The Metabolism and Disposition of Brepocitinib in Humans and Characterization of the Formation Mechanism of an Aminopyridine Metabolite.

Brepocitinib is an oral once-daily Janus kinase 1 and Tyrosine kinase 2 selective inhibitor currently in development for the treatment of several autoimmune disorders. Mass balance and metabolic profiles were determined using accelerator mass spectrometry in six healthy male participants following a single oral 60 mg dose of 14C-brepocitinib (∼300 nCi). The average mass balance recovery was 96.7% ± 6.3%, with the majority of dose (88.0% ± 8.0%) recovered in urine and 8.7% ± 2.1% of the dose recovered in feces. Absorption of brepocitinib was rapid, with maximal plasma concentrations of total radioactivity and brepocitinib achieved within 0.5 hours after dosing. Circulating radioactivity consisted primarily of brepocitinib (47.8%) and metabolite M1 (37.1%) derived from hydroxylation at the C5' position of the pyrazole ring. Fractional contributions to metabolism via cytochrome P450 enzymes were determined to be 0.77 for CYP3A4/5 and 0.14 for CYP1A2 based on phenotyping studies in human liver microsomes. However, additional clinical studies are required to understand the potential contribution of CYP1A1. Approximately 83% of the dose was eliminated as N-methylpyrazolyl oxidative metabolites, with 52.1% of the dose excreted as M1 alone. Notably, M1 was not observed as a circulating metabolite in earlier metabolic profiling of human plasma from a multiple ascending dose study with unlabeled brepocitinib. Mechanistic studies revealed that M1 was highly unstable in human plasma and phosphate buffer, undergoing chemical oxidation leading to loss of the 5-hydroxy-1-methylpyrazole moiety and formation of aminopyrimidine cleavage product M2. Time-dependent inhibition and trapping studies with M1 yielded insights into the mechanism of this unusual and unexpected instability. SIGNIFICANCE STATEMENT: This study provides a detailed understanding of the disposition and metabolism of brepocitinib, a JAK1/TYK2 inhibitor for atopic dermatitis, in humans as well as characterization of clearance pathways and pharmacokinetics of brepocitinib and its metabolites.

Apixaban plasma concentrations in patients with obesity.

PURPOSE: Routine therapeutic drug monitoring of apixaban is currently not recommended but may however be warranted in some situations and for some patient groups to provide better and safer treatment. Due to limited data on apixaban concentrations in different subpopulations, it is still unclear which group of patients could possibly gain from monitoring. The purpose of this study was to examine apixaban exposure in patients with obesity compared with normal-weight patients. METHODS: Forty patients with obesity (mean BMI 39.4 kg/m2) and 40 controls with normal weight (mean BMI 23.4 kg/m2), treated with apixaban 5 mg twice daily were included. The patients were matched for age, sex, and renal function. Trough and peak apixaban concentrations were measured with LC‒MS/MS methodology. RESULTS: The median trough concentrations in patients with obesity (58.7, range 10.7-200.7 ng/ml) were slightly higher than those in patients with normal weight (52.0, range 31.0-150.9 ng/ml) (p < 0.05). Notably, the variability in trough concentration was considerably higher in patients with obesity. Peak concentrations were similar in both groups, with a median of 124.5 ng/ml (range 82.0-277.5) and 113.5 ng/ml (range 75.5-334.6) in patients with obesity and normal weight, respectively. CONCLUSION: Apixaban exposure did not vary substantially between obese and normal weight matched controls, implying that general dose adjustments are not required. However, vast interindividual variability was observed in patients with obesity, suggesting that measuring the concentrations could be valuable for specific patients. Further research is needed to identify which specific patients may benefit from this approach.

Retrospective study of clinical settings, indications and consequences of measurement of direct oral anticoagulant plasma levels in Northern Tasmania, Australia.

BACKGROUND: Routine monitoring of direct oral anticoagulant (DOAC) levels is not recommended but may be useful in certain clinical situations. There is a knowledge gap regarding the clinical use of DOAC levels in Australian hospitals. AIMS: To evaluate the clinical settings, indications and changes to anticoagulant management associated with DOAC levels in a tertiary hospital in Northern Tasmania, Australia. METHODS: Patients with one or more DOAC levels (dabigatran, rivaroxaban or apixaban) requested between January 2017 and December 2022 were identified. Retrospective chart review was performed to evaluate the clinical settings, indications, adequacy of request information and changes to clinical management associated with the measurement of DOAC levels. RESULTS: One hundred and twenty-nine DOAC measurements (54 rivaroxaban, 66 apixaban and nine dabigatran) were performed in 98 patients between January 2017 and December 2022. Annual requests for DOAC levels increased significantly between 2017 and 2019 and remained stable between 2020 and 2021 but declined in 2022. Overall, the most common indication for a DOAC level was renal impairment, followed by bleeding and recurrent thrombosis. Approximately 25% of requests were for acute bleeding with a reversal/haemostatic agent given in 45% of patients, while 10% were prior to urgent surgery. Measurement of DOAC levels was associated with a change in management in 50% of cases. 10% of requests did not specify anticoagulant history. CONCLUSION: Trends in requests for DOAC levels have changed over time. Clinician education regarding the importance of providing specific anticoagulant history is essential. Future prospective studies investigating the clinical utility of DOAC levels in different clinical settings are needed.

Assessment of exposure to direct oral anticoagulants in elderly hospitalised patients.

It is unclear whether elderly patients established on direct oral anticoagulants (DOACs) have greater exposure to these drugs, which could subsequently increase their risk of bleeding. We assessed DOAC exposure and factors affecting it in a real-world elderly cohort of patients. For this, 151 medically stable hospital inpatients (76 established on apixaban, 61 on rivaroxaban, 14 on dabigatran) with a median [interquartile range (IQR)] age of 84 (78-89) years were recruited. Patients provided blood samples for measurement of peak and trough plasma DOAC concentrations. There was up to 48-fold and 13-fold variation in trough and peak plasma drug concentrations respectively. A significantly greater proportion of patients on apixaban had peak plasma drug concentrations within the reported ranges compared to those on either rivaroxaban or dabigatran (82·9% vs. 44·3% vs. 64·3% respectively; P < 0·001). A third of the variability in DOAC plasma concentrations was attributed to the influences of DOAC dosage, renal function and gender. To what extent the observed increases in DOAC exposure in the older patients is the cause of their increased risk of bleeding, which could potentially be ameliorated by dosing titration, requires further investigation.

ABCB1 and ABCG2, but not CYP3A4 limit oral availability and brain accumulation of the RET inhibitor pralsetinib.

BACKGROUND AND PURPOSE: Pralsetinib is an FDA-approved oral small-molecule inhibitor for treatment of rearranged during transfection (RET) proto-oncogene fusion-positive non-small cell lung cancer. We investigated how the efflux transporters ABCB1 and ABCG2, the SLCO1A/1B uptake transporters and the drug-metabolizing enzyme CYP3A influence pralsetinib pharmacokinetics. EXPERIMENTAL APPROACH: In vitro, transepithelial pralsetinib transport was assessed. In vivo, pralsetinib (10 mg/kg) was administered orally to relevant genetically modified mouse models. Pralsetinib concentrations in cell medium, plasma samples and organ homogenates were measured using liquid chromatography-tandem mass spectrometry. KEY RESULTS: Pralsetinib was efficiently transported by human (h)ABCB1 and mouse (m)Abcg2, but not hACBG2. In vivo, mAbcb1a/1b markedly and mAbcg2 slightly limited pralsetinib brain penetration (6.3-and 1.8-fold, respectively). Testis distribution showed similar results. Abcb1a/1b;Abcg2-/- mice showed 1.5-fold higher plasma exposure, 23-fold increased brain penetration, and 4-fold reduced recovery of pralsetinib in the small intestinal content. mSlco1a/1b deficiency did not affect pralsetinib oral availability or tissue exposure. Oral coadministration of the ABCB1/ABCG2 inhibitor elacridar boosted pralsetinib plasma exposure (1.3-fold) and brain penetration (19.6-fold) in wild-type mice. Additionally, pralsetinib was a modest substrate of mCYP3A, but not of hCYP3A4, which did not noticeably restrict the oral availability or tissue distribution of pralsetinib. CONCLUSIONS AND IMPLICATIONS: SLCO1A/1B and CYP3A4 are unlikely to affect the pharmacokinetics of pralsetinib, but ABCG2 and especially ABCB1 markedly limit its brain and testis penetration, as well as oral availability. These effects are mostly reversed by oral coadministration of the ABCB1/ABCG2 inhibitor elacridar. These insights may be useful in the further clinical development of pralsetinib.

Direct Acting Oral Anticoagulants Following Gastrointestinal Tract Surgery.

ABSTRACT: Direct-acting oral anticoagulants (DOACs) vary in bioavailability and sites of absorption in the gastrointestinal tract (GIT). Data on DOAC use after major GIT surgery are limited. The aim of this case series was to report the impact of surgical resection or bypass of the GIT on rivaroxaban and apixaban peak plasma concentrations. This was a case series of patients who received rivaroxaban or apixaban after GIT surgery, during the period of July 1, 2019, to December 31, 2020. Peak plasma concentrations of rivaroxaban and apixaban were assessed for the expected concentrations. Of the 27 assessed patients, 18 (66.7%) received rivaroxaban, and 9 (33.3%) received apixaban. After rivaroxaban therapy, 4 of 5 patients (80%) who underwent gastrectomy, and 3 of 3 patients (100%) who underwent duodenum and proximal jejunum exclusion had peak plasma concentrations of rivaroxaban lower than the effective range, whereas 11 of 11 patients (100%) who underwent distal bowel or ileostomy had peak rivaroxaban plasma within the effective range. After apixaban therapy, 5 of 6 patients (83.3%) who underwent total or partial gastrectomy achieved effective peak concentrations. All the patients who underwent proximal and distal bowel resection or bypass had peak concentrations of apixaban within the effective range. In conclusion, surgical resection or bypass of the upper GIT could affect DOAC absorption and subsequently peak plasma concentrations. This effect was more observed among rivaroxaban recipients. An injectable anticoagulant or vitamin K antagonist may be preferred if DOAC concentrations cannot be measured after GIT surgery.

Direct Oral Anticoagulants Plasma Levels in Patients with Atrial Fibrillation at the Time of Bleeding: A Pilot Prospective Study.

ABSTRACT: Patients with atrial fibrillation (AF) on long-term direct oral anticoagulants (DOACs) may be at higher risk of bleeding because of higher anti-Xa or anti-IIa levels. However, there is no postmarketing study investigating these DOAC plasma levels at the time of bleeding. The aim of this study was to evaluate DOAC levels at the time of a bleeding emergency. We analyzed 5440 patients examined at our Emergency Department in from April 1, 2019, to September 30, 2019. During this period, we prospective identified 105 consecutive patients with bleeding while on long-term antithrombotic therapy; 49 patients had AF on DOACs. We compared DOAC levels in patients who bled against a control sample of 55 patients who tolerated long-term high dose DOAC therapy without any emergency. Samples of these patients were tested with drug-specific anti-Xa chromogenic analysis (rivaroxaban and apixaban) and with Hemoclot Thrombin Inhibitor assay (dabigatran). Dabigatran-treated patients who bled had significantly higher anti-IIa levels when compared with trough (261.4 ± 163.7 vs. 85.4 ± 57.2 ng/mL, P < 0.001) and peak samples of controls (261.4 ± 163.7 vs. 138.8 ± 78.7 ng/mL, P < 0.05). Similarly, there were significantly higher anti-Xa levels in rivaroxaban-treated and apixaban-treated patients with bleeding compared with trough control samples (rivaroxaban: 245.9 ± 150.2 vs. 52.5 ± 36.4 ng/mL, P <0.001 and apixaban: 311.8 ± 142.5 vs. 119.9 ± 81.7 ng/mL, P < 0.001), as well as in apixaban-treated patients when compared with peak control samples (311.8 ± 142.5 vs. 210.9 ± 88.7 ng/mL, P < 0.05). Finally, rivaroxaban anti-Xa levels in patients who bled tended to be higher compared with peak control samples (245.9 ± 150.2 vs. 177.6 ± 38.6 ng/mL, P = 0.13). This observational study showed a significant difference in anti-IIa and anti-Xa plasma levels in patients with AF with bleeding complications compared with those who tolerated long-term high-dose DOAC therapy without bleeding complications.

[nat/89Zr][Zr(pypa)]: Thermodynamically Stable and Kinetically Inert Binary Nonadentate Complex for Radiopharmaceutical Applications.

H4pypa is a nonadentate nonmacrocyclic chelator, which previously demonstrated high affinity for scandium-44, lutetium-177, and indium-111. Herein, we report the highly stable binary [Zr(pypa)] complex; the nonradioactive complex was synthesized and characterized in detail using high-resolution electrospray-ionization mass spectroscopy (HR-ESI-MS) and various nuclear magnetic resonance spectroscopies (NMR), which revealed C2v symmetry of the complex. The geometry of [Zr(pypa)] was further detailed via X-ray crystallography and compared with the structure of [Fe(Hpypa)]. Despite a slow complexation rate with an association half-life of 31.4 h at pH 2 and room temperature, the [Zr(pypa)] complex is thermodynamically stable (log KML = 38.92, pZr = 39.4). Radiochemical studies demonstrated quantitative radiolabeling achieved at 10 µM chelator concentration within 2 h at 40 °C and pH = 7, antibody-compatible conditions. Of the utmost importance, [89Zr][Zr(pypa)] is highly kinetically inert upon challenge with excess EDTA and DFO ligands, superior to [89Zr][Zr(DFO)]+, and maintains inertness toward human serum.

Heparin Anti-Xa Activity, a Readily Available Unique Test to Quantify Apixaban, Rivaroxaban, Fondaparinux, and Danaparoid Levels.

BACKGROUND: Despite their usefulness in perioperative and acute care settings, factor-Xa inhibitor-specific assays are scarcely available, contrary to heparin anti-Xa assay. We assessed whether the heparin anti-Xa assay can (1) be used as a screening test to rule out apixaban, rivaroxaban, fondaparinux, and danaparoid levels that contraindicate invasive procedures according to current guidelines (>30 ng·mL-1, >30 ng·mL-1, >0.1 µg·mL-1, and >0.1 IU·mL-1, respectively), (2) quantify the anticoagulant level if found significant, that is, if it exceeded the abovementioned threshold. METHODS: In the derivation cohort then in the validation cohort, via receiver operating characteristics (ROC) curve analysis, we evaluated the ability of heparin anti-Xa assay to detect levels of factor-Xa inhibitors above or below the abovementioned safety thresholds recommended for an invasive procedure (screening test). Among samples with relevant levels of factor-Xa inhibitor, we determined the conversion factor linking the measured level and heparin anti-Xa activity in a derivation cohort. In a validation cohort, the estimated level of each factor-Xa inhibitor was thus inferred from heparin anti-Xa activity. The agreement between measured and estimated levels of factor-Xa inhibitors was assessed. RESULTS: Among 989 (355 patients) and 756 blood samples (420 patients) in the derivation and validation cohort, there was a strong linear relationship between heparin anti-Xa activities and factor-Xa inhibitors measured level (r = 0.99 [95% confidence interval {CI}, 0.99-0.99]). In the derivation cohort, heparin anti-Xa activity ≤0.2, ≤0.3, <0.1, <0.1 IU·mL-1 reliably ruled out a relevant level of apixaban, rivaroxaban, fondaparinux, and danaparoid, respectively (area under the ROC curve ≥0.99). In the validation cohort, these cutoffs yielded excellent classification accuracy (≥96%). If this screening test indicated relevant level of factor-Xa inhibitor, estimated and measured levels closely agreed (Lin's correlation coefficient close to its maximal value: 95% CI, 0.99-0.99). More than 96% of the estimated levels fell into the predefined range of acceptability (ie, 80%-120% of the measured level). CONCLUSIONS: A unique simple test already widely used to assay heparin was also useful for quantifying these 4 other anticoagulants. Both clinical and economic impacts of these findings should be assessed in a specific study.

Evaluation of the in vitro stability of direct oral anticoagulants in blood samples under different storage conditions.

In this study, we evaluated the in vitro stability of direct oral anticoagulants (DOACs) in blood samples of 57 patients under different storage conditions using functional coagulation assays. We determined the analyte concentrations (1) immediately after blood collection (baseline); (2) after storage of citrated whole blood (agitated) at room temperature and citrated plasma at room temperature and at 4 °C for 4, 8, and 24 h, respectively; and (3) after storage of citrated plasma at -20 °C for 30, 60, and 90 days. According to the concept of acceptable change limits (ACL), analytes were considered stable if the mean relative analyte recovery at a given time was >78%. The mean baseline values (range) of dabigatran, rivaroxaban, apixaban, and edoxaban were 115 ng/mL (62-217), 129 ng/mL (31-215), 156 ng/mL (49-362), and 101 ng/mL (33-283), respectively. After applying the analyte stability limit, all four DOACs were stable for 24 h at room temperature and at 4 °C. The mean recovery after 24 h was 102-111% for dabigatran, 88-97% for rivaroxaban, 95-98% for apixaban, and 90-96% for edoxaban. When plasma samples were stored at -20 °C, the mean percentage deviation after 90 days for all four DOACs was ≤10%, even after three freeze-thaw cycles. Thus, for the correct determination of DOAC plasma concentrations, blood samples do not have to be analyzed immediately and can be stored at room temperature for up to 24 h before analysis. In clinical practice, blood sample transport and storage for DOAC measurements appear to be unproblematic.

Interference of eltrombopag with bilirubin measurements on the Vitros 5600 analyzer.

BACKGROUND: Eltrombopag is a thrombopoietin-receptor agonist used to restore platelet count to hemostatic levels in chronic immune thrombocytopenia. The drug has shown to have hepatobiliary adverse effects, but also positive interference with the analytical measurement of bilirubin. Understanding the degree of interference of this drug with bilirubin testing becomes relevant in the clinical management of these patients. METHODS: Eltrombopag at concentrations ranging from 10 to 150 µg/ml was spiked into plasma samples with different baseline concentrations of bilirubin. Total bilirubin, conjugated, and unconjugated bilirubin were measured for each sample using VITROS TBILI and BuBc slides on the Vitros 5600 automated chemistry platform, and interference was assessed. RESULTS: Plasma samples spiked with eltrombopag yielded falsely elevated bilirubin measurements compared to baseline, with the degree of elevation increasing with greater concentrations of eltrombopag. Bilirubin values were increased relative to baseline across all groups, except in conjugated bilirubin measurements in samples with low baseline conjugated bilirubin. For samples with low total bilirubin at baseline, >100 µg/ml of eltrombopag resulted in an error of >+0.6 mg/dl on the measured total bilirubin. For samples with low unconjugated bilirubin at baseline, the error for the same concentrations was >+0.7 mg/dl. CONCLUSION: Our results show that, at supra-physiologically high concentrations, eltrombopag can positively interfere with bilirubin measurements on Vitros 5600 platform.

A simple HPLC assay for determining eltrombopag concentration in human serum.

Eltrombopag, a thrombopoietin receptor agonist, is used for the treatment of idiopathic thrombocytopenic purpura (ITP) and aplastic anemia. We developed a HPLC assay for the determination of serum eltrombopag concentration in ITP patients. An aliquot of a serum sample spiked with diclofenac as the internal standard (IS) was treated with acetonitrile to precipitate the proteins. Eltrombopag and the IS were separated on an octadecylsilyl silica-gel column using a mobile phase consisting of 10 mM 1-pentanesulfonic acid sodium salt, acetonitrile, and acetic acid. The detection wavelength was set at 265 nm. The calibration curve was linear at the concentration range of 0.15-12.5 µg/mL for eltrombopag (r = 0.9987). The recoveries of eltrombopag from the serum samples were greater than 95.9% with coefficients of variation (CVs) being less than 2.8%. The CVs for the intra-day and inter-day assays were 1.9-11.8% and 1.0-11.8%, respectively. This assay method could be used for therapeutic drug monitoring of eltrombopag in ITP patients.

Determination of selpercatinib, a RET kinase inhibitor, in rat plasma and its application to a pharmacokinetic study.

Selpercatinib (LOXO-292) is a selective and potent RET inhibitor. A highly sensitive, rapid and specific high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for quantification of selpercatinib in rat plasma is reported. The method was validated in terms of selectivity, linearity, accuracy and precision, extraction recovery and matrix effect, stability and carryover as per the US Food and Drug Administration guidelines for bioanalytical method validation. Selpercatinib was detected by an electrospray ionization interface under selective reaction monitoring conditions in the positive ion mode. The calibration curve was linear over the concentration range from 1 to 2000 ng/ml with r2 = 0.9951. The intra- and inter-batch accuracy values ranged from 97.45 to 100.97% and from 98.70 to 100.74% with coefficients of variation of 2.45-6.99% and 5.89-7.99%, respectively. The extraction recovery and IS-normalized matrix factor were acceptable for the bioanalysis of selpercatinib. Additionally, selpercatinib was found to be stable under the detected conditions. It showed linear pharmacokinetic characteristics following oral administration to rats at 2.0-18.0 mg/kg. The results showed that the novel method for detecting selpercatinib in rat plasma could be successfully applied for quantification of selpercatinib in biosamples from nonclinical studies.

Physiologically-based pharmacokinetic model for alectinib, ruxolitinib, and panobinostat in the presence of cancer, renal impairment, and hepatic impairment.

Renal (RIP) and hepatic (HIP) impairments are prevalent conditions in cancer patients. They can cause changes in gastric emptying time, albumin levels, hematocrit, glomerular filtration rate, hepatic functional volume, blood flow rates, and metabolic activity that can modify drug pharmacokinetics. Performing clinical studies in such populations has ethical and practical issues. Using predictive physiologically-based pharmacokinetic (PBPK) models in the evaluation of the PK of alectinib, ruxolitinib, and panobinostat exposures in the presence of cancer, RIP, and HIP can help in using optimal doses with lower toxicity in these populations. Verified PBPK models were customized under scrutiny to account for the pathophysiological changes induced in these diseases. The PBPK model-predicted plasma exposures in patients with different health conditions within average 2-fold error. The PBPK model predicted an area under the curve ratio (AUCR) of 1, and 1.8, for ruxolitinib and panobinostat, respectively, in the presence of severe RIP. On the other hand, the severe HIP was associated with AUCR of 1.4, 2.9, and 1.8 for alectinib, ruxolitinib, and panobinostat, respectively, in agreement with the observed AUCR. Moreover, the PBPK model predicted that alectinib therapeutic cerebrospinal fluid levels are achieved in patients with non-small cell lung cancer, moderate HIP, and severe HIP at 1-, 1.5-, and 1.8-fold that of healthy subjects. The customized PBPK models showed promising ethical alternatives for simulating clinical studies in patients with cancer, RIP, and HIP. More work is needed to quantify other pathophysiological changes induced by simultaneous affliction by cancer and RIP or HIP.

P-glycoprotein (MDR1/ABCB1) controls brain accumulation and intestinal disposition of the novel TGF-ß signaling pathway inhibitor galunisertib.

Galunisertib (LY2157299), a promising small-molecule inhibitor of the transforming growth factor-beta (TGF-ß) receptor, is currently in mono- and combination therapy trials for various cancers including glioblastoma, hepatocellular carcinoma and breast cancer. Using genetically modified mouse models, we investigated the roles of the multidrug efflux transporters ABCB1 and ABCG2, the OATP1A/1B uptake transporters and the drug-metabolizing CYP3A complex in galunisertib pharmacokinetics. In vitro, galunisertib was vigorously transported by human ABCB1, and moderately by mouse Abcg2. Orally administered galunisertib (20 mg/kg) was very rapidly absorbed. Galunisertib brain-to-plasma ratios were increased by ~24-fold in Abcb1a/1b-/- and Abcb1a/1b;Abcg2-/- mice compared to wild-type mice, but not in single Abcg2-/- mice, whereas galunisertib oral availability was not markedly affected. However, recovery of galunisertib in the small intestinal lumen was strongly reduced in Abcb1a/1b-/- and Abcb1a/1b;Abcg2-/- mice. Oral coadministration of the ABCB1/ABCG2 inhibitor elacridar boosted galunisertib brain accumulation in wild-type mice to equal the levels seen in Abcb1a/1b;Abcg2-/- mice. Oatp1a/1b deficiency did not alter oral galunisertib pharmacokinetics or liver distribution. Cyp3a-/- mice showed a 1.9-fold higher plasma AUC0-1 hr than wild-type mice, but this difference disappeared over 8 hr. Also, transgenic human CYP3A4 overexpression did not significantly alter oral galunisertib pharmacokinetics. Abcb1 thus markedly restricts galunisertib brain penetration and affects its intestinal disposition, possibly through biliary excretion. Elacridar coadministration could fully inhibit both processes, without causing acute toxicity. Moreover, mouse Cyp3a, but not human CYP3A4, may eliminate galunisertib at high plasma concentrations. These insights may help to guide the further clinical development and application of galunisertib.

Sensitive CE-MS method for monitoring of riociguat and desmethylriociguat levels in human serum.

Riociguat is novel antihypertensive drug for treatment of pulmonary hypertension. As such, it is still being tested in many clinical and pharmacokinetic trials. Existing methods that determine serum riociguat and desmethylriociguat (DMR) are based solely on liquid chromatography with mass spectrometry. Therefore, we present a novel capillary electrophoresis with mass spectrometry method (CE-MS) for their determination in human serum as alternative method for ongoing trials. Complete resolution of both analytes was achieved by means of pH optimization of ammonium formate background electrolytes that are fully compatible with ESI/MS detection. Simple liquid-liquid extraction was used as sample pretreatment. The calibration dependence of the method was linear (in the range of 10-1000 ng/mL), with adequate accuracy (90.1-114.9%) and precision (13.4%). LOD and LOQ were arbitrarily set at 10 ng/mL for both analytes. Clinical applicability was validated using serum samples from patients treated with riociguat in pharmacokinetic study and the results corresponded with reference HPLC-MS/MS values. Capillary electrophoresis proved to be sensitive and selective tool for the analysis of riociguat and DMR.

A phase 1 study of oral ASP5878, a selective small-molecule inhibitor of fibroblast growth factor receptors 1-4, as a single dose and multiple doses in patients with solid malignancies.

ASP5878 is a selective small-molecule inhibitor of fibroblast growth factor receptors (FGFRs). This study investigated safety, tolerability, and antitumor effect of single and multiple oral doses of ASP5878 in patients with solid tumors. This phase 1, open label, first-in-human study comprised dose-escalation and dose-expansion parts. Primary objectives of the dose-escalation part were to identify the dose-limiting toxicity (DLT), maximum tolerated dose, and recommended dose of ASP5878 for the dose-expansion part. Nine dose cohorts of ASP5878 were evaluated (0.5─2 mg once daily; 2─40 mg twice daily [BID]). A single dose of ASP5878 was followed by a 2-day pharmacokinetic collection, and then either 28-day cycles of daily dosing (ASP5878 ≤ 10 mg BID) or 5-day dosing/2-day interruption (ASP5878 ≥ 20 mg BID). The primary objective of the dose-expansion part was to determine the safety of ASP5878 (16 mg BID) administered in 28-day cycles of 5-day dosing/2-day interruption in patients with urothelial carcinoma, hepatocellular carcinoma, or squamous cell lung carcinoma with FGFR genetic alterations. Safety was assessed by monitoring adverse events (AEs). Thirty-five patients were enrolled and 31 discontinued in the dose-escalation part; 51 patients were enrolled and 51 discontinued in the dose-expansion part. In the dose-escalation part, 66.7% of patients in the 20 mg BID 5-day dosing/2-day interruption group reported DLTs of hyperphosphatemia. The recommended dose for the dose-expansion part was 16 mg BID. Common AEs included retinal detachment, diarrhea, and increased alanine aminotransferase. One death occurred that was not related to ASP5878. ASP5878 was well tolerated with manageable toxicities including hyperphosphatemia.

Simultaneous Determination of Dabigatran, Rivaroxaban, and Apixaban in Human Plasma by Liquid Chromatography/Tandem Mass Spectrometry.

BACKGROUND: Pharmacokinetic studies and therapeutic drug monitoring of anticoagulants require a simple, rapid, and reliable analytical method for monitoring plasma concentrations. The aims of the current work were to develop and validate a liquid chromatography/tandem mass spectrometry method for the simultaneous determination of 3 direct oral anticoagulants (dabigatran, rivaroxaban, and apixaban) in human plasma that is suitable for pharmacokinetic studies and routine therapeutic drug monitoring in busy hospital laboratories. METHODS: This method included a hydrolysis step to account for the active acylglucuronide metabolites of dabigatran that demonstrate an equivalent anticoagulant effect as dabigatran. After hydrolysis, a simple one-step protein precipitation was used for sample preparation. Total dabigatran (the sum of free dabigatran and the contribution from dabigatran acylglucuronides), rivaroxaban, and apixaban, and their corresponding isotopically labeled internal standards were resolved on a C18(2) column. All compounds were detected using electrospray ionization liquid chromatography/tandem mass spectrometry in the positive mode. RESULTS: For all 3 anticoagulants, standard curves were linear over the concentration range of 1.0-1000 mcg/L (r > 0.99), bias was < ±10%, and intraday and interday coefficients of variation (imprecision) were <10%. The limit of quantification was 1.0 mcg/L. For all 3 anticoagulants and corresponding isotopically labeled internal standards, the absolute recoveries were similar and consistent, with mean values of 93%-102%. No significant matrix effects were observed. CONCLUSIONS: This method is simple, rapid, robust, and reliable and can be used to analyze the plasma concentrations of the drugs in patients on dabigatran or rivaroxaban therapy.

Comprehensive characterization and resolution of discrepant spectrophotometric bilirubin results in patients on eltrombopag therapy.

Background Eltrombopag is a thrombopoietin receptor agonist used for the treatment of thrombocytopenic conditions. It can cause pH-dependent discoloration of plasma/serum. Eltrombopag is potentially hepatotoxic. It can affect the assessment of hyperbilirubinemia because of its (i) absorbance at ~450 nm (bilirubin), (ii) absorbance at ~550 nm (diazo-bilirubin) and (iii) it can cause yellowish discoloration of the eyes at normal circulating bilirubin levels. Methods We collected 66 samples from patients on a range of eltrombopag dosages up to 150 mg daily. Bilirubin was measured using multiple routine spectrophotometric analyzers, the Doumas reference method and high-performance liquid chromatography (HPLC). Plasma/serum eltrombopag concentrations were determined using liquid chromatography tandem mass spectrometry (LC-MS/MS). Spike-in and admixture experiments delineated the effects of eltrombopag and its metabolites. Results Forty-nine of 52 samples from patients on ≥50 mg daily eltrombopag therapy showed significantly discrepant inter-analyzer total bilirubin results, a difference up to 64 µmol/L (3.7 mg/dL). In one sample, total bilirubin varied from 8 to 65 µmol/L (0.4-3.8 mg/dL) by different routine analyzers, with direct bilirubin ≤4 µmol/L (0.2 mg/dL). There was a positive correlation between total bilirubin difference and plasma eltrombopag concentration (r = 0.679), and spike-in experiments demonstrated that Beckman AU and Doumas reference methods were susceptible to positive interference. HPLC can quantify bilirubin after separating eltrombopag, and results suggest different analyzers are affected to varying degrees by eltrombopag and its metabolites. Conclusions Eltrombopag and its metabolites can cause positive interference to the spectrophotometric measurements of total bilirubin. Accurate measurements of total bilirubin may improve our understanding of the prevalence of hyperbilirubinemia in patients on eltrombopag therapy.

Clinical implications of assessment of apixaban levels in elderly atrial fibrillation patients: J-ELD AF registry sub-cohort analysis.

PURPOSE: To investigate the distribution of plasma apixaban levels and their relationships with clinical outcomes in elderly patients with atrial fibrillation (AF). METHOD: The J-ELD AF Registry is a multicenter prospective observational study of Japanese non-valvular AF patients aged ≥75 years taking an on-label dose of apixaban (3015 patients from 110 institutions). Among them, plasma apixaban levels at trough were estimated by anti-Xa assay (Api-AXA) in 943 patients. Patients with standard (5 mg bid; n = 431) and reduced (2.5 mg bid; n = 512) dose were further divided into two groups with low and high Api-AXA levels (boundary: median value). RESULTS: The incidence rates (per 100 person-years) of events in the low- and high-Api-AXA groups were as follows: 1.48 and 1.99 (log-rank test, P = 0.695) for stroke or systemic embolism, 0.98 and 1.49 (P = 0.652) for bleeding requiring hospitalization, and 0.49 and 0.99 (P = 0.565) for total deaths in patients with standard dose, versus 0.84 and 1.68 (P = 0.414), 0.42 and 4.64 (P = 0.004), and 2.52 and 6.65 (P = 0.035) in patients with a reduced dose, respectively. In multivariate Cox regression analysis among patients with a reduced dose, a high Api-AXA level was independently associated with bleeding requiring hospitalization (HR 12.12, 95% CI: 1.56-94.22) and nonsignificantly with total deaths. CONCLUSIONS: A high trough apixaban level in patients indicated for standard dose was not associated with adverse events, while a high apixaban level in patients indicated for a reduced dose was associated with bleeding requiring hospitalization.