Linearity Characterization and Uncertainty Quantification of Spectroradiometers via Maximum Likelihood and the Non-parametric Bootstrap.

A technique for characterizing and correcting the linearity of radiometric instruments is known by the names the "flux-addition method" and the "combinatorial technique". In this paper, we develop a rigorous uncertainty quantification method for use with this technique and illustrate its use with both synthetic data and experimental data from a "beam conjoiner" instrument. We present a probabilistic model that relates the instrument readout to a set of unknown fluxes via a set of polynomial coefficients. Maximum likelihood estimates (MLEs) of the unknown fluxes and polynomial coefficients are recommended, while a non-parametric bootstrap algorithm enables uncertainty quantification including standard errors and confidence intervals. The synthetic data represent plausible outputs of a radiometric instrument and enable testing and validation of the method. The MLEs for these data are found to be approximately unbiased, and confidence intervals derived from the bootstrap replicates are found to be consistent with their target coverage of 95%. For the polynomial coefficients, the observed coverages range from 91% to 99%. The experimental data set illustrates how a complete calibration with uncertainties can be achieved using the method plus one well-known flux level. The uncertainty contribution attributable to estimation of the instrument's nonlinear response is less than 0.025% over most of its range.

Quantification of azacitidine incorporation into human DNA/RNA by accelerator mass spectrometry as direct measure of target engagement.

We report an accelerator mass spectrometry (AMS) assay to quantify azacitidine (Aza) incorporation into DNA and RNA from human acute myeloid leukemia (AML) cells, mouse bone marrow (BM) and peripheral blood mononuclear cells (PBMCs). Aza, a cytidine nucleoside analogue, is a disease modifying pharmacological agent used for treatment of myelodysplastic syndromes (MDS) and AML. Our assay was able to directly quantify the complex of Aza incorporated into DNA/RNA, via isolation of DNA/RNA from matrix (i.e., cancer cells, BM and PBMC) and subsequent measurement of total radioactivity (i.e., 14C-Aza) by using AMS. The sensitivity of the method was able to quantify as little as a single Aza molecule incorporated into DNA with approximately 2 × 107 nucleotides from PBMCs. An in vivo mouse model was used for establishing the lower limits of quantification (LLOQs) for Aza incorporated into DNA/RNA in mouse PBMCs (∼ 3.7 × 105) and BM (∼27.8 mg) collected 24 h post-dose after total exposure of 18 nCi/mouse (Aza 1 mg/kg). The LLOQs for PBMC analysis were 2.5 picogram equivalents per microgram (pgEq/µg) DNA and 0.22 pgEq/µg RNA, and for BM analysis were 1.7 pgEq/µg DNA and 0.22 pgEq/µg RNA. A linear relationship (i.e., ∼10-fold) was established of radioactive dose from 14C-Aza 17 nCi/mouse to 188 nCi/mouse and AMS response (i.e., 14C/12C ratio ranging from 2.45 × 10-11 to 2.50 × 10-10), as Aza was incorporated into DNA in mouse BM. The current method enables the direct measurement of Aza incorporation into DNA and RNA from patient PBMCs and BM to provide dosing optimization, and to assess target engagement with as little as ∼5 mL whole blood and ∼3 mL of BM from patients.

Population Pharmacokinetics and Exposure-Response Relationship of Luspatercept, an Erythroid Maturation Agent, in Anemic Patients With ß-Thalassemia.

ß-Thalassemia is an inherited blood disorder resulting from defects in hemoglobin production, leading to premature death of red blood cells (RBCs) or their precursors. Patients with transfusion-dependent ß-thalassemia often need lifelong regular RBC transfusions to maintain adequate hemoglobin levels. Frequent transfusions may lead to iron overload and organ damage. Thus, there is a large unmet need for alternative therapies. Luspatercept, a first-in-class erythroid maturation agent, is the first approved therapy in the United States for the treatment of anemia in adult patients with ß-thalassemia who require regular RBC transfusions. The population pharmacokinetics and exposure-response relationship of luspatercept were evaluated in 285 patients with ß-thalassemia. Luspatercept displayed linear and time-invariant pharmacokinetics when administered subcutaneously once every 3 weeks. Body weight was the only clinically relevant covariate of luspatercept clearance, favoring weight-based dosing. Magnitude and frequency of hemoglobin increase, if not influenced by RBC transfusions, was positively correlated with luspatercept area under the serum concentration-time curve (AUC), 0.2-1.25 mg/kg, whereas a significant reduction in RBC units transfused was observed in frequently transfused patients. The probability of achieving ≥33% or ≥50% reduction in RBC transfusion burden was similar across the time-averaged AUC (0.6-1.25 mg/kg), with the 1 mg/kg starting dose sufficient for most early responders (71%-80%). Increasing luspatercept AUC (0.2-1.25 mg/kg) did not increase incidence or severity of treatment-emergent adverse events. These results provide a positive benefit-risk profile for the recommended luspatercept doses (1-1.25 mg/kg) in treating adult patients with ß-thalassemia who require regular RBC transfusions.

Population Pharmacokinetics and Exposure-Response of Luspatercept, an Erythroid Maturation Agent, in Anemic Patients With Myelodysplastic Syndromes.

Luspatercept is a recombinant fusion protein that enhances late-stage erythroid maturation. This report describes the population pharmacokinetics and exposure-response relationship of luspatercept in 260 patients with anemia due to myelodysplastic syndromes. Luspatercept displayed linear and time-invariant pharmacokinetics over a dose range of 0.125-1.75 mg/kg administered subcutaneously once every 3 weeks. Body weight was the only clinically relevant covariate of luspatercept exposure, supporting the weight-based dosing. The probability of achieving transfusion independence ≥ 8 weeks increased with time-averaged luspatercept serum exposure, reaching the plateau at doses 1.0-1.75 mg/kg. The probability of achieving multiple efficacy end points increased with slower luspatercept clearance, independent of effects of luspatercept exposure or disease characteristics. The probability of experiencing severe treatment-emergent adverse events decreased with increasing luspatercept exposure, especially during long-term treatment. These results provide a positive benefit-risk profile for the titration-to-response dose regimen (1.0-1.75 mg/kg) recommended for this population.

Population Pharmacokinetics of an Anti-PD-L1 Antibody, Durvalumab in Patients with Hematologic Malignancies.

BACKGROUND AND OBJECTIVES: Durvalumab, a human monoclonal antibody targeting programmed cell death ligand 1, has been approved for urothelial carcinoma and stage III non-small cell lung cancer by the US Food and Drug Administration and is being evaluated in various malignancies. The objective of this study was to develop a population-pharmacokinetic model of durvalumab in patients with various hematologic malignancies and to investigate the effects of demographic and disease factors on the pharmacokinetics in this population. METHODS: A total of 1812 concentrations from 267 patients with myelodysplastic syndromes, acute myeloid leukemia, multiple myeloma, non-Hodgkin lymphoma, or Hodgkin lymphoma were included in the analysis. RESULTS: The pharmacokinetics of durvalumab was adequately described by a two-compartment model with first-order elimination. A decrease in durvalumab clearance over time was mainly explained by incorporation of time-dependent changes in albumin (in all patients) and immunoglobulin G (in patients with multiple myeloma) into the model. For multiple myeloma, patients with immunoglobulin G ≥ 20 g/L showed a 30% lower area under the concentration-time curve at cycle 1 compared with patients with immunoglobulin G < 20 g/L. The impact of any baseline covariates on durvalumab pharmacokinetics did not appear to be clinically relevant. The pharmacokinetics of durvalumab in hematologic malignancies was generally consistent with previously reported pharmacokinetics in solid tumors. CONCLUSIONS: These results support the same dosing regimen (1500 mg every 4 weeks) for both solid tumors and hematologic malignancies from the perspective of adequate exposure. Additionally, total immunoglobulin G level could be a critical covariate for the pharmacokinetics of monoclonal antibodies in patients with multiple myeloma.

Quantification of XRCC and DNA-PK proteins in cancer cell lines and human tumors by LC-MS/MS.

BACKGROUND: The x-ray repair cross-complementing (XRCC) proteins and a catalytic subunit of nuclear DNA-dependent serine/threonine protein kinase (DNA-PK) play important roles in cancer biology. Understanding the protein expression levels allows us to reconstruct in vivo functionality and to qualify protein biomarkers. METHODS & RESULTS: XRCC and DNA-PK proteins in human cancer cells and tumor tissues have been identified and quantified by selected peptides using NanoLC and high-resolution mass spectrometry. The stable isotope-labeled full-length protein XRCC4 ([(13)C6, (15)N4]-arginine and [(13)C6, (15)N2]-lysine) uses as the internal standard. CONCLUSION: The assay range is 0.140-450 fmol (coefficient of variation: 25%) for XRCC4 in bovine serum albumen. The quantitative protein expression levels for XRCC and DNA-PK in HeLa, Ramos and HEK-293 cells and tumor tissues (lung and lymphoma) are reported.

No differences in the pharmacodynamics and pharmacokinetics of the thrombin receptor antagonist vorapaxar between healthy Japanese and Caucasian subjects.

BACKGROUND: Vorapaxar, a novel antiplatelet agent in advanced clinical development for the prevention and treatment of atherothrombotic disease, is a potent, orally bioavailable thrombin receptor antagonist selective for the protease-activated receptor 1 (PAR-1). METHODS: Since race/ethnicity may affect the safety, efficacy and dosage of drugs, this study was conducted to evaluate potential differences in the pharmacodynamics, pharmacokinetics and safety of vorapaxar after single (5, 10, 20, or 40 mg) or multiple (0.5, 1, or 2.5 mg once daily) doses in healthy Japanese and matched (gender, age, height, and weight) Caucasian volunteers. RESULTS: Vorapaxar was well tolerated in both Japanese and Caucasian subjects. Pharmacodynamic and pharmacokinetic profiles of vorapaxar in the two racial/ethnic groups were similar. In both racial groups, complete inhibition of platelet aggregation was achieved most rapidly with vorapaxar 40 mg and was consistently achieved and maintained with a 2.5 mg daily maintenance dose. CONCLUSION: There were no substantial differences in the safety, pharmacokinetics or pharmacodynamics of vorapaxar between Japanese and Caucasian subjects.

Interaction of single-dose ezetimibe and steady-state cyclosporine in renal transplant patients.

This open-label, single-period study evaluated the single-dose pharmacokinetics of ezetimibe (EZE) 10 mg in the setting of steady-state cyclosporine (CyA) dosing in renal transplant patients. A single 10-mg dose of EZE was coadministered with the morning dose of CyA (75-150 mg twice a day). Total EZE (sum of unconjugated, parent EZE and EZE-glucuronide; EZE-total) AUC(0-last) and Cmax were compared to values derived from a prespecified database of healthy volunteers. Geometric mean ratios (90% CIs) for (EZE + CyA)/EZE alone for EZE-total AUC((0-last)) and Cmax were 3.41 (2.55, 4.56) and 3.91 (3.13, 4.89), respectively. Compared to healthy controls, EZE-total AUC((0-last)) was 3.4-fold higher in transplant patients receiving CyA; similar exposure levels were seen in a prior multiple-dose study in which EZE 50 mg was administered to healthy volunteers without dose-related toxicity. Because the long-term safety implications of both higher EZE exposures and undetermined effect on CyA are not yet understood, the clinical significance of this interaction is unknown.

Pharmacodynamic interaction between ezetimibe and rosuvastatin.

BACKGROUND: Ezetimibe is a lipid-lowering drug indicated for the treatment of hypercholesterolemia as co-administration with HMG-CoA reductase inhibitors (statins) or as monotherapy. The primary objectives of this study were to evaluate the pharmacodynamic effects and safety of the co-administration of ezetimibe and the new statin rosuvastatin. A secondary objective was to examine the potential for a pharmacokinetic interaction between ezetimibe and rosuvastatin. METHODS: This was a randomized, evaluator (single)-blind, placebo-controlled, parallel-group study in healthy hypercholesterolemic subjects (untreated low-density lipoprotein cholesterol [LDL-C] > or = 130 mg/dL [3.37 mmol/L]). After the outpatient screening and NCEP Step I diet stabilization periods, 40 subjects were randomized to one of the 4 following treatments: rosuvastatin 10 mg plus ezetimibe 10 mg (n = 12); rosuvastatin 10 mg plus placebo (matching ezetimibe 10 mg) (n = 12); ezetimibe 10 mg plus placebo (matching ezetimibe 10 mg) (n = 8); or placebo (2 tablets, matching ezetimibe 10 mg) (n = 8). All study treatments were administered once daily in the morning for 14 days as part of a 16-day inpatient confinement period. Fasting serum lipids were assessed pre-dose on days 1 (baseline), 7, and 14 by direct quantitative assay methods. Safety was evaluated by monitoring laboratory tests and recording adverse events. Blood samples were collected for ezetimibe and rosuvastatin pharmacokinetic evaluation prior to the first and last dose and at frequent intervals after the last dose (day 14) of study treatment. Plasma ezetimibe, total ezetimibe (ezetimibe plus ezetimibe-glucuronide) and rosuvastatin concentrations were determined by validated liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) assay methods. RESULTS: All active treatments caused statistically significant (p < or = 0.02) decreases in LDL-C concentration versus placebo from baseline to day 14. The co-administration of ezetimibe and rosuvastatin caused a significantly (p < 0.01) greater reduction in LDL-C and total cholesterol than either drug alone. In this 2-week inpatient study with restricted physical activity there was no apparent effect of any treatment on high-density lipoprotein cholesterol (HDL-C) or triglycerides. The co-administration of ezetimibe and rosuvastatin caused a significantly (p < 0.01) greater percentage reduction in mean LDL-C (-61.4%) than rosuvastatin alone (-44.9%), with a mean incremental reduction of -16.4% (95%CI -26.3 to -6.53). Reported side effects were generally mild, nonspecific, and similar among treatment groups. There were no significant increases or changes in clinical laboratory tests, particularly those assessing muscle and liver function. There was no significant pharmacokinetic drug interaction between ezetimibe and rosuvastatin. CONCLUSIONS: Co-administration of ezetimibe 10 mg with rosuvastatin 10 mg daily caused a significant incremental reduction in LDL-C compared with rosuvastatin alone. Moreover, co-administering ezetimibe and rosuvastatin was well tolerated in patients with hypercholesterolemia.

Pharmacodynamic and pharmacokinetic interaction between fenofibrate and ezetimibe.

OBJECTIVE: The cholesterol absorption inhibitor, ezetimibe, significantly decreases low-density lipoprotein-cholesterol (LDL-C) levels in patients with primary hypercholesterolemia. The pharmacodynamic, pharmacokinetic, and safety profiles of ezetimibe and fenofibrate were evaluated alone and after co-administration in 32 subjects with primary hypercholesterolemia. RESEARCH DESIGN AND METHODS: This was a randomized, evaluator (single)-blind, placebo-controlled, parallel-group study. Subjects with untreated LDL-C > or = 130 mg/dL (3.37 mmol/L) were randomized to receive one of four oral treatments each morning for 14 days: fenofibrate 200 mg + ezetimibe 10 mg, fenofibrate 200 mg, ezetimibe 10 mg, or placebo. Serum lipids were assessed before drug administration on day 1, day 7, and day 14. Pharmacokinetic parameters were assessed on day 14. MAIN OUTCOME MEASURES: The primary pharmacodynamic parameter was percentage change from baseline in LDL-C concentration following co-administration of ezetimibe and fenofibrate vs either drug alone, or placebo. A secondary outcome was the potential for a pharmacokinetic interaction between ezetimibe and fenofibrate. RESULTS: Ezetimibe and fenofibrate co-administration was well tolerated and produced statistically significant mean percentage reductions from baseline in LDL-C (p < or = 0.05 vs either drug alone or placebo), total cholesterol and triglycerides (p < or = 0.05 vs either fenofibrate or placebo), apolipoprotein C-III (p < or = 0.05 vs placebo), and LDL-III (p < or = 0.05 vs either drug alone or placebo). Ezetimibe did not significantly affect the pharmacokinetics of fenofibrate. Concomitant fenofibrate administration significantly increased the mean C(max) and AUC of total ezetimibe approximately 64% and 48%, respectively. However, based on the established safety profile and flat dose-response of ezetimibe, this effect is not considered to be clinically significant. CONCLUSION: Co-administration of ezetimibe and fenofibrate produced significantly greater reductions in LDL-C than either drug alone and greater reductions in triglycerides than fenofibrate. These effects were accompanied by improvements in the lipid/lipoprotein profile, suggesting that co-administration therapy with ezetimibe and fenofibrate may be an effective therapeutic option for patients with mixed dyslipidemia.

Effects of ezetimibe on the pharmacodynamics and pharmacokinetics of lovastatin.

BACKGROUND: Ezetimibe is a cholesterol absorption inhibitor which decreases low-density lipoprotein cholesterol (LDL-C) in patients with hypercholesterolemia. This study investigated the potential for pharmacodynamic and/or pharmacokinetic interactions between ezetimibe and lovastatin. METHODS: In a randomized, evaluator (single)-blind, placebo-controlled, parallel-group study, 48 healthy men with hypercholesterolemia (screening LDL-C >or= 130 mg/dL) who were stabilized and maintained on a National Cholesterol Education Program (NCEP) Step I diet were randomized to one of the following six oral treatments once daily for 14 days: lovastatin 20 mg; lovastatin 20 mg plus ezetimibe 5, 10, or 20 mg; lovastatin 40 mg plus ezetimibe 10mg; or placebo. RESULTS: Reported adverse events were generally mild, nonspecific, and similar among treatments. There were no significant changes in safety laboratory test results, including those for enzymes indicative of muscle or liver injury. Coadministration of ezetimibe and lovastatin did not increase the plasma concentrations of lovastatin or beta-hydroxylovastatin. In this parallel comparison study there was an apparent decrease in lovastatin exposure, however, the reduction in lovastatin or beta-hydroxylovastatin concentrations was not related to the ezetimibe dose and is not considered to be clinically important. Ezetimibe 5, 10, or 20 mg combined with lovastatin 20 mg caused a significantly (p < 0.01) greater reduction in LDL-C than lovastatin 20 mg alone, with no apparent effect on HDL-C or triglycerides. LDL-C was reduced by 51.0% with ezetimibe 10 mg plus lovastatin 20 mg, 56.0% with ezetimibe 10 mg plus lovastatin 40 mg, 33.2% with lovastatin alone, and 17.3% with placebo. CONCLUSIONS: The co-administration of ezetimibe and lovastatin was well tolerated and resulted in a significantly greater percentage reduction in serum LDL-C concentrations than with lovastatin alone, with an average incremental reduction of 16-18%. Ezetimibe 10mg appears to be the optimal dose when co-administered with lovastatin 20mg once daily. Further incremental reductions in LDL-C from the co-administration of ezetimibe and lovastatin are expected only when the dose of lovastatin is increased. The co-administration of ezetimibe and lovastatin has the potential to produce clinically significant reductions in LDL-C compared to either drug alone, with favorable safety and tolerability.

Pharmacodynamic interaction between the new selective cholesterol absorption inhibitor ezetimibe and simvastatin.

AIMS: The primary aims of these two single-centre, randomized, evaluator-blind, placebo/positive-controlled, parallel-group studies were to evaluate the potential for pharmacodynamic and pharmacokinetic interaction between ezetimibe 0.25, 1, or 10 mg and simvastatin 10 mg (Study 1), and a pharmacodynamic interaction between ezetimibe 10 mg and simvastatin 20 mg (Study 2). Evaluation of the tolerance of the coadministration of ezetimibe and simvastatin was a secondary objective. METHODS: Eighty-two healthy men with low-density lipoprotein cholesterol (LDL-C) >or=130 mg dl-1 received study drug once daily in the morning for 14 days. In Study 1 (n=58), five groups of 11-12 subjects received simvastatin 10 mg alone, or with ezetimibe 0.25, 1, or 10 mg or placebo. In Study 2 (n=24), three groups of eight subjects received simvastatin 20 mg alone, ezetimibe 10 mg alone, or the combination. Blood samples were collected to measure serum lipids in both studies. Steady-state pharmacokinetics of simvastatin and its beta-hydroxy metabolite were evaluated in Study 1 only. RESULTS: In both studies, reported side-effects were generally mild, nonspecific, and similar among treatment groups. In Study 1, there were no indications of pharmacokinetic interactions between simvastatin and ezetimibe. All active treatments caused statistically significant (P<0.01) decreases in LDL-C concentration vs placebo from baseline to day 14. The coadministration of ezetimibe and simvastatin caused a dose-dependent reduction in LDL-C and total cholesterol, with no apparent effect on high-density lipoprotein cholesterol (HDL-C) or triglycerides. The coadministration of ezetimibe 10 mg and simvastatin 10 mg or 20 mg caused a statistically (P<0.01) greater percentage reduction (mean -17%, 95% CI -27.7, -6.2, and -18%, -28.4, -7.4, respectively) in LDL-C than simvastatin alone. CONCLUSIONS: The coadministration of ezetimibe at doses up to 10 mg with simvastatin 10 or 20 mg daily was well tolerated and caused a significant additive reduction in LDL-C compared with simvastatin alone. Additional clinical studies to assess the efficacy and safety of coadministration of ezetimibe and simvastatin are warranted.

Disposition of the selective cholesterol absorption inhibitor ezetimibe in healthy male subjects.

Ezetimibe [SCH 58235; 1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone], a selective cholesterol absorption inhibitor, is being developed for the treatment of primary hypercholesterolemia. The absorption, metabolism, and excretion of ezetimibe were characterized in eight healthy male volunteers in this single-center, single-dose, open-label study. Subjects received a single oral 20-mg dose of [14C]ezetimibe (approximately 100 microCi) with 200 ml of noncarbonated water after a 10-h fast. Concentrations of radioactivity and/or ezetimibe (conjugated and unconjugated) were determined in plasma, urine, and fecal samples. Ezetimibe was rapidly absorbed and extensively conjugated following oral administration. The main circulating metabolite in plasma was SCH 60663 [1-O-[4-[trans-(2S,3R)-1-(4-fluorophenyl)-4-oxo-3-[3(S)-hydroxy-3-(4-fluorophenyl)propyl]-2-azetidinyl]phenyl]-beta-D-glucuronic acid], the glucuronide conjugate of ezetimibe. Plasma concentration-time profiles of unconjugated and conjugated drug exhibited multiple peaks, indicating enterohepatic recycling. Approximately 78 and 11% of the administered [14C]ezetimibe dose were excreted in feces and urine, respectively, by 240 h after drug administration. Total recovery of radioactivity averaged 89% of the administered dose. The main excreted metabolite was the glucuronide conjugate of ezetimibe. The primary metabolite in urine (0- to72-h composite) was also the glucuronide conjugate (about 9% of the administered dose). Significant amounts (69% of the dose) of ezetimibe were present in the feces, presumably as a result of SCH 60663 hydrolysis and/or unabsorbed drug. No adverse events were reported in this study. A single 20-mg capsule of [(14)C]ezetimibe was safe and well tolerated after oral administration. The pharmacokinetics of ezetimibe are consistent with extensive glucuronidation and enterohepatic recirculation. The primary metabolic pathway for ezetimibe is by glucuronidation of the 4-hydroxyphenyl group.