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Charles Frost,1 Yan Song,1 Yu Chen Barrett,1 Jessie Wang,2 Janice Pursley,3 Rebecca A Boyd,4 Frank LaCreta1 1Exploratory Clinical and Translational Research, 2Exploratory Development Global Biometric Sciences, 3Analytical and Bioanalytical Development, Bristol-Myers Squibb Company, Princeton, NJ, USA; 4Global Innovative Pharma Business Clinical Pharmacology, Pfizer Inc., Groton, CT, USA Background: Currently, there are no direct comparisons of apixaban and rivaroxaban, two new oral direct factor Xa inhibitors approved for management of thromboembolic disorders. Objective: Compare the pharmacokinetics and anti-factor Xa activity (AXA) of apixaban and rivaroxaban. Methods: In this randomized, open-label, two-period, two-treatment crossover study, healthy subjects (N=14) received apixaban 2.5 mg twice daily (BID) and rivaroxaban 10 mg once daily (QD) for 4 days with a ≥4.5-day washout. Plasma samples were obtained for pharmacokinetic and AXA assessments; parameters were calculated using noncompartmental methods. Results: Median time-to-maximum concentration was 2 hours for both compounds, and the mean half-life was 8.7 and 7.9 hours for apixaban and rivaroxaban, respectively. Daily exposure, the area under the curve (AUC(0–24)), appeared similar for rivaroxaban (1,094 ng · h/mL) and apixaban (935 ng · h/mL), whereas mean peak-to-trough plasma concentration ratio was 3.6-fold greater for rivaroxaban (16.9) than apixaban (4.7). Coefficient of variation for exposure parameters (AUC0–24, Cmax, Cmin) was 20%–24% for apixaban versus 29%–46% for rivaroxaban. Peak AXA, AXA AUC(0–24), and AXA fluctuation were ~2.5-, 1.3-, and 3.5-fold higher for rivaroxaban than apixaban, respectively. Trough concentrations and AXA were lower for rivaroxaban (10 ng/mL and 0.17 IU/mL vs 17 ng/mL and 0.24 IU/mL for apixaban, respectively). Rivaroxaban exhibited a steeper concentration–AXA response (slope: 0.0172 IU/ng vs 0.0134 IU/ng for apixaban, P

Frost C, Song Y, Barrett YC, et al. Clin Pharmacol. 2014;6:179–187.Page 183, Figure 2, the arithmetic mean apixaban trough concentration on Day 4 prior to the morning dose (ie, 0-hr Figure 2) was not correct. The arithmetic mean (SD) apixaban trough concentration on Day 4 prior to the morning dose was 24.6 (6.1) ng/mL. Read the original article.

Yimin Cui,1 Yan Song,2 Jessie Wang,2 Zhigang Yu,2 Alan Schuster,2 Yu Chen Barrett,2 Charles Frost2 1Peking University First Hospital, Beijing, People's Republic of China; 2Bristol-Myers Squibb, Princeton, NJ, USA Background: The pharmacokinetics (PK), pharmacodynamics (PD), and safety of apixaban were assessed in healthy Chinese subjects in this randomized, placebo-controlled, double-blind, single-sequence, single- and multiple-dose study. Subjects and methods: Eighteen subjects 18–45 years of age were randomly assigned (2:1 ratio) to receive apixaban or matched placebo. Subjects received a single 10 mg dose of apixaban or placebo on day 1, followed by 10 mg apixaban or placebo twice daily for 6 days (days 4–9). The PK and PD of apixaban were assessed by collecting plasma samples for 72 hours following the dose on day 1 and the morning dose on day 9, and measuring apixaban concentration and anti-Xa activity. Safety was assessed via physical examinations, vital sign measurements, electrocardiograms, and clinical laboratory evaluations. Results: PK analysis showed similar characteristics of apixaban after single and multiple doses, including a median time to maximum concentration of ~3 hours, mean elimination half-life of ~11 hours, and renal clearance of ~1.2 L/hour. The accumulation index was 1.7, consistent with twice-daily dosing and the observed elimination half-life. Single-dose data predict multiple-dose PK, therefore apixaban PK are time-independent. The relationship between anti-Xa activity and plasma apixaban concentrations appears to be linear. Apixaban was safe and well tolerated, with no bleeding-related adverse events reported. Conclusion: Apixaban was safe and well tolerated in healthy Chinese subjects. Apixaban PK and PD were predictable and consistent with findings from previous studies in Asian and non-Asian subjects. The administration of apixaban does not require any dose modification based on race. Keywords: apixaban, oral anticoagulant, factor Xa inhibitor, pharmacokinetics, pharmacodynamics

Cui Y, Song Y, Wang J, et al. Clinical Pharmacology: Advances and Applications. 2013;5(1):177–184.Note that on page 182, Figure 3 should be corrected as follows, with the line for mean anti-Xa activity extended through to 72 hours post dose on Day 9, and the Day 1 plasma apixaban concentration-time profile rendering error bars and symbols more visually apparent.Read the original article

This randomized, double-blind, placebo-controlled, ascending single intravenous (IV) bolus-dose study evaluated safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of apixaban, a direct factor Xa (FXa) inhibitor approved for multiple indications. Eight healthy subjects were randomized 3:1 (apixaban:placebo) within each IV dose cohort (0.5, 1.25, 2.5, 3.75, and 5 mg). The 2.5-mg IV panel also received 5 mg of oral apixaban or placebo. Blood samples were collected for PK and PD, including international normalized ratio, modified prothrombin time (mPT), and anti-FXa activity. Apixaban had 66.2% oral bioavailability, dose-proportional exposure, 17 to 26 L steady-state volume of distribution, and 3.2 to 3.5 L/h total plasma clearance. Renal clearance was ≈27%. Anti-FXa activity and mPT changes followed the apixaban plasma concentration-time profile; both were highly correlated with concentration (R 2 = 0.99 and R 2 = 0.93 for anti-FXa activity and mPT, respectively). International normalized ratio remained within reference range (0.9-1.3). There were no serious or bleeding-related adverse events. Overall, an apixaban single IV bolus was safe and well tolerated over a 10-fold dose range by these subjects. Apixaban had good oral bioavailability, dose-proportional exposure, and constant plasma clearance over a broad dose range, with modest renal clearance. Apixaban PD were consistent with reversible FXa inhibition., (© 2021, The American College of Clinical Pharmacology.)

Background: FEV 1 as a percentage of predicted (FEV 1 %pred) is commonly measured in asthma clinical studies; however, reports vary on its association with asthma control instruments evaluating symptoms., Objective: Assess the association between FEV 1 %pred and Asthma Control Questionnaire (ACQ) scores in a managed-care population with persistent asthma., Methods: Retrospective analysis of survey responses and spirometry results of patients (aged ≥12 years) with persistent asthma from the Observational Study of Asthma Control and Outcomes was done. Eligible patients received 4 identical surveys including the 5-item ACQ (ACQ-5)/6-item ACQ (ACQ-6) and completed spirometry in parallel. Longitudinal analyses, comparisons of change over time, and fixed- and random-effects regression analyses were conducted, with/without adjustment for covariates., Results: There were 1748 survey responses with valid spirometry results. In unadjusted models, coefficients for ACQ-5/ACQ-6 scores were not statistically significant and coefficient of determination (R 2 ) was low (0.03). When adjusted for covariates, ACQ-5 and ACQ-6 scores were significantly associated with FEV 1 %pred (P < .001) and R 2 increased to 0.11 and 0.12, respectively. In adjusted models, every 1-point increase in ACQ-5 and ACQ-6 scores was associated with a 1.7% and 1.9% decrease, respectively, in FEV 1 %pred. Change in FEV 1 %pred and change in ACQ-5/ACQ-6 scores were not significantly associated in regressions with/without covariates., Conclusions: The weak and statistically insignificant association between FEV 1 %pred and ACQ-5/ACQ-6 scores in unadjusted models suggests a high degree of unexplained variation between these measures. Results support the use of both symptoms and pulmonary function, rather than relying on one measure alone, to assess asthma control in clinical care and outcomes studies., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The objective was to characterise apixaban pharmacodynamic (PD) activity in umbilical cord (UC), paediatric, and adult plasma. Plasma was obtained from blood samples from six UC donors, 70 paediatric (neonates [birth-≤1 month], infants [>1-≤6 months], toddlers [>6 months-≤2 years], young children [>2-≤6 years], children [>6-≤12 years], adolescents [>12-≤18 years]), and six adult (19-45 years) subjects. Plasma spiked with apixaban 0 (baseline), 30, or 110 ng/ml was analysed for anti-factor Xa activity, factor X levels, prothrombin time (PT), and modified PT (mPT). Apixaban had similar concentration-related effects on anti-factor Xa activity across groups (30 ng/ml: 0.223-0.295 IU/ml; 110 ng/ml: 1.212-1.474 IU/ml). Endogenous baseline factor X levels were 43 %-68 % lower in plasma from UC and subjects ≤6 months versus adults. Factor Xa inhibition (percentage change from baseline in apparent factor X levels) was similar for both apixaban concentrations across groups, except UC, neonate, and infant groups, which showed greater inhibition vs adults for apixaban 110 ng/ml. Baseline PT and mPT were similar across groups. Apixaban had no effect on PT at the concentrations tested. Apixaban 110 ng/ml prolonged mPT similarly across groups (44.4-53.2 s to 64.5-70.0 s); no prolongation was found with apixaban 30 ng/ml. Apixaban demonstrated consistent concentration-related effects on other PD endpoints in plasma samples from all age groups, except factor Xa inhibition.

The development of LXR agonists for the treatment of coronary artery disease has been challenged by undesirable properties in animal models. Here we show the effects of an LXR agonist on lipid and lipoprotein metabolism and neutrophils in human subjects. BMS-852927, a novel LXRβ-selective compound, had favorable profiles in animal models with a wide therapeutic index in cynomolgus monkeys and mice. In healthy subjects and hypercholesterolemic patients, reverse cholesterol transport pathways were induced similarly to that in animal models. However, increased plasma and hepatic TG, plasma LDL-C, apoB, apoE, and CETP and decreased circulating neutrophils were also evident. Furthermore, similar increases in LDL-C were observed in normocholesterolemic subjects and statin-treated patients. The primate model markedly underestimated human lipogenic responses and did not predict human neutrophil effects. These studies demonstrate both beneficial and adverse LXR agonist clinical responses and emphasize the importance of further translational research in this area., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background and Objectives: The effects of age and sex on apixaban pharmacokinetics and pharmacodynamics were studied., Methods: This was an open-label, single-dose, 2 × 2 factorial study. Healthy young (aged 18-40 years) and elderly (aged ≥ 65 years) male and female subjects received a single oral 20 mg dose of apixaban. Blood and urine samples were collected for pharmacokinetic and pharmacodynamic (blood only) analyses. Subjects were monitored for adverse events throughout the study., Results: Seventy-nine subjects were enrolled into four groups: young males (n = 20), elderly males (n = 20), young females (n = 20) and elderly females (n = 19). Age did not affect the maximum observed plasma concentration (C max). The mean area under the concentration-time curve from time zero extrapolated to infinite time (AUC∞) was 32% greater in elderly subjects than in young subjects. The mean C max and AUC∞ values were 18 and 15% higher, respectively, in females than in males. The time course of the mean international normalized ratio (INR), modified prothrombin time (mPT) and anti-Xa activity tracked the apixaban concentration-time curve. All three pharmacodynamic measures exhibited a positive linear correlation with the plasma apixaban concentration. Differences in the mean INR, mPT and anti-Xa activity between age and sex groups were small (<15% at the maximum mean values) and were generally related to pharmacokinetic differences. However, anti-Xa activity demonstrated less variability than the INR or mPT, and may have utility as a bioassay for apixaban. Apixaban was well tolerated, with no serious adverse events., Conclusion: There were no clinically meaningful age- or sex-related differences in the pharmacokinetics and pharmacodynamics of apixaban that would require dose modification on the basis of age or sex alone.

Aim: To assess pharmacokinetic and pharmacodynamic interactions between naproxen (a non-steroidal anti-inflammatory drug) and apixaban (an oral, selective, direct factor-Xa inhibitor)., Method: In this randomized, three period, two sequence study, 21 healthy subjects received a single oral dose of apixaban 10 mg, naproxen 500 mg or co-administration of both. Blood samples were collected for determination of apixaban and naproxen pharmacokinetics and pharmacodynamics (anti-Xa activity, international normalized ratio [INR] and arachidonic acid-induced platelet aggregation [AAI-PA]). Adverse events, bleeding time and routine safety assessments were also evaluated., Results: Apixaban had no effect on naproxen pharmacokinetics. However, following co-administration, apixaban AUC(0,∞), AUC(0,t) and Cmax were 54% (geometric mean ratio 1.537; 90% confidence interval (CI) 1.394, 1.694), 55% (1.549; 90% CI 1.400, 1.713) and 61% (1.611; 90% CI 1.417, 1.831) higher, respectively. Mean (standard deviation [SD]) anti-Xa activity at 3 h post-dose was approximately 60% higher following co-administration compared with apixaban alone, 4.4 [1.0] vs. 2.7 [0.7] IU ml(-1) , consistent with the apixaban concentration increase following co-administration. INR was within the normal reference range after all treatments. AAI-PA was reduced by approximately 80% with naproxen. Co-administration had no impact beyond that of naproxen. Mean [SD] bleeding time was higher following co-administration (9.1 [4.1] min) compared with either agent alone (5.8 [2.3] and 6.9 [2.6] min for apixaban and naproxen, respectively)., Conclusion: Co-administration of naproxen with apixaban results in higher apixaban exposure and appears to occur through increased apixaban bioavailability. The effects on anti-Xa activity, INR and inhibition of AAI-PA observed in this study were consistent with the individual pharmacologic effects of apixaban and naproxen., (© 2014 The British Pharmacological Society.)

Aim: Apixaban is an oral, direct, factor-Xa inhibitor approved for thromboprophylaxis in patients who have undergone elective hip or knee replacement surgery and for prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation. This open label, parallel group study investigated effects of extremes of body weight on apixaban pharmacokinetics, pharmacodynamics, safety and tolerability., Method: Fifty-four healthy subjects were enrolled [18 each into low (≤50 kg), reference (65-85 kg) and high (≥120 kg) body weight groups]. Following administration of a single oral dose of 10 mg apixaban, plasma and urine samples were collected for determination of apixaban pharmacokinetics and anti-factor Xa activity. Adverse events, vital signs and laboratory assessments were monitored., Results: Compared with the reference body weight group, low body weight had approximately 27% [90% confidence interval (CI): 8-51%] and 20% (90% CI: 11-42%) higher apixaban maximum observed plasma concentration (Cmax) and area under the concentration-time curve extrapolated to infinity (AUC(0,∞)), respectively, and high body weight had approximately 31% (90% CI: 18-41%) and 23% (90% CI: 9-35%) lower apixaban Cmax and AUC(0,∞) , respectively. Apixaban renal clearance was similar across the weight groups. Plasma anti-factor Xa activity showed a direct, linear relationship with apixaban plasma concentration, regardless of body weight group. Apixaban was well tolerated in this study., Conclusion: The modest change in apixaban exposure is unlikely to require dose adjustment for apixaban based on body weight alone. However, caution is warranted in the presence of additional factors (such as severe renal impairment) that could increase apixaban exposure., (© 2013 Bristol-Myers Squibb Co. British Journal of Clinical Pharmacology © 2013 The British Pharmacological Society.)

Aim: Apixaban is an oral factor Xa inhibitor approved for stroke prevention in atrial fibrillation and thromboprophylaxis in patients who have undergone elective hip or knee replacement surgery and under development for treatment of venous thromboembolism. This study examined the safety, pharmacokinetics and pharmacodynamics of multiple dose apixaban., Method: This double-blind, randomized, placebo-controlled, parallel group, multiple dose escalation study was conducted in six sequential dose panels - apixaban 2.5, 5, 10 and 25 mg twice daily and 10 and 25 mg once daily- with eight healthy subjects per panel. Within each panel, subjects were randomized (3:1) to oral apixaban or placebo for 7 days. Subjects underwent safety assessments and were monitored for adverse events (AEs). Blood samples were taken to measure apixaban plasma concentration, international normalized ratio (INR), activated partial thromboplastin time (aPTT) and modified prothrombin time (mPT)., Results: Forty-eight subjects were randomized and treated (apixaban, n = 36; placebo, n = 12); one subject receiving 2.5 mg twice daily discontinued due to AEs (headache and nausea). No dose limiting AEs were observed. Apixaban maximum plasma concentration was achieved ~3 h post-dose. Exposure increased approximately in proportion to dose. Apixaban steady-state concentrations were reached by day 3, with an accumulation index of 1.3-1.9. Peak : trough ratios were lower for twice daily vs. once daily regimens. Clotting times showed dose-related increases tracking the plasma concentration-time profile., Conclusion: Multiple oral doses of apixaban were safe and well tolerated over a 10-fold dose range, with pharmacokinetics with low variability and concentration-related increases in clotting time measures., (© 2013 Bristol-Myers Squibb Co. British Journal of Clinical Pharmacology © 2013 The British Pharmacological Society.)

Conventional prothrombin time (PT) assays have limited sensitivity and dynamic range in monitoring the anticoagulant activity of direct factor Xa inhibitors. Hence, new assays are needed. We modified a PT assay by adding calcium chloride (CaCl2) to the thromboplastin reagent to increase assay dynamic range and improve sensitivity. Effects of calcium and sodium ion concentrations, and sample handling, were evaluated to optimize assay performance. Increasing concentrations of calcium ions produced progressive increases in PT across the factor Xa inhibitor concentrations of 0 to 2500 nmol/L for razaxaban and apixaban. The greatest effect was seen when the thromboplastin reagent was diluted 1:2.25 with 100 mmol/L CaCl2 (thus selected for routine use). The optimized assay showed an interassay precision of 1.5 to 9.3 percentage coefficient of variation (%CV) for razaxaban and 3.1 to 4.6 %CV for apixaban. We conclude that the modified PT assay is likely to be suitable as a pharmacodynamic marker for activity at therapeutic concentrations of factor Xa inhibitors.

In contrast to vitamin K antagonists, which reduce the functional levels of several coagulation factors, the new oral anticoagulants specifically target either thrombin or factor Xa. These new agents have such predictable pharmacokinetics and pharmacodynamics that routine coagulation monitoring is unnecessary. However, there are still some situations in which measurement of anticoagulant effect may be required. The coagulation assays that are used to monitor heparin derivatives or vitamin K antagonists may not always accurately reflect the anticoagulant activity of the new oral anticoagulants, and specialized assays may be needed. In this article, we: (i) identify situations in which assessment of anticoagulant effect may aid treatment decisions; (ii) describe the effects of the new oral anticoagulants on the various coagulation tests; (iii) review the specialized coagulation assays that have been developed to measure the anticoagulant effects of the new oral anticoagulants; and (iv) provide a clinical perspective on the role of coagulation testing in the clinical management of patients treated with the new oral anticoagulants., (© 2012 International Society on Thrombosis and Haemostasis.)

Aims: To evaluate apixaban single dose safety, tolerability, pharmacokinetics and pharmacodynamics and assess the effect of food on apixaban pharmacokinetics., Methods: A double-blind, placebo-controlled, single ascending-dose, first-in-human study assessed apixaban safety, pharmacokinetics and pharmacodynamics in healthy subjects randomized to oral apixaban (n = 43; 0.5-2.5 mg as solution or 5-50 mg as tablets) or placebo (n = 14) under fasted conditions. An open label, randomized, two treatment crossover study investigated apixaban pharmacokinetics/pharmacodynamics in healthy subjects (n = 21) administered apixaban 10 mg in fasted and fed states. Both studies measured apixaban plasma concentration, international normalized ratio (INR), activated partial thromboplastin time (aPTT) and prothrombin time (PT) or a modified PT (mPT)., Results: In the single ascending-dose study increases in apixaban exposure appeared dose-proportional. Median t(max) occurred 1.5-3.3 h following oral administration. Mean terminal half-life ranged between 3.6 and 6.8 h following administration of solution doses ≤2.5 mg and between 11.1 and 26.8 h for tablet doses ≥5 mg. Concentration-related changes in pharmacodynamic assessments were observed. After a 50 mg dose, peak aPTT, INR and mPT increased by 1.2-, 1.6- and 2.9-fold, respectively, from baseline. In the food effect study: 90% confidence intervals of geometric mean ratios of apixaban C(max) and AUC in a fed vs. fasted state were within the predefined no effect (80-125%) range. Apixaban half-life was approximately 11.5 h. The effect of apixaban on INR, PT and aPTT was comparable following fed and fasted administration., Conclusions: Single doses of apixaban were well tolerated with a predictable pharmacokinetic/pharmacodynamic profile and a half-life of approximately 12 h. Apixaban can be administered with or without food., (© 2012 Bristol-Myers Squibb Co.. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.)

Following major orthopaedic surgery, guidelines usually recommend continued thromboprophylaxis after hospitalisation. The availability of an effective oral anticoagulant with an acceptable safety profile that does not require routine clinical monitoring may lead clinicians to switch patients from subcutaneous to an oral therapy either during hospitalisation or at discharge. The purpose of this study was to assess the effect of enoxaparin on the pharmacokinetics, pharmacodynamics and safety of apixaban, an oral, direct inhibitor of coagulation factor Xa. In this four-period, crossover study, 20 healthy subjects were randomised to receive single doses of apixaban 5 mg orally; enoxaparin 40 mg subcutaneously; apixaban 5 mg and enoxaparin 40 mg concomitantly; and apixaban 5 mg followed 6 hours (h) after by enoxaparin 40 mg. Pharmacokinetics of apixaban were not affected by enoxaparin. Average peak pharmacodynamic effect, measured by anti-Xa activity, was 1.36 U/ml after administration of apixaban and was 0.42 U/ml after enoxaparin. Following co-administration of apixaban and enoxaparin, peak anti-Xa activity was 42% higher than for apixaban alone. Following administration of enoxaparin 6 h after apixaban, peak anti-Xa activity was 15% higher than for apixaban alone. In conclusion, enoxaparin had no effect on the pharmacokinetics of apixaban. The increase in anti-Xa activity after co-administration was modest and appeared to be additive. Peak anti-Xa activity increases are mitigated by separating administration of subcutaneous anticoagulation and apixaban when switching between therapies; the potential for pharmacodynamic interaction may be further mitigated by transitioning at the next scheduled dose (12 h).

In patients with acute deep-vein thrombosis (DVT), apixaban, a direct oral factor Xa inhibitor, showed efficacy and safety similar to low-molecular-weight heparin followed by vitamin K antagonist (LMWH/VKA). We evaluated biomarkers of coagulation activity in relation to treatment dose, duration and clinical outcome. Patients (N = 520) with symptomatic DVT were randomised to receive apixaban (5 mg bid, 10 mg bid or 20 mg qd) or LMWH/VKA for 12 weeks. Plasma D-dimer, prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin complex (TAT) levels were measured at baseline, and weeks 3 and 12 after treatment. Median plasma levels of D-dimer, F1+2 and TAT were elevated at baseline. At weeks 3 and 12, biomarker levels were normalised in most patients in all treatment groups, consistent with the low rate of venous thromboembolism (VTE) observed. Median reduction in D-dimer was similar in all treatment groups; percentage of patients with D-dimer above upper limit of normal decreased from 95% to 24-40% at week 12. F1+2 decline was greater with LMWH/VKA than apixaban. F1+2 in the apixaban groups changed to a similar extent (>84% of patients had F1+2 within reference range at week 12). Magnitude of TAT reduction was not quantifiable. In conclusion, levels of coagulation biomarkers decreased over 12 weeks of treatment with apixaban or LMWH/VKA in most patients with acute VTE. Baseline D-dimer and F1+2 were higher in patients with recurrent symptomatic VTE than in those without. Plasma levels of coagulation biomarkers did not appear to correlate with total bleeding events.

Apixaban and other factor Xa (FXa) inhibitors are in late-stage clinical development for prevention and treatment of thromboembolic diseases. Although routine monitoring will not be required, in certain situations assessment of drug level may be helpful. This study evaluated the suitability of commercially available prothrombin time/international normalised ratio (PT/INR) and anti-FXa activity assays to measure FXa inhibitors in plasma. Twelve PT (ISI 0.89-1.88) and three anti-Xa assays were evaluated in vitro using human plasma spiked with four FXa inhibitors (0-2,000 ng/ml). Assay variability and correlation with drug plasma exposure were evaluated in patients with venous thromboembolism (VTE) treated with apixaban. All FXa inhibitors prolonged PT; however, assay sensitivity was dependent on thromboplastin reagents used and FXa inhibitors tested. To achieve a doubling of PT, the concentration of each FXa inhibitor varied 2.6- to 8-fold between thromboplastin reagents. The rank order of a FXa inhibitor's effect on PT ratio varied across thromboplastin reagents. Conversion to INR increased variability. Different anti-Xa assays showed different dynamic ranges for each FXa inhibitor; however, their rank order was consistent. For apixaban, the dynamic range of <7.8-240 ng/ml, and inter- and intra-assay precision of <6% coefficient of variation by Rotachrom assay appeared suitable for the anticipated apixaban plasma concentrations with 2.5 and 5 mg bid clinical doses. The stronger correlation between apixaban plasma concentration and anti-Xa activity (r2 = 0.88-0.89) compared with PT/INR (r2 = 0.36) in patients undergoing VTE treatment suggested that anti-Xa activity was the better indicator of apixaban plasma concentrations.

Most biological drug products elicit some level of anti-drug antibody (ADA) response. This antibody response can, in some cases, lead to potentially serious side effects and/or loss of efficacy. In humans, ADA often causes no detectable clinical effects, but in the instances of some therapeutic proteins these antibodies have been shown to cause a variety of clinical consequences ranging from relatively mild to serious adverse events. In nonclinical (preclinical) studies, ADA can affect drug exposure, complicating the interpretation of the toxicity, pharmacokinetic (PK) and pharmacodynamic (PD) data. Therefore, the immunogenicity of therapeutic proteins is a concern for clinicians, manufacturers and regulatory agencies. In order to assess the immunogenic potential of biological drug molecules, and be able to correlate laboratory results with clinical events, it is important to develop reliable laboratory test methods that provide valid assessments of antibody responses in both nonclinical and clinical studies. For this, method validation is considered important, and is a necessary bioanalytical component of drug marketing authorization applications. Existing regulatory guidance documents dealing with the validation of methods address immunoassays in a limited manner, and in particular lack information on the validation of immunogenicity methods. Hence this article provides scientific recommendations for the validation of ADA immunoassays. Unique validation performance characteristics are addressed in addition to those provided in existing regulatory documents pertaining to bioanalyses. The authors recommend experimental and statistical approaches for the validation of immunoassay performance characteristics; these recommendations should be considered as examples of best practice and are intended to foster a more unified approach to antibody testing across the biopharmaceutical industry.

The appropriate evaluation of the immunogenicity of biopharmaceuticals is of major importance for their successful development and licensure. Antibodies elicited by these products in many cases cause no detectable clinical effects in humans. However, antibodies to some therapeutic proteins have been shown to cause a variety of clinical consequences ranging from relatively mild to serious adverse events. In addition, antibodies can affect drug efficacy. In non-clinical studies, anti-drug antibodies (ADA) can complicate interpretation of the toxicity, pharmacokinetic (PK) and pharmacodynamic (PD) data. Therefore, it is important to develop testing strategies that provide valid assessments of antibody responses in both non-clinical and clinical studies. This document provides recommendations for antibody testing strategies stemming from the experience of contributing authors. The recommendations are intended to foster a more unified approach to antibody testing across the biopharmaceutical industry. The strategies proposed are also expected to contribute to better understanding of antibody responses and to further advance immunogenicity evaluation.

Thrombocytopenia exposes patients to increased bleeding risk. This serious adverse event was observed with a frequency of approximately 2% in early clinical trials with the potent, orally bioavailable glycoprotein (GP) IIb/IIIa receptor antagonist roxifiban. We previously reported that drug-dependent antibodies (DDAbs) to GP IIb/IIIa are the main cause of thrombocytopenia with roxifiban. Two ELISA assays for detection of free DDAbs (in citrate plasma) and total DDAbs (in EDTA plasma to elute platelet bound DDAbs) were developed and analytically validated. These tests served two purposes during the clinical development program, to pre-screen patients for pre-existing antibodies and monitor patients for increasing antibody titers as a surrogate for eminent thrombocytopenia. The free DDAb assay showed inter and intra-assay precision of 5-12 and 12-14% CV, respectively. The total DDAb assay showed a precision of 5-10 and 4-12% CV, respectively. Three cycles of freeze-thaw did not significantly alter DDAb values in citrate plasma, EDTA plasma or extraction solution. The clinical qualifications of the two assays were conducted in two phase II clinical trials in coronary arterial disease (CAD) patients dosed with roxifiban. Both assays have demonstrated clinical sensitivity of nearly 99-100% and clinical specificity of nearly 95%.

Despite major advances in modern drug discovery and development, the number of new drug approvals has not kept pace with the increased cost of their development. Increasingly, innovative uses of biomarkers are employed in an attempt to speed new drugs to market. Still, widespread adoption of biomarkers is impeded by limited experience interpreting biomarker data and an unclear regulatory climate. Key differences preclude the direct application of existing validation paradigms for drug analysis to biomarker research. Following the AAPS 2003 Biomarker Workshop (J. W. Lee, R. S. Weiner, J. M. Sailstad, et al. Method validation and measurement of biomarkers in nonclinical and clinical samples in drug development. A conference report. Pharm Res 22:499-511, 2005), these and other critical issues were addressed. A practical, iterative, "fit-for-purpose" approach to biomarker method development and validation is proposed, keeping in mind the intended use of the data and the attendant regulatory requirements associated with that use. Sample analysis within this context of fit-for-purpose method development and validation are well suited for successful biomarker implementation, allowing increased use of biomarkers in drug development.

A sensitive method for quantitation of urinary 6beta-hydroxycortisol (6beta-HC) and cortisol using on-line SPE and LC-MS/MS was developed and validated. Human urine samples were injected directly onto an on-line solid phase extraction apparatus, Prospekt-2, followed by HPLC separation and electrospray triple quadrupole LC-MS/MS detection. The inter-day precision for the 6beta-HC:cortisol ratio was 7-9%. The lower limit of quantitation was 1 and 0.2 ng/mL for 6beta-HC and cortisol, respectively. Using the method we observed a diurnal variation on the 6beta-HC:cortisol ratio in healthy volunteers with the maximal ratio observed in the 2-10 pm urine collection period.

Most biopharmaceutical therapeutics elicit some level of antibody response against the product. This antibody response can, in some cases, lead to potentially serious side effects and/or loss of efficacy. Therefore, the immunogenicity of therapeutic proteins is a concern for clinicians, manufacturers and regulatory agencies. In order to assess immunogenicity of these molecules, appropriate detection, quantitation and characterization of antibody responses are necessary. Inadequately designed antibody assays have led to the hampering of product development or, during licensure, post-marketing commitments. This document provides scientific recommendations based on the experience of the authors for the development of anti-product antibody immunoassays intended for preclinical or clinical studies. While the main focus of this document is assay design considerations, we provide scientific focus and background to the various assay performance parameters necessary for developing a valid assay. Sections on assay performance parameters, including those that appear in regulatory guidances, are contained in this manuscript.

A radioimmunoassay (RIA) was developed for the determination of XV459, the active hydrolysis metabolite of the oral prodrug roxifiban (DMP 754), in human plasma. XV459 is a potent antagonist of the glycoprotein IIb/IIIa receptor. The method utilizes a competitive double antibody format employing an 125I-labeled XV459 analogue tracer which competes with XV459 for antibody binding sites and a second antibody precipitation step to separate antibody bound analyte from free analyte. The method has a validated lower quantification limit of 0.35 ng/ml (0.81 nM) using 12.5 microl of plasma and with dilution can accommodate clinical plasma samples ranging up to 48.0 ng/ml (110.7 nM). Cross-validation to an existing quantitative liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method showed good correlation (r(2)=0.98). The RIA has been successfully used to assay over 10000 clinical samples with sensitivity and specificity comparable to the LC-MS/MS method, but with faster turn around time and at greatly reduced costs.

Thrombocytopenia is a relatively common side effect observed during glycoprotein (GP) IIb/IIIa antagonist therapy. With the oral antagonist roxifiban, we observed thrombocytopenia, defined as 50% reduction of platelets over predose values or below 90 000/microL (9 x 10(10)/L), with a frequency of 2% (8 of 386). Thrombocytopenia occurred either early (days 2 to 4) or delayed (days 11 to 16). No additional cases were observed with up to 6 months of treatment. Retrospective analysis provided evidence for drug-dependent antibodies (DDABs) to GP IIb/IIIa in 5 of 6 subjects, suggestive of an immune etiology of thrombocytopenia. The hypothesis that excluding patients based on positive DDAB reaction would reduce the frequency of thrombocytopenia was tested. Patients were screened for DDABs during the study qualification period and, overall, 3.9% of the patients were excluded based on pre-existing DDAB concentrations above a statistically defined medical decision limit. An additional 2.6% were excluded based on therapy-related antibody production during the first 2 weeks. With antibody testing, 0.2% of patients (2 of 1044) developed immune-mediated thrombocytopenia. One case developed a rapidly increasing antibody concentration and presented with thrombocytopenia despite discontinuation of roxifiban therapy. The second case was related to a false-negative test result. The frequency of thrombocytopenia was statistically significantly reduced from 2% to 0.2% (P =.0007) comparing nonscreened and screened patients. Testing for DDABs can reduce the frequency of thrombocytopenia in patients treated with roxifiban and, by analogy, other GP IIb/IIIa antagonists. Thus, DDAB testing may be employed to increase the safety of GP IIb/IIIa antagonists.