More specific quantitative assays for

More specific quantitative assays for DiXaIs are based on the inhibition of chromogenic or clotting activity of Factor Xa. During drug development, Kubitza developed an endogenous Anti-Factor Xa assay [45], which is very sensitive to Rivaroxaban drug concentration. This assay was based on Factor Xa generation in Diclazuril and measurement of residual factor Xa activity with the use of a chromogenic substrate. Undiluted, or poorly diluted plasma was used, and hirudin (a potent Thrombin irreversible inhibitor) was used to prevent clot formation, and to block the coagulation cascade at the factor Xa activity. This assay was very useful for drug development, and validation of anti-Factor Xa activity of Rivaroxaban, but it was difficult to optimize and standardize for more current laboratory use in treated patients. With MM Samama we worked on that method for some time, as this assay was thought to be the closest to the physiological process of Factor Xa generation and inhibition and it was expected to actually reflect the anticoagulant activity targeted to Factor Xa. We then switched to a more pharmacological assay, based on a two-stage method, for which the principle was the inhibition of a constant and limited amount of human Factor Xa by Rivaroxaban, in the presence of a high sodium chloride concentration (1 M NaCl). In these conditions, heparins can no longer interact with AT, and the assay is then specific for DiXaIs [38]. Only a low and negligible activity due to β-AT, still active at this ionic strength, remained, but the assay was highly specific for DiXaI/Rivaroxaban activity in plasma. This two-stage assay was also correctly and successfully applied for measuring Apixaban and Edoxaban concentrations in plasma. As will be discussed in the next paragraphs, this 2-stage Anti-Factor Xa assay has to be calibrated with drug specific plasma calibrators and controls, obtained by spiking drug into plasma, distributing it into vials and lyophilizing it. This 2-stage assay was compared in various studies to the reference method for measuring DOACs, which is Liquid Chromatography: Mass Spectrometry (LC:MS) [44], [46]. In most of the clinical trials, for measuring DiXaIs kinetics in treated subjects, the peak and through concentrations were measured using this reference LC:MS technique, and some Anti-Factor Xa assays were evaluated in some cases. This LC:MS reference method cannot be used in clinical laboratories, as it is complex, time consuming, needs specific equipment, and is poorly adapted to individual testing. More conventional laboratory methods, based on inhibition of Factor Xa enzymatic activity were then required for measuring drug in some patients when needed. Although LC:MS measures drug mass in plasma, and Anti-Factor Xa assays the Anti-Factor Xa activity, there is usually an excellent correlation between both types of assays [43], [44], [46]. DiXaIs are mainly present in the active form, and possible metabolites, such as M4 for Edoxaban, keep a similar Anti-Factor Xa activity to that of the parent molecule [16], [17], [18]. When the DiXaI therapy has entered into clinical practice, practitioners and laboratories who wished to measure the drug concentration in treated patients' plasma, expected to use an already available Anti-Factor Xa assay. The assay considered was the conventional Anti-Factor Xa kinetics method designed for measuring UFH/LMWH, Sodium Danaparoid (Orgaran®) and Fondaparinux (Arixtra®). This technique is used on automated instruments and is available in many laboratories at any time of the day or night, including wards and emergencies (24/24 and 7/7). Applications for DiXaIs measurements were then successfully developed with this kinetics, 1-stage assay. As for the 2-stage assay, dedicated drug specific plasma calibrators and controls are required. For DiXaIs, results cannot be given in Anti-Factor Xa International Units (IU) as for heparins (UFH, LMWH), because the mode of action of DiXaIs is quite different from that of heparin (Fig. 2), and inhibitory units cannot be extrapolated. Concentrations of DiXaIs are given in ng/ml by reference to the specific DiXaI drug used for calibration, and assigned calibrator concentrations are validated with the LC:MS reference method. Both 1-stage and 2-stages Anti-Factor Xa methods, used with drug-specific plasma calibrators and controls, are convenient laboratory tools for measuring DiXaI drug concentration/activity (Anti-Factor Xa) in plasma, and can be used in any laboratory and on any coagulation instrument. Fig. 5 presents the usual dose response curves obtained with both Anti-Factor Xa assays for Rivaroxaban or Apixaban. Although both methods can be used for all DiXaIs, we present the 1-stage assay only for Rivaroxaban, and the 2-stage assay only for Apixaban. No specific skill is needed for performing these assays, other than a normal training to standard laboratory coagulation assays and their use on automated instruments (semi-automated instruments or manual methods are also possible). The sole caution to consider in interpreting results is to analyze DiXaIs concentration in respect to the latest drug intake time, and consider the drugs kinetics and clearance. In a normal population, peak concentrations are reached 2 to 4 hours after intake and through concentrations are measured just before the next drug intake, i.e. after 12 hours for a therapeutic protocol with two doses per day (the usual case for Apixaban), or after 24 hours for a single dose per day (the usual case for Rivaroxaban). Peak concentrations are useful for detecting overdose, and through concentrations permit identification of any risk of progressive drug accumulation in patients with decreased liver or kidney (mainly for Edoxaban) clearance [19], [20].