Pharmacokinetics of S-epacadostat, an indoleamine 2,3-dioxygenase 1 inhibitor, in dog plasma and identification of its metabolites in vivo and in vitro.

S-epacadostat (S-EPA) is an efficient and selective small-molecule inhibitor of indoleamine 2,3-dioxygenase 1. It is an EPA analog with a sulfur atom instead of a nitrogen atom at the furazan C3 position. This study documents the pharmacokinetics of S-EPA in dogs and its metabolic pathway. After an oral administration of 15 mg/kg of S-EPA in dogs, the time to peak concentration was 0.80 h, the mean elimination half-life was 7.3 h, and the absolute bioavailability was 55.8%. Furthermore, we identified S-EPA metabolites in dog plasma and dog liver microsomes by UPLC-Q Exactive Orbitrap HRMS. In dog plasma, we found five metabolites, which came from glucuronidation (M1 and M2), deoxygenation (the amidine M4), glucuronidation of M4 (M3), and desulfonamidation and oxidation of M4 (the carboxylic acid M5). In dog liver microsomes, we identified three major metabolites, namely, the glucuronide conjugate (M6), a mono-oxidation product (M7), and a desulfonamidation and oxidation product (M8). Gut microbiota may cause the differences between in vivo and in vitro oxidation metabolisms. Contrary to EPA, S-EPA did not undergo dealkylation, suggesting that substituting the nitrogen with sulfur affects the metabolism of the adjacent alkyl side chain.

Surface polymer imprinted optical fibre sensor for dose detection of dabrafenib.

Dabrafenib is one of the most widely used of the new generation of targeted anti-cancer drugs. However, its therapeutic window varies for different patients and so there is an unmet need for methods to monitor the dose of drug which the patient receives and at the specific site where it acts. In the case of cancers, it is critical to measure the concentration of drug not just in the bloodstream overall, but in or near tumours, as these will not be the same over multiple time periods. A novel sensor based on an optical fibre long period grating (LPG) modified with a molecular imprinted polymer (MIP) has been developed with the ultimate aim of achieving minimally invasive measurements of Dabrafenib at the tumour site. A molecularly imprinted polymer specific for Dabrafenib was coated on a methacryloylalkoxysilane-functionalised optical fibre long period grating. In vitro experimental results demonstrate that the Dabrafenib sensitivity is 15.2 pm (µg mL-1)-1 (R2 = 0.993) with a limit of detection (LoD) of 74.4 µg mL-1 in serum solution. Moreover, the proposed sensor shows selective response to Dabrafenib over structurally similar 2-Aminoquinoline.

A novel high-performance liquid chromatography with diode array detector method for the simultaneous quantification of the enzyme-reactivating oximes obidoxime, pralidoxime, and HI-6 in human plasma.

Oximes such as pralidoxime (2-PAM), obidoxime (Obi), and HI-6 are the only currently available therapeutic agents to reactivate inhibited acetylcholinesterase (AChE) in case of intoxications with organophosphorus (OP) compounds. However, each oxime has characteristic agent-dependent reactivating efficacy, and therefore the combined administration of complementary oximes might be a promising approach to improve therapy. Accordingly, a new high-performance liquid chromatography method with diode-array detection (HPLC-DAD) was developed and validated allowing for simultaneous or single quantification of 2-PAM, Obi, and HI-6 in human plasma. Plasma was precipitated using 5% w/v aqueous zinc sulfate solution and subsequently acetonitrile yielding high recoveries of 94.2%-101.0%. An Atlantis T3 column (150 × 2.1mm I.D., 3 µm) was used for chromatographic separation with a total run time of 15 min. Quantification was possible without interferences within a linear range from 0.12 to 120 µg/mL for all oximes. Excellent intra-day (accuracy 91.7%-98.6%, precision 0.5%-4.4%) and inter-day characteristics (accuracy 89.4%-97.4%, precision 0.4%-2.2%) as well as good ruggedness were found. Oximes in processed samples were stable for at least 12 h in the autosampler at 15°C as well as in human plasma for at least four freeze-thaw cycles. Finally, the method was applied to plasma samples of a clinical case of pesticide poisoning.

Pharmacokinetics of S-EPA, and its inhibition on indoleamine 2,3-dioxgenase: a case of sulfur-substitution affecting distributions in blood cells.

1. S-EPA is a sulfur-substitution analog of epacadostat (EPA), an effective small molecule indoleamine 2,3-dioxgenase1 (IDO) inhibitor. By in vitro and in vivo experiments, pharmacokinetic differences of two closely related analogs, S-EPA and EPA was investigated in this study. 2. Liver microsomes clearance experiments showed S-EPA had comparable metabolic stability with EPA in rat and human liver microsomes. The whole blood distribution experiments showed the distribution ratio of S-EPA in blood cells to plasma in mice, rats, dogs and monkey was 1.2, 4.8, 2.2 and 40.6, respectively. While the distribution ratio of EPA ranged from 0.94 to 1.30 in mice, rats, dogs and was 3.1 in monkeys. 3. The pharmacokinetic study in rats showed the exposure (AUClast) of S-EPA in plasma and blood cells was 1.7-fold and 3.9-fold higher than that of EPA, respectively. Moreover, the exposure ratio of S-EPA in blood cells to plasma was 3.7, while the ratio of EPA was 1.6. 4. In CT26 tumor bearing mice, the IDO inhibition of S-EPA and EPA on plasma or tumor kynurenine was generally consistent. And the inhibition ratio could reach at more than 50% at 3 h after single dose, at least lasting up to 8 h.

Combination delivery of two oxime-loaded lipid nanoparticles: Time-dependent additive action for prolonged rat brain protection.

A novel approach for brain protection against poisoning by organophosphorus agents is developed based on the combination treatment of dual delivery of two oximes. Pralidoxime chloride (2-PAM) and a novel reactivator, 6-(5-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)pentyl)-3-hydroxy picolinaldehyde oxime (3-HPA), have been loaded in solid-lipid nanoparticles (SLNs) to offer distinct release profile and systemic half-life for both oximes. To increase the therapeutic time window of both oximes, SLNs with two different compartments were designed to load each respective drug. Oxime-loaded SLNs of hydrodynamic diameter between 100 and 160 nm and negative zeta potential (-30 to -25 mV) were stable for a period of 10 months at 4 °C. SLNs displayed longer circulation time in the bloodstream compared to free 3-HPA and free 2-PAM. Oxime-loaded SLNs were suitable for intravenous (iv) administration. Paraoxon-poisoned rats (0.8 × LD50) were treated with 3-HPA-loaded SLNs and 2-PAM+3-HPA-loaded SLNs at the dose of 3-HPA and 2-PAM of 5 mg/kg. Brain AChE reactivation up to 30% was slowly achieved in 5 h after administration of 3-HPA-SLNs. For combination therapy with two oximes, a time-dependent additivity and increased reactivation up to 35% were observed.

Behavioral, Pharmacokinetic, and Cardiovascular Evaluation of Candidate Oximes in African Green Monkeys.

Oxime reactivators are critical antidotes after organophosphate pesticide or nerve agent poisoning, directly restoring the function of inhibited acetylcholinesterase. In the continuing search for more broad-spectrum acetylcholinesterase reactivators, this study evaluated one of the leading next-generation oxime reactivators: methoxime, (1,1'-trimethylene bis[4-(hydroxyimino)methyl]pyridinium dichloride (MMB-4). The pharmacokinetics of both salts of MMB-4 (dichloride [2Cl] and dimethanesulphonate [DMS]) were characterized across a range of relevant doses (19, 58, and 116 µmol/kg, intramuscular) in a nonhuman primate model (male African green monkeys), and only subtle differences were observed between the salts. Additionally, the behavioral and cardiovascular safety of these MMB-4 salts was compared directly to other available oximes (HI-6 2Cl, HI-6 DMS, and pyridine-2-aldoxime chloride (2-PAM Cl)) at comparable projected doses. Automated operant behavioral tests were used to examine attention, motivation, visual discrimination, concept execution, and fine motor coordination after high doses of all oxime salts, and of all oximes studied, only the highest dose of 2-PAM Cl (447 µmol/kg) disrupted behavioral performance. Likewise, the effects of a range of doses of MMB-4 2Cl or DMS, HI-6 2Cl or DMS, or 2-PAM Cl on cardiovascular parameters were measured in African green monkeys implanted with telemetry devices. Only a small transient decrease in pulse pressure was observed following administration of the highest dose of MMB-4 DMS (116 µmol/kg). Thus, MMB-4 salts, up to the 9× equivalent of a projected autoinjector dose in humans, did not produce behavioral or cardiovascular toxicity in African green monkeys in the current study, and the pharmacokinetic parameters were orderly and predictable.

Quantification of the next-generation oral anti-tumor drugs dabrafenib, trametinib, vemurafenib, cobimetinib, pazopanib, regorafenib and two metabolites in human plasma by liquid chromatography-tandem mass spectrometry.

A sensitive and selective method of high performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS/MS) has been developed for the simultaneous quantification of six anticancer protein kinase inhibitors (PKIs), dabrafenib, trametinib, vemurafenib, cobimetinib, pazopanib, regorafenib, and two active metabolites (regorafenib-M2 and regorafenib-M5) in human plasma. Plasma protein precipitation with methanol enables the sample extraction of 100 µL aliquot of plasma. Analytes are detected by electrospray triple-stage quadrupole mass spectrometry and quantified using the calibration curves with stable isotope-labeled internal standards. The method was validated based on FDA recommendations, including assessment of extraction yield (74-104%), matrix effects, analytical recovery (94-104%) with low variability (<15%). The method is sensitive (lower limits of quantification within 1 to 200 ng/mL), accurate (intra- and inter-assay bias: -0.3% to +12.7%, and -3.2% to +6.3%, respectively) and precise (intra- and inter-assay CVs within 0.7-7.3% and 2.5-8.0%, respectively) over the clinically relevant concentration range (upper limits of quantification 500 to 100,000 ng/mL). This method is applied in our laboratory for both clinical research programs and routine therapeutic drug monitoring service of PKIs.

Physiologically Based Pharmacokinetic Modeling to Identify Physiological and Molecular Characteristics Driving Variability in Drug Exposure.

Prospectively defining the physiological and molecular characteristics most likely driving between-subject variability (BSV) in drug exposure provides the opportunity to inform the assessment of biomarkers to account for this variability. A physiologically based pharmacokinetic (PBPK) model was constructed and verified for dabrafenib. This model was then used to evaluate the physiological and molecular characteristics driving BSV in dabrafenib exposure. The capacity to discriminate a steady-state dabrafenib trough concentration >48 ng/mL was also evaluated. The mean simulated/observed ratios for the parameters describing dabrafenib exposure in single-dose, multiple-dose, and drug-drug interaction studies were between 0.78 and 1.23. Multivariable analysis indicated that consideration of baseline weight, body mass index, and CYP2C8, CYP3A4, and P-gp abundance strongly predicts steady-state dabrafenib trough concentration above 48 ng/mL (ROC AUC = 0.94; accuracy = 86%). This is the first study to use a verified PBPK model to identify baseline physiological and molecular characteristics driving BSV in drug exposure.

Evaluation of Potential Drug-Drug Interaction Between Delayed-Release Dimethyl Fumarate and a Commonly Used Oral Contraceptive (Norgestimate/Ethinyl Estradiol) in Healthy Women.

Delayed-release dimethyl fumarate (DMF) is an oral therapy for relapsing multiple sclerosis with anti-inflammatory and neuroprotective properties. This 2-period crossover study was conducted to evaluate the potential for drug-drug interaction between DMF (240 mg twice daily) and a combined oral contraceptive (OC; norgestimate 250 µg, ethinyl estradiol 35 µg). Forty-six healthy women were enrolled; 32 completed the study. After the lead-in period (OC alone), 41 eligible participants were randomized 1:1 to sequence 1 (OC and DMF coadministration in period 1; OC alone in period 2) or sequence 2 (regimens reversed). Mean concentration profiles of plasma norelgestromin (primary metabolite of norgestimate) and ethinyl estradiol were superimposable following OC alone and OC coadministered with DMF, with 90% confidence intervals of geometric mean ratios for area under the plasma concentration-time curve over the dosing interval and peak plasma concentration contained within the 0.8-1.25 range. Low serum progesterone levels during combined treatment confirmed suppression of ovulation. The pharmacokinetics of DMF (measured via its primary active metabolite, monomethyl fumarate) were consistent with historical data when DMF was administered alone. No new safety concerns were identified. These results suggest that norgestimate/ethinyl estradiol-based OCs may be used with DMF without dose modification.

Trough dabrafenib plasma concentrations can predict occurrence of adverse events requiring dose reduction in metastatic melanoma.

INTRODUCTION: Dabrafenib and trametinib bitherapy provides significant benefits in BRAFV600mut metastatic melanoma patients; however, adverse events (AE) occur, leading to dose reduction in 33% of patients. We aimed to investigate a relation between plasma dabrafenib and trametinib concentrations and occurrence of AE. METHODS: Plasma samples from metastatic BRAFV600mut melanoma patients treated with dabrafenib±trametinib were prospectively collected at trough concentration before any dose reduction. Dabrafenib and trametinib were measured by UPLC-MS/MS. Plasma threshold of concentration associated with dose reduction for AE was studied by ROC-curve analysis. RESULTS: Twenty-seven patients (13M/14F) were included. Dabrafenib trough plasma concentrations displayed high interindividual variability, ranging from 15.4 to 279.6ng/ml, mean±SD 58.7±61.1ng/ml. Trough trametinib plasma concentrations ranged from 4.1 to 23.8ng/ml, mean±SD 11.9±4.1ng/ml. Mean trough dabrafenib plasma concentration was higher in patients with AE requiring dose reduction (30%) than in other patients: 118.6ng/ml and 33.5ng/ml respectively (P<0.0001). Adverse events leading to dabrafenib dose reduction were all grade≥2. No differences in mean trametinib trough plasma concentrations were observed in patients requiring or not dose reduction. A dabrafenib trough plasma threshold of 48ng/ml can predict the occurrence of adverse events requiring dose reduction.

Simple and cost-effective liquid chromatography-mass spectrometry method to measure dabrafenib quantitatively and six metabolites semi-quantitatively in human plasma.

Dabrafenib is an inhibitor of BRAF V600E used for treating metastatic melanoma but a majority of patients experience adverse effects. Methods to measure the levels of dabrafenib and major metabolites during treatment are needed to allow development of individualized dosing strategies to reduce the burden of such adverse events. In this study, an LC-MS/MS method capable of measuring dabrafenib quantitatively and six metabolites semi-quantitatively is presented. The method is fully validated with regard to dabrafenib in human plasma in the range 5-5000 ng/mL. The analytes were separated on a C18 column after protein precipitation and detected in positive electrospray ionization mode using a Xevo TQ triple quadrupole mass spectrometer. As no commercial reference standards are available, the calibration curve of dabrafenib was used for semi-quantification of dabrafenib metabolites. Compared to earlier methods the presented method represents a simpler and more cost-effective approach suitable for clinical studies. Graphical abstract Combined multi reaction monitoring transitions of dabrafenib and metabolites in a typical case sample.

An UPLC-MS/MS method for the quantification of BRAF inhibitors (vemurafenib, dabrafenib) and MEK inhibitors (cobimetinib, trametinib, binimetinib) in human plasma. Application to treated melanoma patients.

Targeted therapies for cancers are fast-growing therapies. For instance kinase inhibitors such as BRAF inhibitors (BRAFi) and MEK inhibitors (MEKi) are increasingly used to treat malignant melanoma. The metabolic profile of these drugs can result in great interindividual variability, justifying therapeutic drug monitoring (TDM). We describe a rapid and specific method for quantification of 2 BRAFi (vemurafenib, dabrafenib) and 3 MEKi (cobimetinib, trametinib and binimetinib). Chromatography was performed on a Waters Acquity-UPLC system with CORTECS C18+ column, under a gradient of 10% acetic acid in water/acetonitrile. An Acquity-TQD® with electrospray ionization was used for detection. Samples were prepared by solid phase extraction (Oasis® MCX microElution) before injection in the system. Calibration curves ranges from 0.4 to 100µg/ml for vemurafenib, from 1 to 1000ng/ml for dabrafenib, from 0.5 to 500ng/ml for cobimetinib and binimetinib, and from 0.75 to 250ng/ml for trametinib. At all concentrations the bias was within ±15% of the nominal concentrations and precision was ≤15%. All results were within the acceptance criteria of the EMA guidelines on method validation. This rapid, sensitive and specific UPLC-MS/MS method can perform simultaneous quantification of targeted therapies used in malignant melanoma and is usable for routine TDM.

The pharmacokinetics of 12-week continuous contraceptive patch use.

OBJECTIVES: We sought to assess the change in serum ethinyl estradiol (EE2) and norelgestromin (NGMN) levels over 12 weeks of continuous contraceptive patch use. STUDY DESIGN: We asked participants (n=30) to apply consecutive patches to be worn continuously (without a patch-free interval) for 12 weeks. We collected blood samples at the end of each patch week and two times during weeks 4, 8, and 12 (with the additional blood draw occurring mid-week). Liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS/MS) was utilized to assess EE2 and NGMN levels. RESULTS: Twenty-seven women completed the study; 26 were compliant with patch use. Ethinyl estradiol levels ranged from 0 to 193 pg/mL over the period. We observed an accumulation over the 12-week time at an estimated rate of 2.15 pg/mL per week (95% confidence interval 0.95-3.35, p<.001). The change in NGMN levels ranged from 0 to 2.52 ng/mL over the 12 weeks (95% confidence interval 0.021-0.019, p=.915). The most common side effects reported were vaginal spotting, breast tenderness and abdominal pain/cramping. There were no serious adverse events reported. CONCLUSION: While the range of weekly EE2 values was quite wide, the absolute values remain low and generally within the expected range described in product labeling. Providers may consider prescribing continuous use of the patch, but given the slow accumulation of EE2 over time, 12 weeks should not be exceeded in the absence of safety data.

Pharmacokinetic profile of promising acetylcholinesterase reactivators K027 and K203 in experimental pigs.

Standard treatment of organophosphorus compounds (OPs) poisoning includes administration of an anti-muscarinic (atropine), anticonvulsive (diazepam) and acetylcholinesterase reactivator (oxime). From a wide group of newly synthesized oximes, oxime K027 and oxime K203 seem to be perspective compounds in some specific OPs intoxication. The available in vitro and in vivo preclinical data indicate that both oximes may be considered for potential human use. The main aim of this study was to establish plasmatic concentration curves of both oximes after intramuscular (i.m.) and intragastric (i.g.) application with subsequent pharmacokinetic analysis and study distribution after (i.m.) application on a non-rodent animal model (experimental pigs; 1500mg/animal). According to the results, both oximes had similar Cmax (K027: 106±19µg/mL and K203: 111±8µg/mL) in Tmax 19±5min, respectively, in 22±3min. Bioavailability of oxime K027 calculated as AUCtotal (8389±1024minµg/mL) was halved compared to oxime K203 (16938±795minµg/mL). The highest concentration from peripheral tissues was found in the kidney and lung, but the brain concentrations stay very low, the plasma/brain ratio being approximately 1%. The applied doses were derived from the recommendation where it is possible to use three autoinjectors to save human life. The results provide us with knowledge about the pharmacokinetics and distribution of these new oximes and may help us to better estimate the human pharmacokinetic profile.

Population Pharmacokinetic and Pharmacodynamic Modeling of Epacadostat in Patients With Advanced Solid Malignancies.

Epacadostat (EPA, INCB024360) is a selective inhibitor of the enzyme indoleamine 2,3-dioxygenase 1 (IDO1) and is being developed as an orally active immunotherapy to treat advanced malignancies. In the first clinical study investigating the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of EPA in oncology patients, increasing doses of EPA ranging from 50 mg once daily to 700 mg twice daily were administered as a monotherapy to 52 subjects with advanced solid tumors. The EPA plasma concentration-time profiles were adequately described by a population PK model comprised of the first-order kinetics of oral absorption with 2-compartment distribution and constant clearance from the central compartment. Body weight was the only significant covariant to influence EPA PK. Determination of EPA's on-target potency, ie, its half-maximal inhibitory concentration (IC50 ) against IDO1, is important for dose selection but complicated by the bioconversion of tryptophan (TRP) to kynurenine (KYN) catalyzed by both IDO1 and TRP 2,3-dioxygenase (TDO). In vitro and ex vivo, the IC50 was estimated following the selective induction of IDO1, rendering the TDO activity relatively insignificant; however, it was desirable to determine the in vivo IC50 without inducing an IDO1 abundance. A mechanistic population PD model was developed based on time-matched EPA, TRP, and KYN plasma concentrations in 44 oncology patients, and EPA in vivo IC50 was estimated to be ∼70 nM, consistent with the ex vivo value independently determined. The model suggests that ∼60% and 40% of TRP→KYN bioconversion was mediated by IDO1 and TDO, respectively, in the cancer patients at baseline. For this study population of limited numbers of subjects, neither age nor sex was a significant covariate for EPA PK or PD.

Determination of epacadostat, a novel IDO1 inhibitor in mouse plasma by LC-MS/MS and its application to a pharmacokinetic study in mice.

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.

Simultaneous quantification of dabrafenib and trametinib in human plasma using high-performance liquid chromatography-tandem mass spectrometry.

Dabrafenib (Tafinlar(®)) and trametinib (Mekinist(®)) are registered for the treatment of patients with BRAF V600 mutation positive unresectable or metastatic melanoma. To support therapeutic drug monitoring (TDM) and clinical pharmacological trials, an assay to simultaneously quantify dabrafenib and trametinib in human plasma using liquid chromatography tandem mass spectrometry was developed and validated. Human plasma samples were collected on an outpatient base and stored at nominally -20°C. Analytes and internal standards (stable isotope labeled compounds) were extracted with TBME. After snap freezing the samples in a dry ice-ethanol bath, the organic layer was transferred to a clean tube and evaporated under a gentle stream of nitrogen gas. The dry extract was then reconstituted with 100µL acetonitrile and 5µL of the final extract was injected and separated on a C18 column with gradient elution, and analyzed with triple quadrupole mass spectrometry in positive-ion mode. The validated assay ranges from 50 to 5000ng/mL for dabrafenib and 0.5-50ng/mL for trametinib were linear, and correlation coefficient (r(2)) of 0.996 or better. At all concentrations of both analytes the biases were within ±15% of the nominal concentrations and precisions were ≤15%. All results were within the acceptance criteria of the latest US FDA guidance and EMA guidelines on method validation. Dabrafenib was found to degrade under the influence of light in different organic solvents and at least seven degradation products were detected. In conclusion, the described method to simultaneously quantify dabrafenib and trametinib in human plasma was successfully validated and applied for therapeutic drug monitoring in cancer patients treated with dabrafenib and trametinib.

Potential Underprediction of Warfarin Drug Interaction From Conventional Interaction Studies and Risk Mitigation: A Case Study With Epacadostat, an IDO1 Inhibitor.

Drug-drug interaction (DDI) studies involving warfarin are typically conducted with subtherapeutic doses of warfarin to ensure the safety of volunteers. However, this approach may potentially have a systemic bias of underestimating pharmacodynamic (PD) DDI effect on warfarin at therapeutic levels of anticoagulation. We demonstrate here the utility of model-based DDI prediction for a clinically relevant warfarin regimen, using the example of epacadostat (INCB024360), the first-in-class indoleamine 2,3-dioxygenase 1 inhibitor in clinical development as a novel orally active immuno-oncological therapy. Observed data from a dedicated clinical DDI study using subtherapeutic warfarin suggested warfarin pharmacokinetics (PK), but not PD (anticoagulation), was significantly affected by concomitant epacadostat. However, subsequent PK/PD modeling and simulations indicated a clinically important DDI effect on warfarin PD at a higher baseline of the international normalization ratio (INR) and enabled recommendation of warfarin dose adjustment that is dependent on epacadostat dosing regimen and target INR.

Micellar electrokinetic chromatographic method for the dabrafenib determination in biological samples.

Two different micellar electrokinetic chromatographic methods to determine dabrafenib in urine and serum, both using borate buffer (pH 9.2, 20 mM) and SDS as separation electrolyte, are developed and validated. The analyses were carried out in a fused-silica capillary of 75 µm of internal diameter and total length of 47 and 37 cm for urine and serum determination, respectively. The detection of the target compound was performed at 227 nm in urine samples and at 251 nm in serum samples. The linearity range was from 1 to 21 mg/L of dabrafenib in urine and from 2 to 40 mg/L in serum. In all cases, inter- and intraday RSDs were <4%. Sample preparation of serum samples consists of an only step of 1:1 dilution with water before its injection in the electrophoretic system. These simple, sensitive, accurate, and cost-effective methods can be used in routine clinical practice to monitor dabrafenib concentrations in urine and serum of metastatic melanoma skin cancer patients.

Validation of a liquid chromatography-triple quadrupole mass spectrometric method for the determination of 5-nitro-5'-hydroxy-indirubin-3'-oxime (AGM-130) in human plasma and its application to microdose clinical trial.

A liquid chromatography-triple quadrupole mass spectrometric (LC-MS/MS) method was developed and validated for the determination of 5-nitro-5'-hydroxy-indirubin-3'-oxime (AGM-130) in human plasma to support a microdose clinical trial. The method consisted of a liquid-liquid extraction for sample preparation and LC-MS/MS analysis in the positive ion mode using TurboIonSpray(TM) for analysis. d3 -AGM-130 was used as the internal standard. A linear regression (weighted 1/concentration) was used to fit calibration curves over the concentration range of 10-2000 pg/mL for AGM-130. There were no endogenous interference components in the blank human plasma tested. The accuracy at the lower limit of quantitation was 96.6% with a precision (coefficient of variation, CV) of 4.4%. For quality control samples at 30, 160 and 1600 pg/mL, the between run CV was ≤5.0 %. Between-run accuracy ranged from 98.1 to 101.0%. AGM-130 was stable in 50% acetonitrile for 168 h at 4°C and 6 h at room temperature. AGM-130 was also stable in human plasma at room temperature for 6 h and through three freeze-thaw cycles. The variability of selected samples for the incurred sample reanalysis was ≤12.7% when compared with the original sample concentrations. This validated LC-MS/MS method for determination of AGM-130 was used to support a phase 0 microdose clinical trial.