraction. The analytes were chromatographically separated on Cadenza CD-C18 column with gradient elution, then detected with electrospray positive ionization in multiple reaction monitoring mode. The established method showed linearity ranging 0.5-100 ng/mL for donepezil and 0.2-40 ng/mL for all three metabolites and was fully validated in accordance with bioanalytical guidelines. Selectivity, clear peak separation and no carryover were ensured for all the analytes. The intra- and inter-batch reproducibility assessments demonstrated that accuracy and precision were within the acceptance criteria. Minimal matrix effects and consistent extraction recovery were noted. Stability assessment demonstrated that all the analytes were stable for at least 184 days at -20°C. Assay of post-dose samples also showed clear peak separation of the analytes, indicating successful clinical application.

Validation of a UPLC-MS/MS method for the simultaneous determination of E6005, a phosphodiesterase 4 inhibitor, and its metabolite in human plasma.

E6005, a novel phosphodiesterase 4 inhibitor, is currently under clinical development for the treatment of atopic dermatitis. As ER-392710 (M11), a hydrolyzed metabolite, is a main metabolite, a simultaneous assay method for quantification of E6005 and M11 in human plasma has been developed and validated using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). E6005, M11, and each deuterium-labeled compound used as internal standard were extracted from 100µL human plasma by solid phase extraction then chromatographed on an Acquity UPLC BEH C18 column (100mm×2.1mm i.d., 1.7µm) under gradient elution. The analytes were detected by selected reaction monitoring in the positive ion mode with the mass transition of m/z 473.1/163.0 and m/z 459.1/149.0 for E6005 and M11, respectively. E6005 and M11 were quantifiable ranging from 1 to 200ng/mL with no carryover. Accuracy and precision in intra- and inter-batch reproducibility assays were within the acceptance criteria recommended by the regulatory bioanalytical guidelines. Various stability assessments including possible conversion of E6005 to M11 were thoroughly performed to demonstrate the stability of E6005 and M11 in human blood and plasma. The method was successfully applied to support clinical trials.

Validation of a hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of gemcitabine in human plasma with tetrahydrouridine.

A simple and reproducible bioanalytical method for the determination of gemcitabine in human plasma treated with tetrahydrouridine (THU) was developed and validated using a hydrophilic interaction ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). To prevent deamination of gemcitabine, blood was treated with THU, and the plasma samples obtained after centrifugation were used in this study. Gemcitabine and gemcitabine-(13)C, (15)N2 used as an internal standard, were extracted from human plasma treated with THU using a 96-well Hybrid SPE-Precipitation plate. Extracts were chromatographed on a hydrophilic interaction chromatography column with isocratic elution. Detection was performed using Quattro Premier with positive electrospray ionization multiple reaction monitoring mode. The standard curve ranged from 10 to 10,000 ng/mL without carryover. No significant interferences were detected in blank plasma and no interferences by 2'-2'-difluoro-2'-deoxyuridine, a metabolite of gemcitabine. Accuracy and precision in the intra-batch reproducibility study using quality control samples with three THU levels did not exceed ±5.4 and 7.3%, respectively, and the inter-batch reproducibility results also met the criteria. Stability of gemcitabine was ensured in whole blood and plasma as well as stability of THU in solutions. The UPLC-MS/MS method developed was successfully validated and can be applied for gemcitabine bioanalysis in clinical studies.

Validation of an ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of flecainide in human plasma and its clinical application.

A simple and reproducible bioanalytical method for the determination of flecainide in human plasma was developed and validated using an ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) to obtain higher sensitivity than the current available methods. After simple protein precipitation, flecainide and a stable isotope-labeled internal standard (IS) were chromatographed on an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 µm) with isocratic elution of mobile phase consisting of 45% methanol containing 0.1% formic acid at a flow rate 0.25 mL/min. Detection was performed in positive electrospray ionization by monitoring the selected ion transitions at m/z 415.4/301.1 for flecainide and m/z 419.4/305.1 for the IS. The method was validated according to current bioanalytical method validation guidelines. The calibration standard curve was linear from 2.5 to 1000 ng/mL using 0.1 mL of plasma. No significant interferences were detected in blank human plasma. Accuracy and precision in the intra- and inter-batch reproducibility study were within acceptance criteria. Neither hemolysis effects nor matrix effects were observed. The UPLC-MS/MS method developed was successfully applied to determine plasma flecainide concentrations to support clinical studies and incurred sample reanalysis also ensured the reproducibility of the method.

Application of liquid chromatography-tandem mass spectrometry for the determination of opioidmimetics in the brain dialysates from rats treated with opioidmimetics intraperitoneally.

We have determined three opioidmimetics (compounds I-III) in the rat brain dialysates after intraperitoneal (i.p.) administration of compounds I-III using a liquid chromatography/mass spectrometry with tandem mass spectrometry (LC-MS/MS). The dialysate samples with methanol were directly analyzed by online column-switching liquid chromatography. Using multiple reaction monitoring (MRM, product ions m/z 421 of m/z 657 for compound I, m/z 421 of m/z 643 for compound II, and m/z 407 of m/z 629 for compound III) on LC-MS/MS with electrospray ionization (ESI), opioidmimetics in rat brain dialysates were determined. Calibration curves of the method showed a good linearity in the range of 10-100 ng/ml for each compound. The limit of determination was estimated to be ca. 1 ng/ml for compounds II and III, and ca. 5 ng/ml for compound I, respectively. The precision of analysis showed coefficients of variation ranging from 4.7 to 10.4% at compound III concentration (10-100 ng/ml) in Ringer's solution. As a result, the procedure proved to be very suitable for routine analysis. The method was applied to the analysis of three opioidmimetics in the brain dialysate samples from rats treated with these compounds.

Determination of cabergoline and L-dopa in human plasma using liquid chromatography-tandem mass spectrometry.

We determined cabergoline and L-dopa in human plasma using liquid chromatography-mass spectrometry with tandem mass spectrometry (LC-MS-MS). The deproteinized plasma samples with organic solvent or acid were analyzed directly by reversed-phase liquid chromatography. Using multiple reaction monitoring (MRM, product ions m/z 381 of m/z 452 for cabergoline and m/z 152 of m/z 198 for L-dopa) on LC-MS-MS with electrospray ionization (ESI), cabergoline and L-dopa in human plasma were determined. Calibration curves of the method showed a good linearity in the range 5-250 pg/ml for cabergoline and 1-200 ng/ml for L-dopa, respectively. The limit of determination was estimated to be approximately 2 pg/ml for cabergoline and approximately 0.1 ng/ml for L-dopa, respectively. The method was applied to the analysis of cabergoline and L-dopa in plasma samples from patients treated with these drugs. The precision of analysis showed coefficients of variation ranging from 3.8% to 10.5% at cabergoline concentration of 13.8-26.2 pg/ml and from 2.9% to 8.9% at an L-dopa concentration of 302.5-522.1 ng/ml in patient plasma. As a result, the procedure proved to be very suitable for routine analysis.