Yu Chen Barrett

Fit-for-Purpose Method Development and Validation for Successful Biomarker Measurement

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 Res22:499–511, 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.

Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products

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.

Clinical laboratory measurement of direct factor Xa inhibitors: Anti-Xa assay is preferable to prothrombin time assay

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 inhibitors 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.

Apixaban, an oral, direct factor Xa inhibitor: single dose safety, pharmacokinetics, pharmacodynamics and food effect in healthy subjects

To evaluate apixaban single dose safety, tolerability, pharmacokinetics and pharmacodynamics and assess the effect of food on apixaban pharmacokinetics.

Safety, pharmacokinetics and pharmacodynamics of multiple oral doses of apixaban, a factor Xa inhibitor, in healthy subjects.

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.

Effect of extremes of body weight on the pharmacokinetics, pharmacodynamics, safety and tolerability of apixaban in healthy subjects

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.

A randomised assessment of the pharmacokinetic, pharmacodynamic and safety interaction between apixaban and enoxaparin in healthy subjects

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).

A randomized direct comparison of the pharmacokinetics and pharmacodynamics of apixaban and rivaroxaban.

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<0.0001). Conclusion Apixaban 2.5 mg BID demonstrated less intersubject variability in exposure, lower AXA AUC, and higher trough and smaller peak-to-trough fluctuations in plasma concentration and AXA, suggesting more constant anticoagulation compared with rivaroxaban 10 mg QD. However, the clinical impact of these differences on the relative efficacy and safety of apixaban and rivaroxaban remains to be determined.

A Novel Prothrombin Time Assay for Assessing the Anticoagulant Activity of Oral Factor Xa Inhibitors

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.

Beneficial and Adverse Effects of an LXR Agonist on Human Lipid and Lipoprotein Metabolism and Circulating Neutrophils

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.