Assays for Measuring Rivaroxaban: Their Suitability and Limitations

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1 REVIEW ARTICLE Assays for Measuring Rivaroxaban: Their Suitability and Limitations Edelgard Lindhoff-Last, MD,* Meyer Michel Samama, MD, Thomas L. Ortel, MD, Jeffrey I. Weitz, MD, k and Theodore E. Spiro, MD Abstract: Several new oral anticoagulants such as rivaroxaban (which targets Factor Xa) and dabigatran etexilate (which targets thrombin) are in advanced stages of clinical development and are already available for clinical use in some countries. Although these agents do not require routine coagulation monitoring, assays to assess the level of anticoagulation may be of assistance in certain circumstances such as in case of overdose, in patients with a hemorrhagic or thromboembolic event during treatment, or to assess compliance. Moreover, the influence of the new oral anticoagulants on routine coagulation tests must be recognized. The prothrombin time is not suitable for rivaroxaban measurement for several reasons, and the routinely used international normalized ratio for monitoring the vitamin K antagonists cannot be applied to rivaroxaban. Development of universal assays is challenging because the new oral anticoagulants have different targets, and even those with the same target have variable effects on routine coagulation assays. Focusing on rivaroxaban, there is emerging evidence that an anti-factor Xa assay that uses rivaroxaban-containing plasma calibrators may provide the optimal method for determining plasma rivaroxaban concentrations. Key Words: anticoagulants, coagulation assays, monitoring, rivaroxaban (Ther Drug Monit 2010;32: ) Received for publication April 29, 2010; accepted July 20, From the *Division of Vascular Medicine and Haemostaseology, Department of Internal Medicine, University Hospital Frankfurt, Frankfurt, Germany; Hôtel-Dieu University Hospital, Paris, France; Biomnis Laboratories R&D, Ivry-sur-Seine, France; Hemostasis and Thrombosis Center, Duke University Medical Center, Durham, NC; k Departments of Medicine and Biochemistry and Biomedical Sciences, McMaster University and Thrombosis and Atherosclerosis Research Institute, Hamilton, Ontario, Canada; and {Bayer HealthCare Pharmaceuticals Inc, Montville, NJ. The authors received editorial support from Yong-Ling Liu with funding from Bayer Schering Pharma AG and Johnson & Johnson Pharmaceutical Research & Development, LLC. Theodore E. Spiro is an employee of Bayer HealthCare Pharmaceuticals Inc. E. Lindhoff-Last has acted as a consultant and is a member of the national advisory board of Bayer Schering Pharma AG in Germany. M. M. Samama and J. I. Weitz have acted as consultants for Bayer. T. Ortel has no conflict of interest to disclose. Correspondence: Edelgard Lindhoff-Last, MD, University Hospital Frankfurt, Theodor-Stern-Kai 7, Frankfurt, Germany ( Lindhoff-last@ em.uni-frankfurt.de). Copyright Ó 2010 by Lippincott Williams & Wilkins INTRODUCTION Anticoagulant therapy is widely used for the prevention and treatment of venous and arterial thromboembolism. The limitations of currently available anticoagulants such as vitamin K antagonists (VKAs) and heparins have prompted the search for new oral anticoagulants that target a single enzyme within the coagulation pathway. Rivaroxaban is an oral, direct Factor Xa inhibitor 1 approved in more than 90 countries worldwide, including the European Union, for the prevention of venous thromboembolism (VTE) after elective hip or knee arthroplasty in adult patients. It is currently being investigated in Phase III studies for the prevention of VTE in medical patients, treatment of VTE, stroke prevention in patients with atrial fibrillation, and for the prevention of recurrent ischemia in patients with acute coronary syndrome. Rivaroxaban is a selective inhibitor of Factor Xa and inhibits free and prothrombinase-bound Factor Xa as well as clot-associated Factor Xa. 2,3 Rivaroxaban has been found to have predictable pharmacokinetics and pharmacodynamics and minimal clinically relevant drug drug interactions. 4 6 Consequently, rivaroxaban can be given in fixed doses without the need for routine coagulation monitoring. However, an assay to measure rivaroxaban levels would be valuable to assess compliance. Such an assay may also be of assistance in other circumstances such as in the case of overdose, in patients with a hemorrhagic or thromboembolic event during treatment with rivaroxaban, in those with deteriorating renal function, or in patients who require urgent surgery. Therapeutic drug monitoring is usually performed by measuring either the plasma concentration of a drug or by using pharmacologic or biochemical assays. Assay techniques for measuring anticoagulants include clot-based assays, chromogenic assays, enzyme-linked immunosorbent assays, and highperformance liquid chromatography. 7 Of these techniques, clotbased assays and chromogenic assays are most commonly used for clinical laboratory monitoring. Clotting assays provide a global assessment of the coagulation pathways and are prolonged by most anticoagulants. By contrast, chromogenic assays, which use specific enzyme-directed substrates, can be used to measure the levels of an anticoagulant. 7 There is emerging evidence that the new oral anticoagulants have variable effects on routine tests of coagulation. 8 For example, the direct thrombin inhibitor dabigatran etexilate has little effect on the prothrombin time (PT) and its effect on the activated partial thromboplastin time (aptt) is not dose-linear. 9,10 At therapeutic doses, apixaban, another oral, direct Factor Xa inhibitor, has little effect on either the PT or the aptt. 11,12 It is Ther Drug Monit Volume 32, Number 6, December

2 Lindhoff-Last et al Ther Drug Monit Volume 32, Number 6, December 2010 clear, therefore, that these routine tests of coagulation will not be useful to measure these agents. Although rivaroxaban prolongs the PT, its effect is modest and dependent on the sensitivity of the thromboplastin reagent used in the assay. 13 Although the international normalized ratio (INR; developed for the monitoring of VKA therapy) corrects for varying sensitivities of thromboplastin reagents to reductions in the levels of vitamin K- dependent clotting proteins, this correction is not useful with rivaroxaban, a direct Factor Xa inhibitor. Consequently, other assays are needed to measure rivaroxaban concentrations. Measuring plasma drug concentration has long been established as a standard for therapeutic drug monitoring, and measuring the intensity of coagulation does not necessarily quantify or translate to plasma concentrations of an anticoagulant, especially in patients who have hepatic insufficiency or develop disseminated intravascular coagulation. It is becoming apparent that measuring the plasma concentrations of rivaroxaban (likes for many other therapeutic drugs) may be the optimal method to assess the level of anticoagulation if required. In plasma, rivaroxaban is highly bound to albumin and, therefore, the high protein binding of rivaroxaban must be taken into account when measuring plasma concentrations and when dilution of plasma samples is required. This article discusses the suitability and limitations of several assays for measuring both the anticoagulant effect of rivaroxaban as well as its plasma concentrations and the potential candidate assays that may be suitable if plasma concentration measurement is required. THE COAGULATION CASCADE Vascular injury and the resulting disruption of the endothelium can lead to the initiation of complex hemostatic responses, including vasoconstriction, the formation of a platelet plug, and activation of the coagulation system. Activation of coagulation results in a burst of thrombin generation that rapidly converts fibrinogen to fibrin, resulting in the formation of a thrombus (see Fig. 1). Coagulation is activated when blood comes in contact with tissue factor (TF)-bearing cells. TF binds to circulating Factor VIIa to form the TF VIIa complex, which activates Factor IX and Factor X. Activated Factor X (Factor Xa) converts small amounts of prothrombin to thrombin, which then feeds back to activate Factors VIII and V (key cofactors in coagulation). The coagulation cascade is amplified by further generation of Factor Xa by the Factor IXa Factor VIIIa Ca 2+ phospholipid complex (the tenase complex). Factor Xa is incorporated into the prothrombinase complex with Factor Va, phospholipid, and Ca 2+ and converts prothrombin to thrombin (Factor IIa). Thrombin plays a central role in the clotting process: it converts soluble fibrinogen to fibrin, activates Factors V, VIII, and XI (which generates more thrombin), activates Factor XIII (which crosslinks the fibrin), and activates platelets. 14,15 METHODS FOR COAGULATION MONITORING Prothrombin Time and the International Normalized Ratio The PT assay is a widely available global clotting test that can be used to measure the integrity of the extrinsic and final common pathways of coagulation involving Factors II, V, VII, and X. It represents the time, in seconds, for plasma to clot after the addition of TF and Ca 2+ (thromboplastin). 16 Inhibitors or deficiencies of clotting factors within the extrinsic and common pathways result in a prolongation of the PT. Commercially available thromboplastins vary in their TF source and methods of preparation leading to different FIGURE 1. Schematic representation of the coagulation cascade demonstrating the inhibiting effects of traditional and new anticoagulants on coagulation factors. AT, antithrombin; LMWH, low-molecularweight heparin; UFH, unfractionated heparin. 674 q 2010 Lippincott Williams & Wilkins

3 Ther Drug Monit Volume 32, Number 6, December 2010 Measurement of Rivaroxaban Levels in Plasma sensitivities to the anticoagulant effect of VKAs. 17 To standardize the PT test, the World Health Organization introduced the international sensitivity index/inr (ISI/INR) system in ,18 TheINRisamathematicalconversionofan individual s PT that accounts for the sensitivity of the thromboplastin used in a given laboratory by factoring in the ISI supplied by its manufacturer. The INR is calculated as follows: INR = (patient s PT/the mean normal PT) ISI in which the geometric mean normal PT is the PT of at least 20 healthy subjects of both sexes tested at the testing laboratory. 18 This system has been developed for the monitoring of VKA therapy (such as warfarin) in patients under stable conditions. VKAs exert their anticoagulant effect by interfering with the cyclic interconversion of vitamin K and its 2,3-epoxide, leading to the hepatic production of partially carboxylated and decarboxylated coagulation factors (II, VII, IX, and X) with reduced coagulant activity. 19 The ISI value used in the calculation indicates the overall sensitivity of the PT reagent to reductions in all of those vitamin K-dependent clotting factors (II, VII, and X), although the PT reagents are generally more sensitive to deficiencies of Factor VII within the extrinsic pathway and less sensitive to deficiencies within the final common pathway. 16 Prothrombin Time and Rivaroxaban The mechanisms of action of rivaroxaban and other new oral anticoagulants, however, differ from that of VKAs. Rivaroxaban selectively and directly inhibits Factor Xa, one of the pivotal enzymes in the coagulation system (see Fig. 1). 3 In healthy subjects, rivaroxaban prolongs the PT in a dosedependent fashion when the test is performed with Neoplastin Plus (Diagnostica Stago, Asnières-sur-Seine, France). 5 The same is true in patients undergoing major orthopedic surgery using Neoplastin (Diagnostica Stago). 20 The prolongation of PT correlates with rivaroxaban plasma concentrations. 6,20 These studies suggested that the PT may be a useful laboratory test platform for measuring rivaroxaban. However, accumulating evidence suggests that the PT assay is not a suitable test and the INR system cannot be used for measuring rivaroxaban. First, the PT assay is a global clotting test and may be prolonged for a number of reasons. In patients with hepatic impairment, sepsis, vitamin K deficiency, disseminated intravascular coagulation, various types of cancers, and other systemic diseases, the basal levels are increased before taking rivaroxaban. This may influence the prolongation of PT mediated by rivaroxaban, thus interfering with the ability of the test to accurately measure rivaroxaban blood concentrations. This is also the case for other anticoagulants. Plasma determination of the direct thrombin inhibitor hirudin with the ecarin clotting assay, for example, has been shown to be strongly influenced by the prothrombin level. This was not the case with a chromogenic anti-factor IIa inhibition assay in which an excess of exogenous Factor IIa is included. 26 Second, the effect of rivaroxaban on PT is transient 27,28 and changes over time after administration according to the half-life of 7 to 11 hours. 4 Warfarin has a much longer half-life (36 42 hours), 19 and its effect on PT is indirect because it requires the synthesis of clotting factors with reduced coagulant activity. Thus, its disappearance from plasma does not restore the normal function of the coagulation factors; the restoration of normal coagulation is dependent on the synthesis of new, fully functional clotting factors. Third, in Phase II studies for the prevention of VTE, the mean concentration range (minimum plasma concentration [C trough ] to maximum plasma concentration) for a total daily rivaroxaban dose of 5 to 20 mg was 12 to 152 ng/ml in the hip study and 19 to 201 ng/ml in the knee study. 6,20 The PT test does not have the sensitivity to measure C trough of the dose range of rivaroxaban that is currently used in the clinical setting or under development for new indications (ie, mg). In human plasma spiked with rivaroxaban, the effect on PT (using STA Néoplastine CI Plus; Diagnostica Stago) can be observed at rivaroxaban plasma concentrations between approximately 50 and 700 ng/ml. The test lacks accuracy and precision at lower rivaroxaban plasma concentrations. 29 Thus, the PT test may not provide reliable results for checking compliance if the blood samples are collected at a time around C trough or for confirming that there is no residual anticoagulant effect in case of a need for invasive procedures such as spinal punctures or epidural anesthesia. Finally, different thromboplastin reagents used in the PT assays showed variable sensitivities to rivaroxaban at the same concentration; 30 the coefficients of variation between different laboratories are too large to provide a precise measurement over the therapeutic dose range of 2.5 to 20 mg, especially concerning C trough. In addition, expression of PT results in seconds and/or conversion of PT ratios to INR values did not correct the variability in response to rivaroxaban observed between various reagent systems, and in some cases the INR correction even exacerbated the problem. 31 Therefore, the INR monitoring system used for warfarin cannot be used for rivaroxaban and does not provide meaningful information. Furthermore, the calculated INR values for different Factor Xa inhibitors show markedly different results. 32,33 Therefore, the PT and the INR correction might also not be suitable for other new oral anticoagulants such as the direct Factor Xa inhibitors and the direct thrombin inhibitor dabigatran etexilate. 9,10 Dilute Prothrombin Time In routine PT tests, the quantity of thromboplastin is much higher than that under physiological conditions. In the dilute PT test, thromboplastin reagents are diluted to increase the sensitivity of the test and, thus, the conditions of this test might be closer to physiological conditions. 34 The dilute PT assay has been suggested to be one of the potential assays for monitoring apixaban. 12 However, like the PT assay, the results obtained with rivaroxaban varied depending on the reagents used, and the test lacked sensitivity at lower rivaroxaban plasma concentrations. 35 Activated Partial Thromboplastin Time The aptt test is a measure of the integrity of the intrinsic and common pathways of the coagulation cascade involving Factors II, V, VIII, IX, X, XI, and XII. The aptt reagent is called partial thromboplastin because TF is not present in conjunction with the phospholipid as it is in the PT reagent. The aptt is performed by first adding a surface activator (eg, kaolin, celite, or silica) and diluted phospholipid q 2010 Lippincott Williams & Wilkins 675

4 Lindhoff-Last et al Ther Drug Monit Volume 32, Number 6, December 2010 to citrated plasma. After this initial incubation to allow optimal activation of contact factors, Ca 2+ is then added before the measurement of clotting time. The aptt ranges between 22 and 40 seconds, depending on the reagent and coagulometer used. 36 Inhibitors or deficiencies of clotting factors within the intrinsic and final common pathways result in the prolongation of aptt. 16,36 Heparin prolongs aptt, whereas the synthetic pentasaccharide fondaparinux has hardly any effect. 7,37 Dabigatran etexilate prolongs aptt; however, the effect is not linear and the assay is not recommended for monitoring dabigatran etexilate at therapeutic doses. 9 Rivaroxaban has a weaker effect on aptt than on PT. Like PT, the prolongation of aptt is short-lived, 27 and different reagents also exhibit different sensitivities to rivaroxaban at the same concentrations. 38 Thus, aptt is also not suitable for measuring rivaroxaban blood concentrations. Activated Clotting Time The activated clotting time is a simplified version of the aptt test that measures the intrinsic clotting activity of whole blood. A blood sample is collected in a tube containing an activator of coagulation such as celite, kaolin, or glass particles. Clot formation can be detected by the mobility of a magnet inside the test tube that contains the blood. The reference range varies depending on the method used, but it usually falls within 70 to 180 seconds. This test is used to monitor the effectiveness of high-dose heparin therapy or treatment with bivalirudin. 36 The effect of new oral anticoagulants (including rivaroxaban, apixaban, and dabigatran etexilate) on the activated clotting time has not been reported. HepTest The HepTest (American Diagnostica, Stamford, CT) assay is a clot-based anti-factor Xa assay 7 designed as a surrogate measure of heparin-based drug plasma concentrations. It is based on the ability of anticoagulants to catalyze the inhibition of exogenous Factor Xa by plasma antithrombin. The rate of inhibition, although directly proportional to the concentration of the anticoagulant, is measured indirectly as the time it takes for the sample to clot. In human plasma, apixaban produced a concentrationdependent increase in the HepTest, and the test appeared to be more sensitive than the aptt or PT tests. 12 Rivaroxaban has been shown to have a paradoxic effect on the two-step HepTest (incubation time of 120 seconds) in vitro; higher concentrations of rivaroxaban prolonged the clotting time, whereas lower concentrations of rivaroxaban reduced it. However, this paradoxic decrease did not occur with shorter incubation times. 39 In healthy humans, rivaroxaban prolonged the HepTest with a similar profile as that for PT prolongation. 27 A modification of this test has been recently introduced in which the incubation step has been eliminated and the sequence of reagent addition has been modified. The influence of rivaroxaban has not been studied using this modified HepTest and there are currently no data available on the effects of other Factor Xa inhibitors on this modified test either. However, like other clotting assays, the HepTest is not specific for measuring Factor Xa activity, and the assay is not commonly used in some countries. Prothrombinase-Induced Clotting Time Prothrombinase-induced clotting time (PiCT) is a clotting assay based on the activation of coagulation using a combination of a defined amount of bovine Factor Xa, phospholipids, Ca 2+, and Russell s viper venom-v (an enzyme that specifically activates Factor V), which form the prothrombinase complex. 40 The assay is dependent on the activity of Factor Xa in the plasma sample; thus, in the presence of Factor Xa inhibitors, the clotting time will be delayed. The PiCT assay is approved for determining the anticoagulant activity of heparins and fondaparinux 40 and has recently been proposed as a potential laboratory assay suitable for monitoring direct Factor Xa inhibitors. 41 Similar to the HepTest, rivaroxaban has been shown to cause a paradoxic response in the two-step PiCT test when bovine Factor Xa was used, with a decrease in clotting time at low rivaroxaban concentrations but an increase at high concentrations. However, the decrease was not seen with the one-step PiCTwhen human Factor Xa was used or in antithrombin-depleted plasma. 40 In healthy subjects, rivaroxaban prolonged the PiCT (using the one-step method) with maximum effect occurring 2 hours after administration (1.8 and 2.3 times baseline after rivaroxaban 5 mg and 30 mg, respectively) and was more sensitive than the PT assay. At 24 hours after drug administration, the PiCT remained prolonged by 1.2-fold and 1.5-fold after 5 mg and 30 mg, respectively. 42 However, the commercially available PiCT assay, even modified (ie, as a one-step procedure calibrated using rivaroxaban), may not provide the sensitivity required at low rivaroxaban plasma concentrations, eg, for measuring noncompliance. Thrombin Generation Test The thrombin generation test assesses each phase of thrombin generation using a thrombin-generation trigger (TF or collagen). Thrombin generation can be assessed by measuring the cleavage of a fluorogenic or a chromogenic substrate and quantified using a thrombin calibrator. There are many different types of assays for measuring thrombin generation either in whole blood or in citrated plasma. Because there is no standardization of the methods, the different assay systems generate different results. 43,44 The main measurement parameters include: the maximum concentration of thrombin (peak), the endogenous thrombin potential (ETP; which represent thrombin activity multiplied by the time for which it remains active in the plasma), the lag time of thrombin generation, and the time taken to reach the maximum concentration of thrombin. 45 Rivaroxaban has been shown to affect the various parameters of the thrombin generation test in in vitro studies with greater inhibition on the initiation and propagation phases of thrombin generation than the decay phase. 45 In healthy subjects, rivaroxaban (5 mg or 30 mg) inhibited thrombin generation (by measuring the ETP), with maximum inhibition observed 2 hours after drug administration. 42 Although this study shows that the reduction of ETP correlates with rivaroxaban plasma concentration, the assay is not suitable for measuring rivaroxaban because the assay does not have the sensitivity required at low rivaroxaban plasma concentrations. In addition, the ETP method is not suitable for use as a routine laboratory test because it requires a high technical standard and laboratory skills q 2010 Lippincott Williams & Wilkins

5 Ther Drug Monit Volume 32, Number 6, December 2010 Measurement of Rivaroxaban Levels in Plasma Anti-Factor Xa Chromogenic Assays The chromogenic tests involve the enzymatic cleavage of a synthetic substrate (with the enzyme cleavage site of the natural substrate) onto which a chromophore tag p-nitroaniline is attached to one end. When the enzyme cleaves the synthetic substrate, it releases the yellow-colored p-nitroaniline that can be detected with a spectrophotometer by measuring the change in optical density. In anti-factor Xa chromogenic assays, Factor Xa cleaves the chromogenic substrate, and the change in optical density is directly proportional to the amount of Factor Xa present in the sample. 7,36 Anti-Factor Xa chromogenic assays are used for measuring the levels of heparin and low-molecular-weight heparin. 7,36 A modified anti-factor Xa assay has also been developed and evaluated for fondaparinux. 46 Recent studies suggest that commercially available Factor Xa chromogenic assays may be useful for the quantitative determination of rivaroxaban provided that a standard curve is generated with known amounts of rivaroxaban (ie, using rivaroxaban-spiked plasma samples as a calibrator). Controls for both low and high levels of rivaroxaban are also necessary, and the plasma concentration of rivaroxaban is expressed as ng/ml. In preliminary studies, the suitability of a number of chromogenic Factor Xa assays for the quantitative determination of rivaroxaban was evaluated. In these studies, pooled human plasma was incubated with increasing concentrations of rivaroxaban (0 500 ng/ml). The results showed that although the sensitivity differed among the different assays tested, rivaroxaban could be detected either at low or at high plasma levels using rivaroxaban-spiked plasma samples as a calibrator. 47 Further studies also showed that the commercially available Coamatic Heparin (Chromogenix Instrumentation Laboratory, Milan, Italy) assay could be used for measuring plasma levels of rivaroxaban in the range of 0 to 500 ng/ml using a rivaroxaban calibration curve Although these preliminary results are promising, further work is needed in terms of validation and standardization. A simple, reliable, reproducible, and standardized assay with a high sensitivity for the desired concentration range is yet to be made available for routine use in the clinical setting. DISCUSSION Testing of coagulation parameters during the administration of oral and parenteral anticoagulants is a common and accepted practice in clinical medicine. Several tests have been developed for this purpose. The most notable of these are as follows: the PT expressed as INR, which is widely used for monitoring the VKAs; the aptt (expressed in seconds), used for monitoring unfractionated heparin; and anti-factor Xa assays, used to monitor low-molecular-weight heparins, unfractionated heparin, and fondaparinux. Other tests such as the HepTest and PiCT are available, but their use is limited to only small numbers of laboratories. The calibrated automated thrombogram (used to quantify endogenous thrombin generation), thromboelastography (used to quantify thrombus mechanics and reaction time), and endogenous Factor Xa generation tests have all been developed, but they are only performed in highly specialized coagulation laboratories, which are in a very limited number of centers. New oral agents are being developed to replace the parenterally administered anticoagulants (such as the heparins and direct thrombin inhibitors) and orally administered VKAs. The advantages of the new oral agents include no requirement for routine coagulation monitoring as a result of their more predictable pharmacokinetics and pharmacodynamics and fewer food and drug interactions than VKAs. However, in certain clinical situations, measurement of their blood concentration as well as their pharmacodynamic effects would be desirable. Such situations include: patients with low body weights or obese patients, pediatric patients, those with renal or hepatic impairment, those who accidentally or deliberately overdose, or to measure compliance in patients with dementia or psychiatric disorders or those with questionable compliance with medication, and those admitted to emergency units with hemorrhagic or thrombotic complications. Rivaroxaban is a highly selective, oral Factor Xa inhibitor. 51 It has been shown to have predictable pharmacokinetics with little variation of plasma concentrations with age, body weight, or renal function after fixed-dose administration, 20 a wide therapeutic window, few drug drug interactions, and predictable pharmacodynamics without the need for routine coagulation monitoring. 4 The pharmacodynamics of rivaroxaban, as measured by its anticoagulant activity with a variety of methods (such as inhibition of Factor Xa, prolongation of global clotting tests), correlates closely with its plasma concentrations. 5,27 The gold standard method for measuring rivaroxaban concentrations in biologic fluids is high-performance liquid chromatography coupled with tandem mass spectrometry detectors. 52 However, this methodology cannot be practically implemented in routine clinical laboratories. Preclinical and clinical studies have evaluated several different hemostasis assays, which have provided valuable information for further clinical evaluation of the use of these assays. Inhibition of endogenous Factor Xa activity, the PT and aptt assays, the endogenous thrombin generation potential assay, the anti-factor Xa assay, and the HepTest have all been tested in the early stage of clinical development of rivaroxaban (Phases I and II) and, accordingly, these tests were considered to be possible platforms on which a test to measure rivaroxaban might be performed. However, the endogenous Factor Xa activity assay and the HepTest have been eliminated from further assessment as a result of the limited use of these tests in clinical coagulation laboratories. In addition, a modified PiCT test has been developed and is now available for further clinical evaluation. Because rivaroxaban is a highly selective, direct Factor Xa inhibitor, assays that measure anti-factor Xa activity are being further explored, in particular the chromogenic anti-factor Xa assays. Accumulating evidence suggests that the routinely used clotting assays do not reliably reflect the intensity of the anticoagulant effect of rivaroxaban. Moreover, like with other anticoagulants, the intensity of anticoagulation as measured by global clotting tests does not necessarily quantify or translate to its plasma concentrations (as a result of the various limitations of the assays as discussed previously). Therefore, a simple test that is able to detect a wide plasma concentration range would be useful in certain clinical situations to assess whether patients are over- or underdosed (such as in patients with hemorrhagic or thrombotic complications or suspected q 2010 Lippincott Williams & Wilkins 677

6 Lindhoff-Last et al Ther Drug Monit Volume 32, Number 6, December 2010 noncompliance) or to ensure that the plasma concentration of rivaroxaban is below a clinically relevant level before any surgical procedures. CONCLUSIONS New oral anticoagulants such as rivaroxaban do not require routine coagulation monitoring; however, assays to assess the level of anticoagulation or drug concentration in plasma may be of assistance in certain circumstances. Routinely used global clotting assays are not appropriate for measuring the new oral anticoagulants, including rivaroxaban. The INR system has been developed specifically for monitoring the VKAs (such as warfarin); this system cannot be applied to rivaroxaban, which has a totally different mode of action from that of warfarin. Preliminary studies suggest that the anti-factor Xa chromogenic assays are potential candidate assays for measuring rivaroxaban blood concentrations, if required. However, the assays require a standard curve produced with known amounts of rivaroxaban (ie, using rivaroxaban-spiked plasma samples as a calibrator), and the concentrations need to be expressed as ng/ml. Commercially available assays with standard rivaroxaban calibrators and controls are currently under evaluation and development. ACKNOWLEDGMENTS We acknowledge Yong-Ling Liu who provided editorial support with funding from Bayer Schering Pharma AG and Johnson & Johnson Pharmaceutical Research & Development, LLC. REFERENCES 1. Roehrig S, Straub A, Pohlmann J, et al. Discovery of the novel antithrombotic agent 5-chloro-N-([(5S)-2-oxo-3-[4-(3-oxomorpholin-4-yl) phenyl]-1,3-oxazolidin-5-yl]methyl)thiophene-2-carboxamide (BAY ): an oral, direct Factor Xa inhibitor. JMedChem. 2005;48: Depasse F, Busson J, Mnich J, et al. Effect of BAY a novel, oral, direct Factor Xa inhibitor on clot-bound Factor Xa activity in vitro. J Thromb Haemost. 2005;3(Suppl 1). 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