Effect of Age and Renal Function on Idarucizumab Pharmacokinetics and Idarucizumab-Mediated Reversal of Dabigatran Anticoagulant Activity in a Randomized, Double-Blind, Crossover Phase Ib Study.

BACKGROUND AND OBJECTIVES: Idarucizumab is an antibody fragment that specifically reverses dabigatran-mediated anticoagulation. Safety, pharmacokinetics and pharmacodynamics of idarucizumab were investigated in dabigatran-treated, middle-aged, elderly and renally impaired volunteers with characteristics similar to patients receiving anticoagulant therapy. METHODS: In this randomized, double-blind, crossover study, 46 subjects (12 middle-aged, 45-64 years; 16 elderly, 65-80 years; and 18 with mild or moderate renal impairment) received dabigatran etexilate (DE; 220 or 150 mg twice daily) for 4 days. Idarucizumab doses of 1, 2.5 and 5 g or 2 × 2.5 g 1 h apart, or placebo, were administered as a rapid (5 min) infusion ~2 h after DE at steady state. RESULTS: Dabigatran-prolonged diluted thrombin time, ecarin clotting time and activated partial thromboplastin time were reversed to baseline immediately after idarucizumab infusion in all groups. Reversal was sustained with doses ≥2.5 g. Idarucizumab was well tolerated under all conditions. No impact of age on idarucizumab pharmacokinetics was observed; however, subjects with mild or moderate renal impairment demonstrated increased exposure (up to 84 %), decreased clearance and prolonged (by up to 49 %) initial half-life of idarucizumab compared with healthy middle-aged subjects. CONCLUSIONS: Impaired renal function was associated with increased exposure and decreased clearance of idarucizumab. Idarucizumab resulted in immediate, complete and sustained reversal of dabigatran anticoagulant activity, and was safe and well tolerated in middle-aged, elderly and renally impaired volunteers. The results support the clinical use of a 5 g dose of idarucizumab. CLINICAL TRIAL REGISTRATION: http://www.clinicaltrials.gov . Unique identifier: NCT01955720.

Safety, pharmacokinetics and pharmacodynamics of idarucizumab, a specific dabigatran reversal agent in healthy Japanese volunteers: a randomized study.

BACKGROUND: Idarucizumab is a humanized monoclonal antibody fragment that specifically binds with high affinity to dabigatran. OBJECTIVES: This study investigated the safety, tolerability and pharmacokinetics of idarucizumab alone and with dabigatran at steady state, and the effects of idarucizumab on dabigatran-induced anticoagulation. PATIENTS/METHODS: This was a two-part, phase I, randomized, placebo-controlled, double-blind, rising-dose trial in healthy Japanese males. Part 1: 32 subjects (males) received single idarucizumab doses (1, 2, 4 or 8 g [n=6/dose group]) or placebo (n=2/dose group). Part 2: 48 males received dabigatran (220 mg bid) followed by idarucizumab (n=9/dose group) 1, 2, 4 or 5 g (2×2.5 g), or placebo (n=3/dose group). Anti-idarucizumab antibodies (ADAs) and idarucizumab effect on anticoagulation parameters (diluted thrombin time [dTT], ecarin clotting time [ECT], activated partial thromboplastin time [aPTT] and thrombin time [TT]) were assessed. RESULTS: No adverse events were reported in subjects receiving idarucizumab. After single doses of idarucizumab (alone or at steady state of dabigatran), maximum plasma concentration was achieved around the end of each infusion. Mean all anticoagulation parameters fell below the upper limit of normal immediately after idarucizumab infusion in all dose groups; the effect was sustained at 4 and 2×2.5 g over the entire measurement period until 72 h. At 1- and 2-g doses, partial return of the anticoagulant effect occurred. Idarucizumab alone had no effect on coagulation parameters. Treatment-emergent ADAs occurred in 6/60 males receiving idarucizumab. CONCLUSIONS: Idarucizumab infusion achieved immediate, complete and sustained reversal of dabigatran-induced anticoagulation in Japanese volunteers. Idarucizumab was well tolerated with no procoagulant effects. Trial registration number: ClinicalTrials.gov NCT02028780 (completed).

Idarucizumab does not have procoagulant effects: Assessment of thrombosis biomarkers in healthy volunteers.

Idarucizumab, a humanised monoclonal antibody fragment, binds dabigatran with high affinity and immediately, completely and sustainably reverses dabigatran-induced changes on blood coagulation. The present analysis focuses on the evaluation of potential procoagulant properties of idarucizumab when administered in the absence of dabigatran. As part of two Phase I studies conducted in healthy Caucasian and Japanese male volunteers, the effect of idarucizumab (8 g as a 1-hour [h] infusion and 4 g as a 5-minute [min] infusion) and placebo on calibrated automated thrombography (CAT) was assessed using platelet-poor plasma samples. Measures were made before and 15 min after the end of infusion in Caucasian subjects, as well as pre-dose, 15 min, 4 h and 8 h in Japanese subjects. The levels of the thrombosis markers D-dimer and prothrombin fragment 1 + 2 (F1.2) were assessed over time in plasma samples up to 72 h after the end of infusion of idarucizumab and placebo. Idarucizumab had no apparent effect on endogenous thrombin formation as measured by CAT. D-dimer and F1.2 levels were highly variable in all dose groups but did not increase when compared with placebo or pre-dose levels. In conclusion, idarucizumab had no effect on endogenous thrombin generation. Additional markers of thrombosis, F1.2 and D-dimer, did not differ between placebo and idarucizumab, indicating a lack of procoagulant properties of idarucizumab.

Safety, tolerability, and efficacy of idarucizumab for the reversal of the anticoagulant effect of dabigatran in healthy male volunteers: a randomised, placebo-controlled, double-blind phase 1 trial.

BACKGROUND: Idarucizumab is a monoclonal antibody fragment that binds dabigatran with high affinity in a 1:1 molar ratio. We investigated the safety, tolerability, and efficacy of increasing doses of idarucizumab for the reversal of anticoagulant effects of dabigatran in a two-part phase 1 study (rising-dose assessment and dose-finding, proof-of-concept investigation). Here we present the results of the proof-of-concept part of the study. METHODS: In this randomised, placebo-controlled, double-blind, proof-of-concept phase 1 study, we enrolled healthy volunteers (aged 18-45 years) with a body-mass index of 18·5-29·9 kg/m(2) into one of four dose groups at SGS Life Sciences Clinical Research Services, Belgium. Participants were randomly assigned within groups in a 3:1 ratio to idarucizumab or placebo using a pseudorandom number generator and a supplied seed number. Participants and care providers were masked to treatment assignment. All participants received oral dabigatran etexilate 220 mg twice daily for 3 days and a final dose on day 4. Idarucizumab (1 g, 2 g, or 4 g 5-min infusion, or 5 g plus 2·5 g in two 5-min infusions given 1 h apart) was administered about 2 h after the final dabigatran etexilate dose. The primary endpoint was incidence of drug-related adverse events, analysed in all randomly assigned participants who received at least one dose of dabigatran etexilate. Reversal of diluted thrombin time (dTT), ecarin clotting time (ECT), activated partial thromboplastin time (aPTT), and thrombin time (TT) were secondary endpoints assessed by measuring the area under the effect curve from 2 h to 12 h (AUEC2-12) after dabigatran etexilate ingestion on days 3 and 4. This trial is registered with ClinicalTrials.gov, number NCT01688830. FINDINGS: Between Feb 23, and Nov 29, 2013, 47 men completed this part of the study. 12 were enrolled into each of the 1 g, 2 g, or 5 g plus 2·5 g idarucizumab groups (nine to idarucizumab and three to placebo in each group), and 11 were enrolled into the 4 g idarucizumab group (eight to idarucizumab and three to placebo). Drug-related adverse events were all of mild intensity and reported in seven participants: one in the 1 g idarucizumab group (infusion site erythema and hot flushes), one in the 5 g plus 2·5 g idarucizumab group (epistaxis); one receiving placebo (infusion site haematoma), and four during dabigatran etexilate pretreatment (three haematuria and one epistaxis). Idarucizumab immediately and completely reversed dabigatran-induced anticoagulation in a dose-dependent manner; the mean ratio of day 4 AUEC2-12 to day 3 AUEC2-12 for dTT was 1·01 with placebo, 0·26 with 1 g idarucizumab (74% reduction), 0·06 with 2 g idarucizumab (94% reduction), 0·02 with 4 g idarucizumab (98% reduction), and 0·01 with 5 g plus 2·5 g idarucizumab (99% reduction). No serious or severe adverse events were reported, no adverse event led to discontinuation of treatment, and no clinically relevant difference in incidence of adverse events was noted between treatment groups. INTERPRETATION: These phase 1 results show that idarucizumab was associated with immediate, complete, and sustained reversal of dabigatran-induced anticoagulation in healthy men, and was well tolerated with no unexpected or clinically relevant safety concerns, supporting further testing. Further clinical studies are in progress. FUNDING: Boehringer Ingelheim Pharma GmbH & Co KG.

A randomised study in healthy volunteers to investigate the safety, tolerability and pharmacokinetics of idarucizumab, a specific antidote to dabigatran.

Idarucizumab, a monoclonal antibody fragment that binds dabigatran with high affinity, is in development as a specific antidote for dabigatran. In this first-in-human, single-rising-dose study, we investigated the pharmacokinetics, safety and tolerability of idarucizumab. Healthy male volunteers aged 18-45 years received between 20 mg and 8 g idarucizumab as a 1-hour intravenous infusion in 10 sequential dose groups, or 1, 2 or 4 g idarucizumab as a 5-minute infusion. Subjects within each dose group were randomised 3:1 to idarucizumab or placebo. A total of 110 randomised subjects received study drug (27 placebo, 83 idarucizumab). Peak and total exposure to idarucizumab increased proportionally with dose. Maximum plasma concentrations were achieved near the end of infusion, followed by a rapid decline, with an initial idarucizumab half-life of ~45 minutes. For the 5-minute infusions, this resulted in a reduction of plasma concentrations to less than 5 % of peak within 4 hours. Idarucizumab (in the absence of dabigatran) had no effect on coagulation parameters or endogenous thrombin potential. Overall adverse event (AE) frequency was similar for idarucizumab and placebo, and no relationship with idarucizumab dose was observed. Drug-related AEs (primary endpoint) were rare (occurring in 2 placebo and 3 idarucizumab subjects) and were mostly of mild intensity; none of them resulted in study discontinuation. In conclusion, the pharmacokinetic profile of idarucizumab meets the requirement for rapid peak exposure and rapid elimination, with no effect on pharmacodynamic parameters. Idarucizumab was safe and well tolerated in healthy males.

Measurement of dabigatran plasma concentrations by calibrated thrombin clotting time in comparison to LC-MS/MS in human volunteers on dialysis.

INTRODUCTION: Dabigatran etexilate is an oral direct thrombin inhibitor. Although routine anticoagulation monitoring with dabigatran is not usually required, a simple and precise laboratory test to measure dabigatran concentrations in patient plasma may be useful in certain clinical circumstances, such as emergency situations. The HEMOCLOT(®) Thrombin Inhibitors assay has demonstrated accurate and precise determination of dabigatran concentrations within a range of 50-500 ng/ml. The objective of this study was to assess comparability of dabigatran concentrations determined by HEMOCLOT(®) and by liquid chromatography/tandem mass spectrometry (LC-MS/MS) in plasma samples from human volunteers with end-stage renal disease (ESRD) undergoing regular haemodialysis (HD) during a Phase I study. MATERIALS AND METHODS: Overall, 304 plasma samples were obtained from seven ESRD patients in dabigatran steady-state for measurement by HEMOCLOT(®) (calibrated diluted thrombin time [dTT]) and by LC-MS/MS. Agreement of dabigatran concentrations was assessed by regression analysis and difference plots. RESULTS: The measurements of calibration standards of the HEMOCLOT(®) assay showed excellent precision with coefficients of variation <5%. Accuracy determined by analysis of two quality control samples was 90% and 111%. HEMOCLOT(®)-derived dabigatran plasma concentrations paralleled those obtained by LC-MS/MS. The mean ratio of the LC-MS/MS and dTT-derived concentrations was 0.955 (67% limits of agreement: 0.771-1.18). CONCLUSIONS: The HEMOCLOT(®) Thrombin Inhibitors assay is suitable for measuring dabigatran plasma concentrations in volunteers with ESRD undergoing haemodialysis. The agreement between dabigatran concentrations determined by the HEMOCLOT(®) assay and the LC-MS/MS reference method met bioanalytical acceptance criteria.

Immune profiling in blood identifies sTNF-R1 performing comparably well as biomarker panels for classification of Alzheimer's disease patients.

BACKGROUND: Alzheimer's disease (AD) has been linked to a state of cerebral and systemic inflammation. The objective of the present study was to determine whether singular markers or a set of inflammatory biomarkers in peripheral blood allow discrimination between AD patients and healthy controls at the individual level. METHODS: Using bead based multiplexed sandwich immunoassays, 25 inflammatory biomarkers were measured in 164 serum samples from individuals with early AD and age-matched cognitively healthy elderly controls. The data set was randomly split into a training set for feature selection and classification training and a test set for class prediction of blinded samples (1 : 1 ratio) to evaluate the chosen predictors and parameters. Multivariate data analysis was performed with use of a support vector machine (SVM). RESULTS: After selection of sTNF-R1 as most discriminative parameter in the training set, the application of SVM to the independent test dataset resulted in a 90.0% correct classification for individual AD and control subjects. CONCLUSIONS: We identified sTNF-R1 from a marker set consisting of 25 inflammatory biomarkers, which allowed SVM-based discrimination of AD patients from healthy controls on a single-subject classification level comparably well as biomarker panels with a clinically relevant accuracy and validity. Although larger sample populations will be needed to confirm this diagnostic accuracy, our study suggests that sTNF-R1 in serum-either as singular marker or incorporated into a biomarker panel-could be a powerful new biomarker for detection of AD. In addition, selective inhibition of TNF-R1 function may represent a new therapeutic approach in AD.

Characterization of immunologically active drugs in a novel organotypic co-culture model of the human gut and whole blood.

The human immune system represents a highly complex multicellular network that protects the organism against the environment and pathogens. Within this system, different immune cells communicate with each other, as well as with adjacent organs and tissues, using an impressive network of regulatory signals. This inherent complexity makes it rather difficult to mimic these processes in vitro. Unpredictable drug-induced side effects can be the consequence when moving from preclinical animal models into clinical phase. Therefore, there is a demand for more elaborate in vivo like human cell culture models. In this study, an in vitro co-culture model consisting of Caco-2 human gut epithelial cells and human whole blood representing the immune system is applied to investigate the intestinal absorption of anti-inflammatory drugs and the subsequent modulation of the immunoregulatory signaling processes. By using blood of different donors, the individuality of the immune system is integrated into the overall analysis. The anti-inflammatory drugs prednisolone and ibuprofen were applied on top of the Caco-2 epithelial cells and alterations in the extracellular communication via cytokines and chemokines were visualized using miniaturized multiplexed sandwich immunoassays. Optionally, pretreatment of the Caco-2 epithelial cells with pro-inflammatory mediators can be used to modulate the epithelial barrier function similar to the situation observed during inflammatory conditions of the gut. The presented translational test system, consisting of differentiated Caco-2 intestinal epithelial cells and whole blood substantially improves preclinical screening of immunologically active drugs with respect to an approximation of the human "in vivo" conditions.

Superficial wound swabbing: a novel method of sampling and processing wound fluid for subsequent immunoassay analysis in diabetic foot ulcerations.

OBJECTIVE: In diabetic foot ulcers, wound fluid inflammatory mediators have previously been proposed as surrogate markers for nonhealing. However, currently available wound fluid sampling techniques are not suitable for clinical practice due to low levels of exudate and a high logistical effort. The aim of this investigation was to assess 1) the technique of superficial wound swabbing for harvesting wound fluid; and 2) the quality of the collected fluid for immunoassay analysis of inflammatory mediators. RESEARCH DESIGN AND METHODS: Both nylon-flocked swabs and film dressings were used to collect wound fluid from foot ulcers of diabetic patients. In randomly selected patients, levels of wound fluid inflammatory mediators and matrix metalloproteases were determined using multiplexed bead-based sandwich immunoassays with respect to both sampling methods. Wound fluid spike-in experiments were performed to evaluate the impact of different sample processing protocols on subsequent immunoassay analysis. RESULTS: Using the swabbing technique, a median amount of 40 µL (2-120 µL) wound exudate was collected, which allowed the measurement of several multiplex panels. Comparing both sampling methods, a similar qualitative protein recovery was observed with a trend to analyte enrichment by swabbing. Sample processing using swabs did not affect analyte recovery, with the exception of interleukin (IL)-8, thymus and activation-regulated chemokine, IL-17A, interferon-γ-induced protein 10, and IL-4. CONCLUSIONS: The quality of wound fluid collected by superficial swabbing is not inferior to the current standard technique. Combined with subsequent bead-based sandwich immunoassay analysis, this new method offers a noninvasive technique, suitable for daily clinical routines, for assessment of inflammatory activity in diabetic foot ulcers.

Functional analysis of Rho GTPase activation and inhibition in a bead-based miniaturized format.

Extensive knowledge about protein-protein interactions is fundamental to fully understand signaling pathways and for the development of new drugs. Rho GTPases are key molecules in cellular signaling processes and their deregulation is implicated in the development of a variety of diseases such as neurofibromatosis type 2 and cancer. Here, we describe a bead-based protein-protein interaction assay for overexpressed HA-tagged Rho GTPases to study the GTPγS-dependent interaction with the regulatory protein RhoGDIα. This assay provides a useful tool for the analysis of both macromolecular and small molecule activators and inhibitors of the protein-protein interactions of Rho GTPases with their regulatory proteins in a multiplexed miniaturized format.

Protein-protein-interactions in a multiplexed, miniaturized format a functional analysis of Rho GTPase activation and inhibition.

A miniaturized, bead-based protein-protein-interaction assay was developed to study the interaction of Rho GTPases with regulatory proteins. The setup, which uses only minute amounts of sample, was used to analyze small molecules that inhibit the interaction between Rho GTPases and RhoGDI alpha. Prenylcysteine analogues and the replacement of GDP by non-hydrolysable GTP analogues prevented the formation of Rho GTPase-RhoGDI alpha complexes in a concentration-dependent manner.