Rivaroxaban Concentrations Research Articles

The in-vitro anticoagulant effect of rivaroxaban in neonates

The use of anticoagulants in neonates is increasing because of the medical advances improving the long-term survival of very sick infants who are at risk of venous thromboembolism (VTE). Current anticoagulation therapy in neonates is less than ideal, because of the physiological differences compared to children and adults regarding the pathophysiology of thrombosis and pharmacology of the drug. Limitations associated with conventional anticoagulants have prompted the development of novel drugs that specifically target the key proteins in the coagulation system. Rivaroxaban is the first oral, direct Factor Xa inhibitor available for the prevention of VTE in adults. Its predictable pharmacokinetic profile, high oral bioavailability and once-daily dosing make rivaroxaban an optimal anticoagulant that warrants investigation in neonates. This study was designed to determine whether there are age-related differences in the pharmacodynamic effects of rivaroxaban in vitro amongst neonates. Neonatal and adult plasma pools were created and spiked with increasing concentrations of rivaroxaban (0-500 ng/ml). Commercially available prothrombin time (PT), activated partial thromboplastin time (aPTT) and anti-Factor Xa assays as well as a sub-sampling thrombin generation assay were used to measure the rivaroxaban effect. A dose-dependent response was observed for PT, aPTT and lag time in both the age groups. Rivaroxaban caused a significant increase in the clotting time for PT and aPTT as well as an increase in lag time (as measured by thrombin generation) in neonates when compared with adults. In-vivo studies are required to confirm the consistency of dose-response in neonates.

Abstract T P348: Successful Resolution of the Cardiac Thrombus Using Novel Oral Anticoagulants

Objective: We aimed to determine therapeutic effects of novel oral anticoagulants (NOACs) on cardiac thrombi in acute stroke. Methods: We studied consecutive acute ischemic stroke/TIA patients with non-valvular atrial fibrillation who started to take rivaroxaban or dabigatran and was diagnosed as having cardiac thrombi on transesophageal echocardiography (TEE) within 10 days after the admission between July 2012 and July 2013. All continued to take NOACs without additional antithrombotic agents. Plasma concentrations for rivaroxaban were calibrated based on anti-Xa chromogenic assay for rivaroxaban (Stago®) and thrombin clotting time assay for dabigatran (Hemoclot®) just before (trough) and 4h after medication (peak) at steady state. Results: Eight patients (7 men, 52-83 years old) were studied. Their BW ranged 41.0-88.8 kg, CHADS2 ranged 2-5, and NIHSS ranged 2-18. NOACs were initiated at median 3 days and TEE was performed at median 3 days after admission. As a NOAC, one patient took 15 mg o. d. of rivaroxaban, 5 took 10 mg o.d. of rivaroxaban (officially approved dose in Japan) and the other 2 took 110 mg b.i.d. of dabigatran. For all the patients, the thrombus was identified in the left atrial appendage with median size of 14.7 mm; two of them were hyperechoic, being judged as already-organized. In follow-up TEE examinations median 19 days after admission , 5 thrombi disappeared (rivaroxaban in 3, dabigatran in 2), 1 diminished in size (rivaroxaban), and the other 2, all being initially hyperechoic, did not change (rivaroxaban for both). For 6 patients with absolute/partial dissolution of the thrombi, median peak/trough concentrations of rivaroxaban were 69 (19-255)/10 (range10-20) ng/ml, and those of dabigatran were 100 (40-160)/40 (20-60) ng/ml, median peak/trough aPTT was 42/34.5 s in rivaroxaban users and 45/37 s in dabigatran users, and median peak/trough PT was 20.2/12.9 s and 15.3/13.3 s, respectively. Conclusion: Rivaroxaban and dabigatran have potential to resolve non-organized cardiac thrombi. Monotherapy using NOACs may be enough for early prevention of recurrent stroke in patients with cardioembolic stroke/TIA.

Abstract T P346: Anticoagulation Intensity of Low-dose Rivaroxaban for Japanese Patients With Nonvalvular Atrial Fibrillation

Objective: In Japan, low-dose rivaroxaban [15 mg o.d. and 10 mg o.d. only for CCr 30-49 mL/min] was approved for clinical use to NVAF patients, because of its unique pharmacokinetics in Japanese subjects showing higher rivaroxaban concentration than Caucasian subjects when using the same dosage and the results of the J-ROCKET AF trial (Hori M, et al: Circ J 2012). We aimed to determine the anticoagulation intensity of rivaroxaban and its determinant factors in Japanese NVAF patients, especially in those taking crushed tablets. Methods: Consecutive stroke patients with NVAF admitted between July 2012 and July 2013 were studied. Prothrombin time (PT, Recombiplastin/HemosIL®), activated partial thromboplastin time (aPTT, Actin/Sysmex®), and anti-Xa chromogenic assay (Stago®) were measured just before and 4h and 9 h after the administration at the steady state of rivaroxaban (mean 6th day). We calibrated its plasma concentration based on anti-Xa assay. Results: Of 104 patients (32 women, 74±9 y.o., BW 59±11 kg) involving 36 with CCr <50 ml/min (Cockcroft-Gault), 57 took 15 mg o.d. of rivaroxaban and 46 took 10 mg o.d. Median PT, aPTT, and concentration were 12.8s, 31s, and 13.5 ng/ml just before administration, 19.3s, 42s, and 170.5 ng/ml at 4h, and 15.8s, 37s, and 63.0 ng/ml at 9h. Both PT and aPTT were positively correlated with concentration (r=0.88, 0.74, respectively). Age, BW, CCr, and rivaroxaban dosage were not associated with concentrations at 4h and 9h, though they were associated with concentration just before administration. In 10 patients taking crushed tablets, including 5 patients via a nasogastric tube, median PT (15.8s vs. 19.7s, p<0.001), aPTT (33s vs. 42s, p=0.012) and concentration (53 ng/ml vs. 186 ng/ml, p<0.001) at 4h were lower than the remaining 94 patients. Crushed tablets were independently associated with lower anticoagulant intensity. Conclusion: Nearly peak concentration of rivaroxaban was similar or a little lower than that in the ROCKET AF and J-ROCKET AF trials (Kaneko M, et al: Drug Metab Pharmacokinet 2013). Tablet crushing may decrease anticoagulation intensity.

Reversal of rivaroxaban-induced anticoagulation with prothrombin complex concentrate, activated prothrombin complex concentrate and recombinant activated factor VII in vitro

Anticoagulation therapies carry a risk of bleeding; reversal agents may be beneficial in cases of severe bleeding even for anticoagulants with a relatively short half-life, such as the oral factor Xa inhibitor rivaroxaban. We investigated the in vitro reversal effect of prothrombin complex concentrate (PCC; 0.2-1.0U/mL), activated PCC (aPCC; 0.2-1.0U/mL) and recombinant activated factor VII (rFVIIa; 5-50μg/mL) on rivaroxaban-induced (200-1000ng/mL) changes in prothrombin time (PT) and thrombin generation (TG) in plasma, and in thromboelastometry (clotting time [CT]) in whole blood from healthy subjects. All three agents were partially effective in reversing rivaroxaban-induced anticoagulation but showed different profiles. rFVIIa and aPCC were more effective than PCC in reversing prolongations of PT, CT and TG lag time; rFVIIa was more effective than aPCC. However, the reversal effect reached a plateau with a maximal effect of approximately 50%. Inhibition of maximum thrombin concentration was slightly reversed by these agents; aPCC was the most effective. In contrast, inhibition of endogenous thrombin potential (ETP) was strongly reversed by aPCC, with significant increases over baseline at low rivaroxaban concentrations. Compared with aPCC, PCC showed a similar but less effective reversal profile. rFVIIa reversed ETP inhibition by approximately 50%. The extent of reversal by aPCC, PCC and rFVIIa was dependent on the parameter measured in rivaroxaban-anticoagulated plasma or blood. ETP measurements may have predictive power for assessing the reversal potential of PCC or aPCC and may be used to indicate an increased prothrombotic risk.

Performance of coagulation tests in patients on therapeutic doses of rivaroxaban

Knowledge of anticoagulation status during rivaroxaban therapy is desirable in certain clinical situations. It was the study objective to determine coagulation tests most useful for assessing rivaroxaban's anticoagulant effect. Peak and trough blood samples from 29 patients taking rivaroxaban 20 mg daily were collected. Mass spectrometry and various coagulation assays were performed. "On-therapy range" was defined as the rivaroxaban concentrations determined by LC-MS/MS. A "misprediction percentage" was calculated based on how often results of each coagulation assay were in the normal reference range, while the rivaroxaban concentration was in the "on-therapy" range. The on-therapy range was 8.9-660 ng/ml. The misprediction percentages for prothrombin time (PT) and activated partial thromboplastin time (aPTT), using multiple reagents and coagulometers, ranged from 10%-52% and 31%-59%, respectively. PT, aPTT and activated clotting time (ACT) were insensitive to trough rivaroxaban: 59%, 62%, and 80% of samples had a normal result, respectively. Over 95% of PT and ACT values were elevated at peak. Four different rivaroxaban calibrated anti-Xa assays had R² values >0.98, demonstrating strong correlations with rivaroxaban drug levels. In conclusion, PT, aPTT and ACT are often normal in patients on therapeutic doses of rivaroxaban. However, PT and ACT may have clinical utility at higher drug plasma levels. Rivaroxaban calibrated anti-factor Xa assays can accurately identify low and high on-therapy rivaroxaban drug levels and, therefore, have superior utility in all clinical situations where assessment of anticoagulation status may be beneficial.

Rivaroxaban and Hemostasis in Emergency Care

Rivaroxaban is an oral, direct Factor Xa inhibitor, approved for the prevention and treatment of several thromboembolic disorders. Rivaroxaban does not require routine coagulation monitoring and has a short half-life. However, confirmation of rivaroxaban levels may be required in circumstances such as life-threatening bleeding or perioperative management. Here, we explore the management strategies in patients receiving rivaroxaban who have a bleeding emergency or require emergency surgery. Rivaroxaban plasma concentrations can be assessed quantitatively using anti-Factor Xa chromogenic assays, or qualitatively using prothrombin time assays (using rivaroxaban-sensitive reagents). In patients receiving long-term rivaroxaban therapy who require elective surgery, discontinuation of rivaroxaban 20–30 hours beforehand is normally sufficient to minimize bleeding risk. For emergency surgery, we advise against prophylactic use of hemostatic blood products, even with high rivaroxaban concentrations. Temporary rivaroxaban discontinuation is recommended if minor bleeding occurs; for severe bleeding, rivaroxaban withdrawal may be necessary, along with compression or appropriate surgical treatment. Supportive measures such as blood product administration might be beneficial. Life-threatening bleeding demands comprehensive hemostasis management, including potential use of agents such as prothrombin complex concentrate. Patients taking rivaroxaban who require emergency care for bleeding or surgery can be managed using established protocols and individualized assessment.

The Impact Of Dietary Vitamin K On The Pharmacological Activity Of FXa Inhibitor, Rivaroxaban, In Man

Introduction/Background Over several decades, warfarin has been used as the drug of choice for prevention and treatment of thromboembolic disorders. The impact of dietary vitamin K intake on anticoagulation response to warfarin is well established. However, the effect of dietary vitamin K on the activity of the novel oral anticoagulants is less well known. Since the activation of both factor X and thrombin is inextricably linked to the vitamin K cycle in the coagulation cascade, there is the possibility that the pharmacological activity of agents that inhibit these proteins could be influenced by alterations in vitamin K availability. Aim To examine the influence of dietary vitamin K intake on the pharmacological activity of rivaroxaban in man. Methods The pharmacological activity of rivaroxaban was evaluated ex-vivo. Thirty one medically stable elderly inpatients (12 males) with a poor dietary intake in terms of calories and nutrients including vitamin K (12 males) and 28 healthy subjects (7 males) with adequate diets were enrolled into the study. An overnight fasted venous blood sample (20 ml) was taken from each subject. The plasma was stored at -80 degree C for the later measurement of the effects of rivaroxaban on clotting times and clotting factor activity. Each subject completed a validated dietary questionnaire for quantification of vitamin K content of foods eaten in the past week. The plasma samples from each subject were incubated with a range of rivaroxaban concentrations (100 - 500 ng/ml) in order to simulate the therapeutic drug plasma concentrations. Normal prothrombin time (PT), modified prothrombin time (mPT), and vitamin K dependent clotting factors activity were measured according to established in-house methods. Results The mean±SD age of elderly patients and healthy subjects was 87±6 and 36±10 years, respectively. Rivaroxaban produced a greater prolongation of both PT (P=0.01) and m-PT (P=0.02) in the plasma of elderly subjects than the younger healthy subjects over the drug concentration range studied. The mean difference in PT between the two groups ranged from 1.8s to 5.0s at 100 ng/ml and 500 ng/ml plasma rivaroxaban concentrations respectively. The mean difference in m-PT between the two groups ranged from 10.0s to 34.0s at 100 ng/ml and 500 ng/ml plasma rivaroxaban concentrations respectively. There was also a greater inhibition of factor Xa (P=0.005) activity by rivaroxaban in the elderly subjects compared to the healthy subjects, whereas factor IXa (P=0.03) activity was significantly inhibited in the healthy subjects compared to the elderly subjects. However, there were no significant differences between the two groups in factors II and VII activity and fibrinogen concentration. The analysis of dietary questionnaires for vitamin K intake and plasma for fasting vitamin K levels is in progress and the results will be presented. Conclusion We have previously demonstrated in a proof-of-concept study in rats that vitamin K deficiency significantly enhanced the pharmacological activity of the prototype DTI, ximelagatran [1]. The present study findings suggest that the anticoagulation response to rivaroxaban may be influenced by poor dietary vitamin K intake. Assessment of whether dietary vitamin K influences anticoagulation response as well as any influence of age, co morbidities and drug intake in patients taking rivaroxaban is warranted. [1] Kamali F, Wood P, Ward A. Vitamin K deficiency amplifies anticoagulation response to ximelagatran: possible implications for direct thrombin inhibitors and their clinical safety. Annals of Hematology 2009; 88:141-149. Disclosures: No relevant conflicts of interest to declare.

Is Dilute Russell's Viper Venom Time a Useful Assay To Monitor Patients Treated By Rivaroxaban Or Dabigatran Etexilate?

IntroductionThere is still a need for a general test easily implementable and widely available that may be used to screen all patients on DOACs. A recent study has suggested that the dilute Russell Viper Venom Time (DRVV-T) could be used for the monitoring of DOACs but these results have been generated in vitro.The primary objective of this study is to analyse and compare the results obtained with the DRVV Screen and Confirm tests to the plasma drug levels measured by LC-MS/MS. We also aimed at proposing specific cut-off associated with supratherapeutic levels at Ctrough. Finally, a comparison of our results with those obtained with PT for rivaroxaban and aPTT for dabigatran is also provided. MethodsThirty-two rivaroxaban and 31 dabigatran platelet poor plasma samples from real-life patients were included in the study. Dilute Russell's Viper Venom time was measured using STA®-Staclot®DRVV Screen and Confirm reagents. Prothrombin Time and aPTT have been performed with Triniclot PT Excel S® and RecombiPlastin 2G® and with STA®C.K. Prest and SynthasIL®, respectively. The Hemoclot Thrombin Inhibitor® and the Biophen DiXaI® have been performed to estimate plasma concentration of dabigatran and rivaroxaban, respectively. All methodologies were performed on a STA-R Evolution® analyser according with the recommendation of the manufacturer, except for RecombiPlastin 2G® which were performed on an ACL-TOP®. All of these tests have been performed according to the recommendations of the manufacturer.The reference LC-MS/MS measurement of plasma drug concentrations were validated according to FDA Guidelines for Industry for Bioanalytical Method (for rivaroxaban) and to the Validation European Medicines Agency guidelines (for dabigatran). ResultsThe plasma concentrations range from 6 to 426ng/mL for rivaroxaban and from 0 to 386ng/mL for dabigatran as determined by LC-MS/MS.Tables 1 and 2 summarize Spearman correlations and Bland-Atlman analyses for rivaroxaban and dabigatran, respectively. Figures 1 and 2 provide the results of STA®-Staclot®DRVV Screen and Confirm versus LC-MS/MS measurements. Bland-Altman graphs are also provided. DiscussionSTA®-Staclot®DRVV-Screen and Confirm shows a better correlation than PT or aPTT. Bland-Altman analyses reveal an overestimation of approximately 40ng/mL and large 5th-95th limits of agreement with both STA®-Staclot®DRVV-Screen and Confirm.Specific cut-offs associated with supratherapeutic level (>200ng/mL) at Ctrough have been defined. For STA®-Staclot®DRVV-Screen, results below 125 seconds or below a ratio of 3 could exclude plasma concentrations >200ng/mL for rivaroxaban and dabigatran (Figure 1 - A and B). For STA®-Staclot®DRVV-Confirm, cut-offs must be adapted independently (Figure 2 - A and B). Results below 75 seconds or below a ratio of 2, could exclude rivaroxaban plasma concentrations >200ng/mL. For dabigatran, the threshold could be defined at 90 seconds or at a ratio of 2.5. ConclusionThanks to its good correlation with plasma drug level, DRVVT can be more informative than PT and aPTT, to exclude supra-therapeutic level of rivaroxaban and dabigatran at Ctrough. However, due to overestimations in plasma drug level, it cannot be recommended to accurately estimate plasma drug concentrations which require more specific coagulation assays or LC-MS/MS measurements. [Display omitted] [Display omitted] Table 1Reference liquid chromatography coupled to tandem mass spectrometrySpearman Correlation: rs (95%CI)Bland-Altman analyses: mean difference (95% limit of agreement) in ng/mLBiophen Direct Factor Xa Inhibitor® (DiXaI)0.91 (0.81-0.96)-8 (-79 to 63)Calibrated STA®-Staclot® DRVV-Screen0.87 (0.75-0.94)-43 (-164 to 78)Calibrated STA®-Staclot® DRVV-Confirm0.88 (0.76-0.94)-42 (-138 to 54)Triniclot PT Excel S®0.83 (0.67-0.92)-12 (-159 to 134)HemosIL RecombiPlasTin 2G®0.86 (0.72-0.93)-82 (-204 to 41)Table 2Reference liquid chromatography coupled to tandem mass spectrometrySpearman Correlation: rs (95%CI)Bland-Altman analyses: mean difference (95% limit of agreement) in ng/mLHemoclot Thrombin Inhibitor®0.98 (0.96-0.99)-6 (-70 to 58)Calibrated STA®-Staclot® DRVV-Screen0.91 (0.81-0.96)-45 (-156 to 66)Calibrated STA®-Staclot® DRVV-Confirm0.97 (0.93-0.99)-44 (-96 to 7)STA®-C.K.Prest0.84 (0.68-0.92)-23 (-206 to 159)HemosIL SynthasIL®0.89 (0.79-0.95)-28 (-211 to 155) Disclosures:No relevant conflicts of interest to declare.

Novel Point-Of-Use FXa Assay For Monitoring Anticoagulant Therapy By Oral Factor Xa Inhibitors and Heparin

Introduction A highly specific, rapid, robust and sensitive assay for Factor Xa (FXa) activity will greatly improve monitoring and optimization of anticoagulant therapy by direct FXa inhibitors and by low molecular weight (LMW) heparins. We have developed a new point-of-use diagnostic system that will provide results in less than 30 minutes, enabling rapid medical decisions based on quantitation of FXa activity and anticoagulant drugs in patients’ blood. Anticoagulant therapy with unfractionated or LMW heparins needs aggressive monitoring in pregnant women, patients with renal insufficiency, and neonates, in particular in cases of treatment-related side effects, including heparin-induced thrombocytopenia and bleeding tendency (Chest, 2008). New oral anticoagulants rivaroxaban (Xarelto®) and apixaban (Eliquis®) have been effective in reducing risk of stroke and systemic embolism in patients with nonvalvular atrial fibrillation, and in preventing and treating deep vein thrombosis. Both drugs directly inhibit FXa and have significant therapeutic potential as an alternative to warfarin. Unlike warfarin, these drugs do not require constant monitoring and dose adjustment to maintain anticoagulation within the therapeutic interval. However, excessive anticoagulation and consequent hemorrhage risks have been sufficiently common problems to warrant clinical development of specific antidotes for FXa inhibitors. Currently available anti-Xa heparin assays may not be appropriate for rivaroxaban and apixaban measurements. Additionally, recently published data indicate that new oral anticoagulants interfere with the measurement of common test parameters, expanding the need for more specific diagnostic tests (Clinical Chemistry, February 2013). This novel rapid and specific FXa assay will allow monitoring LMW heparins and the new FXa inhibitors, identify heparin-refractory patients as well as those requiring antidotes to oral Factor Xa inhibitors, in addition to directly measuring FXa activity in patients with inherited deficiency of Factor X, congenital antithrombin deficiency, and acquired FXa deficiencies. Method A diagnostic assay to measure FXa activity in human plasma using electrochemiluminescence (ECL) technology was developed at Wellstat Diagnostics, LLC. The assay utilizes a synthetic peptide containing an FXa substrate sequence that is recognized by specific antibodies (labeled with an ECL-active ruthenium chelate) only after specific cleavage by FXa protease. The resulting ECL signal is directly proportional to FXa activity within the physiological range. The assay has been adapted to also provide quantitative measurement of activity of anticoagulant drugs that inhibit FXa. In the modified format the resulting ECL signal is inversely proportional to the concentration of rivaroxaban, apixaban or heparin in plasma. Results The ECL-based assay measures activity of anticoagulant drugs on FXa in human plasma with high intra- and inter-assay reproducibility, accuracy and specificity. Using FX-deficient plasma spiked with Factor X in the range 1-100 ng/mL, we have demonstrated linear increases of ECL signal directly proportional to the concentration of spiked Factor X activated by a specific Russell Viper Venom activator. Serial dilutions of anticoagulant drugs rivaroxaban, apixaban and enoxaparin (LMW heparin) in normal donor plasma showed exponential decreases of ECL signal in the range of concentrations relevant to the therapeutic doses of all three drugs. The dynamic range of the drug detection was 15 - 500 ng/mL for rivaroxaban and apixaban, and 0.2 - 2 IU/mL of enoxaparin. The assay was specific to activity of FXa, while insensitive to variable concentrations of other clotting factors in human plasma, as well as to the presence of other drugs in plasma that don’t directly inhibit FXa. Conclusions The point-of-use FXa assay is shown to be highly specific, robust, rapid and reproducible for measurements of anticoagulant activity of apixaban, rivaroxaban and enoxaparin in human plasma. Use of this point-of-use system for monitoring anticoagulant therapy may be further extended to measure other blood clotting factors (for example, Factor IXa, VIIa, FVIII), for monitoring activity of drugs acting on these factors and for rapid diagnosis of coagulation disorders. Disclosures: No relevant conflicts of interest to declare.

Zymogen-Like FXa Is An Effective Pro-Hemostatic To Reverse The Anticoagulant Effects Of Direct FXa Inhibitors

Oral anticoagulants are the mainstay of treatment for prothrombotic disorders. The emerging oral factor Xa (FXa) inhibitors, which include rivaroxaban and apixaban, have been shown to be highly effective anticoagulants in several clinical scenarios, including venous thromboembolism and non-valvular atrial fibrillation. Compared to warfarin, direct FXa inhibitors have less variable pharmacokinetics, may not require routine monitoring of coagulation parameters, and have comparable to a somewhat lower bleeding risk. Despite these advantages, no approved strategy has been developed to reverse the anticoagulant effects of these drugs in the event of life-threatening bleeding or emergent need for surgery. This represents an urgent unmet clinical need. Our group has recently developed a panel of FXa mutants that are more zymogen-like than wild-type (wt)-FXa. These “zymogen-like” FXa variants have lower activity in in vitro assays compared to wt-FXa due to impaired active site maturation. Furthermore, the variants have longer plasma half-lives (>30 minutes) in vitro compared to wt-FXa (1-2 minutes) due to diminished reactivity with antithrombin III (ATIII) and tissue factor pathway inhibitor (TFPI). Remarkably however, binding to FVa rescues the activity of these zymogen-like FXa variants and as a result they are highly effective procoagulants in vivo in the setting of hemophilia (Nat. Biotech; 2011, 29:1028-33). We hypothesized that these variants could also be effective procoagulants to overcome the effects of direct FXa inhibitors. Furthermore, since direct FXa inhibitors bind the FXa active site, we expect them to compete with ATIII and TFPI for FXa binding and prolong their half-lives. We tested both of these hypotheses in in vitro coagulation studies and in vivo hemostasis models. Rivaroxaban dose-dependently inhibited thrombin generation in thrombin generation assays (TGA) when added to normal human plasma. Specifically, 500 nM rivaroxaban, the expected therapeutic steady-state plasma concentration, decreased peak thrombin generation to ∼10% of normal, and addition of 3 nM of the FXa zymogen-like variant FXaI16L restored peak thrombin generation to 105% of normal. Higher concentrations of rivaroxaban (2.5 µM) completely abrogated thrombin generation in this assay, but 10 nM FXaI16L restored thrombin generation to 72% of normal under these conditions. We compared these data to results obtained with other proposed reversal strategies. Gla-domainless, catalytically inactive FXa (GD-FXaS195A), which has been shown to reverse the effects of rivaroxaban by scavenging the inhibitor, restored thrombin generation in the presence of 500 nM rivaroxaban, but required high concentrations (1 µM; >300-fold greater than FXaI16L) to be effective. In addition, activated prothrombin complex concentrates (FEIBA), which have been shown to have some ex vivo efficacy, were ineffective under our assay conditions. In tail-clip hemostasis studies in mice, rivaroxaban dose-dependently increased blood loss, with 50 mg/kg rivaroxaban resulting in 217% of normal blood loss. Addition of FXaI16L (200 mg/kg) reduced rivaroxaban-induced blood loss to 141% of normal. To examine the effect of rivaroxaban on the half-life of FXa, we pre-incubated FXaI16L or wt-FXa with or without rivaroxaban in normal human plasma and then performed TGA experiments after various incubation times. When wt-FXa or FXaI16L were pre-incubated in plasma in the absence of rivaroxaban, their half-lives were 4.6 minutes and 1.37 hours, respectively. Remarkably, when wt-FXa or FXaI16L were incubated in plasma in the presence of 500 nM rivaroxaban, their respective half-lives were prolonged to 9.4 hours (123-fold increase) and 18.1 hours (13.2-fold increase). These results suggest that a zymogen-like FXa variant, FXaI16L, can reverse the effects of rivaroxaban in vitro and in vivo. Furthermore, FXaI16L is a bypassing agent that only requires catalytic amounts of protein, in contrast to scavengers or “true” antidotes like GD-FXaS195A that require stoichiometric concentrations. This indicates that much lower quantities of FXaI16L may be effective in vivo. We also showed that rivaroxaban dramatically prolongs the half-life of FXa in plasma, possibly by competing with ATIII and TFPI for FXa binding. This work provides a starting point for the development of a long half-life reversal strategy for the emerging FXa inhibitors. Disclosures:Patel-Hett:Pfizer: Employment. Jasuja:Pfizer: Employment. Fruebis:Pfizer: Employment. Pittman:Pfizer: Employment. Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding; Alnylam: Consultancy.

Gastrointestinale Blutungen beim kardiologischen Patienten

Oral anticoagulation and antiplatelet therapy are risk factors for gastrointestinal (GI) bleeding. GI bleeding-especially lower GI bleeding-seems to be associated with a poorer outcome. With the introduction of dabigatrane and rivaroxaban, difficulties in the management of bleeding complications arose. Thus, the goal of the authors was to establish a standard operating procedure (SOP) for the treatment of severe GI bleeding associated with rivaroxaban, dabigatrane, and antiplatelet therapy. Bleeding complications during phenprocoumon treatment should be treated with prothrombin complex concentrates and vitamin K1. Dabigatrane elimination is highly dependent to the renal function. The measurement of drug concentrations of dabigatrane and rivaroxaban is useful to indicate an increased risk of bleeding complications. Severe bleeding associated with dabigatrane or rivaroxaban therapy should trigger prothrombin complex therapy, whereby in cases with severe bleeding associated with antiplatelet therapy platelet transfusion should be initiated. Low-dose aspirin should be continued after 24 h.

Rivaroxaban demonstrates in vitro anticoagulant effects in canine plasma

Rivaroxaban is an oral direct factor X inhibitor used in human thrombotic disorders and its oral administration makes it an attractive potential anticoagulant for dogs. The objective of this study was to evaluate the in vitro anticoagulant effect of rivaroxaban on canine pooled platelet-poor plasma (PPP). Pooled PPP was collected from 20 healthy adult Beagle dogs. Aliquots of pooled citrated PPP were treated in vitro with DMSO solutions of rivaroxaban (98% purity) to obtain 19 final concentrations ranging from 0 to 1000mg/L of drug. Samples were immediately submitted for the following coagulation assays: prothrombin time (PT), activated partial thromboplastin time (aPTT), tissue factor-induced thrombin generation and anti-factor Xa activity. Concentration–effect data were analyzed with various nonlinear regression models for stimulatory or inhibitory effects.Rivaroxaban caused a concentration-dependent prolongation of all coagulation parameters. Rivaroxaban concentration for 50% baseline inhibition of the propagation phase of thrombin (rate index) was 0.024mg/L, and for 50% baseline inhibition of the optical density in the anti-factor Xa activity assay was 0.053mg/L. At these concentrations, PT and aPTT remained within the reference range. Two-fold prolongation from baseline of PT and aPTT was achieved with higher concentrations, i.e. 1.24 and 1.69mg/L, respectively. Thrombin generation was completely suppressed by concentrations ⩾0.8mg/L. In conclusion, rivaroxaban showed an in vitro concentration-dependent anticoagulant effect on canine plasma. Thrombin generation and anti-factor Xa activity were more sensitive and accurate than PT and aPTT in detecting the anticoagulant effect of rivaroxaban.

Measurement and Reversal of Prophylactic and Therapeutic Peak Levels of Rivaroxaban

Rivaroxaban (Xarelto, Bayer HealthCare, Leverkusen, Germany) is a new oral anticoagulant drug. Anticoagulants may cause bleeding, thereby requiring reliable monitoring and efficient therapy. We investigated thromboelastometry versus routine coagulation tests to measure prophylactic and therapeutic concentrations of rivaroxaban and their reversal with prothrombin complex concentrate (PCC) and activated recombinant factor VII (rFVIIa) in vitro. Rivaroxaban was solubilized, and PCC and rFVIIa were added in 2 concentrations to the rivaroxaban-spiked blood samples, and thromboelastometry and measurements were performed. Rivaroxaban increased tissue factor-activated clotting time (CT(ExTEM)) dose dependently. Activated partial prothrombin time (aPTT), prothrombin time ratio (PTR), and prothrombin time (PT) were changed significantly in both concentrations. Reversal with PCC in both dosages caused no significant change in the measured parameters. For prophylactic rivaroxaban dosage, rFVIIa changed the PT significantly but not CT(ExTEM), aPTT, and PTR. For therapeutic rivaroxaban dosage, the CT(ExTEM) was significantly reduced. The other parameters remained unaffected. Thromboelastometry can detect rivaroxaban effects. In vitro rFVIIa seems highly effective for reversal in contrast to PCC.

Case Scenario: Management of Trauma-induced Coagulopathy in a Severe Blunt Trauma Patient

Case Scenario: Management of Trauma-induced Coagulopathy in a Severe Blunt Trauma Patient

Laboratory tests during direct oral anticoagulant treatment? No

The anticoagulant effect of Dabigatran etexilate, Rivaroxaban, and Apixaban is dose-predictable, steady, and little influenced by diet and drugs [1]. Thus, similar to lowmolecular weight heparin, laboratory tests have been unnecessary to evaluate such direct oral anticoagulant drugs (DOACs) in trials on thromboprophylaxis (Table 1). Nor such testing was needed in patients with atrial fibrillation (AF) receiving Dabigatran etexilate (RE-LY trial), Rivaroxaban (ROCKET AF trial) or Apixaban (ARISTOTLE trial). In the AF setting, vis-a-vis an at least as effective prevention of stroke and systemic embolism, the risk of intracranial hemorrhage was lower with DOACs than with INR-adjusted warfarin. Patients at high risk of bleeding are little represented in studies with DOACs [2, 3]. However, from October 2010 to December 2011, vis-avis 3.5 gastrointestinal bleedings and 2.4 intracranial hemorrhages/100,000 days at risk in new users of warfarin, 1.6 gastrointestinal bleedings and 0.8 intracranial hemorrhages/100,000 days at risk occurred among new users of Dabigatran in the every day practice in the USA [4]. At variance with the large majority of cases, there are special conditions and clinical settings (Table 2) in which the anticoagulant effect of DOACs should be measured [5]. However, peak concentrations of Rivaroxaban (&200 ng mL) are determined by HPLC methods that are not suitable for routine practice [6]. On the other hand, in healthy volunteers, a 20 % prolongation of the aPTT has been observed for single Apixaban doses ranging from 25 to 50 mg [7]. Moreover, depending on the reagent employed, the concentrations of Rivaroxaban that double the aPTT range from 389 to 617 ng mL [8]. Thus, the aPTT does not accurately assess the anticoagulant effect of Rivaroxaban or of Apixaban. A concentration-dependent prolongation of the aPTT occurs by spiking Dabigatran in human plasma [9]. However, such prolongation is linear only at Dabigatran concentrations [200 ng mL (expected drug exposure being 50–300 ng mL), and the effects on the aPTT vary up to 26 % between the most and least sensitive reagent [10]. Thus, the aPTT is not suitable for an accurate measurement of the anticoagulant effect of (high) Dabigatran concentrations. At variance with Apixaban, Rivaroxaban prolongs the PT in a dose-dependent manner. However, at approved dose regimens (10–20 mg), the changes observed are small, and the results depend on the reagents employed. Accordingly, rather than quantitative, this accessible clotting method provides qualitative estimates of the anticoagulant effect of Rivaroxaban [11]. Likewise, significant effects in the PT-INR (i.e., INR [1.2) are only found in response to concentrations of Dabigatran [200 ng mL [9]. Moreover, depending on the thromboplastin reagent used, major differences are observed in the results of the assays [10]. Thus, in its current form, the prothrombin time (PT), standardized as the International Normalized Ratio (INR), is not suitable for assessing the anticoagulant effect of Dabigatran. Nor is standardization between reagents and laboratories available for the very sensitive thrombin time (TT). [12] As a whole, while the PT and the aPTT provide little help distinguishing between treatment failure and non-adherence of patients, these routinely available clotting methods may be useful to establish whether the anticoagulant effect of a DOAC is higher than expected [an aPTT value twice the highest normal limit (i.e., [80 s) 12 h after the last administration of Dabigatran argues for G. Di Minno (&) E. Ricciardi A. Scalera Dipartimento di Medicina Clinica e Chirurgia, Clinica Medica, Universita degli Studi di Napoli ‘‘Federico II’’ Napoli, Via S. Pansini 5, 80131 Naples, Italy e-mail: diminno@unina.it

Measurement of dabigatran and rivaroxaban in primary prevention of venous thromboembolism in 106 patients, who have undergone major orthopedic surgery: an observational study

No routine coagulation laboratory test is recommended during rivaroxaban or dabigatran treatment. However measuring drug concentration and/or anticoagulant activity can be desirable in some special clinical settings, such as bleeding, thrombosis recurrence or emergency surgery. The effects of dabigatran etexilate and rivaroxaban on various coagulation assays have been previously studied in normal plasma spiked with increasing concentrations of the drug. In contrast, few data are available in routinely treated patients. In order to perform and to interpret the results of these tests, it is necessary to determine the usual responses of patient's plasma. We have used several coagulation tests in a prospective study including 106 patients receiving thromboprophylactic treatment with dabigatran 150 or 220 mg od and rivaroxaban 10 mg od for major orthopaedic surgery. The most common tests--prothrombin time (PT) and activated partial thromboplastin time (aPTT)--give results, which vary according to the reagent used. To overcome this limitation, we advocate the use of plasma calibrators, which decreases the inter-laboratory heterogeneity of results. Anti-Xa measurement and Hemoclot, a thrombin diluted clotting assay, are specific assays which have been proposed for rivaroxaban and dabigatran respectively. These tests, conventional PT, aPTT and thrombin generation (TG) have been performed. We demonstrated that measurements of both drugs can determine reliably the drug concentration in patients' plasmas. PT is more prolonged with rivaroxaban than with dabigatran. Interestingly, the pattern of TG was clearly different in relation to the difference in the mechanism of action of the two new anticoagulants. A significant inter-individual variability of response is detected. Rivaroxaban--mean Cmax 140 ng/mL (extremes 0-412) induces a greater increase of PT than dabigatran. aPTT is sensitive to dabigatran. Rivaroxaban concentrations were in good agreement with two other studies while unexplained lower than expected concentrations were found in dabigatran patients receiving 220 mg once a day [mean Cmax 60 ng/mL (extremes 0-320)]. An interference by pantoprazole, a drug which reduces dabigatran absorption, could explain the observed lower than expected results.

Ex vivo effects of low-dose rivaroxaban on specific coagulation assays and coagulation factor activities in patients under real life conditions

Global coagulation assays display variable effects at different concentrations of rivaroxaban. The aim of this study is to quantify the ex vivo effects of low-dose rivaroxaban on thrombophilia screening assays and coagulation factor activities based on the administration time, and to show how to mask possible interferences. Plasma samples from 40 patients receiving rivaroxaban 10 mg daily were investigated to measure activities of clotting factor II, V, VII, VIII, IX, XI, XII and XIII; protein C- and protein S-levels; lupus anticoagulants; anticardiolipin IgG and IgM; D-dimer, heparin-platelet factor 4 (HPF4) antibodies and screening tests for von Willebrand disease (VWD). Two hours after rivaroxaban administration, the activities of clotting factors were significantly decreased to different extents, except for factor XIII. Dilution of plasma samples resulted in neutralisation of these interferences. The chromogenic protein C activity assay was not affected by rivaroxaban. Depending on the timing of tablet intake in relation to blood sampling protein S activity was measured falsely high when a clotting assay was used. False-positive results for lupus anticoagulants were observed depending on the assay system used and the administration time of rivaroxaban. ELISA-based assays such as anticardiolipin IgG and IgM, D-dimer, HPF4-antibodies and the turbidimetric assays for VWD were not affected by rivaroxaban. Specific haemostasis clotting tests should be performed directly prior to rivaroxaban intake. Assay optimisation in the presence of rivaroxaban can be achieved by plasma dilution. Immunologic assays are not influenced by rivaroxaban, while chromogenic assays can be used, when they do not depend on factor Xa.

Comparison of calibrated chromogenic anti-Xa assay and PT tests with LC-MS/MS for the therapeutic monitoring of patients treated with rivaroxaban

Possibilities to monitor rivaroxaban therapy could be useful in certain circumstances. Prothrombin time (PT) or chromogenic anti-Xa assays such as the Biophen Direct Factor Xa Inhibitor® (DiXaI) have been proposed to estimate rivaroxaban concentrations but are mainly based on in vitro studies. The study aim was to compare PT and Biophen DiXaI® measurements with liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements in plasma samples from patients treated with Xarelto®. Fifty-two plasma samples were included. PT was performed using Innovin® and Triniclot PT Excel S®. Biophen DiXaI® was performed according to instructions from the manufacturer. The rivaroxaban plasma concentration ranged between 0 and 485 ng/ml as measured by LC-MS/MS. The limits of quantification were 30 ng/ml and 5 ng/ml for Biophen DiXaI® and LC-MS/MS, respectively. The linear correlation between Biophen DiXaI® and LC-MS/MS analyses was high for all rivaroxaban concentrations (r² = 0.95). For concentrations ≤100 ng/ml, r²-value was 0.83. The Bland-Altman analysis showed a mean difference of -16 ng/ml (SD: 25 ng/ml). The PT methods did not correlate well with plasma concentrations measured by LC-MS/MS (r² ≈ 0.60). In conclusion, the important inter-individual variability and the poor correlation with LC-MS/MS preclude the use of PT to estimate rivaroxaban concentrations. Thanks to its small inter-individual variability and good agreement with LC-MS/MS measurements, we recommend the use of Biophen DiXaI® assays to estimate concentrations of rivaroxaban >30 ng/ml. Quantification of low rivaroxaban levels (<30 ng/ml) requires the LC-MS/MS method.

The discovery of rivaroxaban: translating preclinical assessments into clinical practice.

Direct oral anticoagulants that target a single coagulation factor (such as factor Xa or thrombin) have been developed in recent years in an attempt to address some of the limitations of traditional anticoagulants. Rivaroxaban is an oral, direct factor Xa inhibitor that inhibits free and clot-bound factor Xa and factor Xa in the prothrombinase complex. Preclinical studies demonstrated a potent anticoagulant effect of rivaroxaban in plasma as well as the ability of this agent to prevent and treat venous and arterial thrombosis in animal models. These studies led to an extensive phase I clinical development program that investigated the pharmacological properties of rivaroxaban in humans. In these studies, rivaroxaban was shown to exhibit predictable pharmacokinetics and pharmacodynamics and to have no clinically relevant interactions with many commonly prescribed co-medications. The pharmacodynamic effects of rivaroxaban (for example, inhibition of factor Xa and prolongation of prothrombin time) were closely correlated with rivaroxaban concentrations in plasma. The encouraging findings from preclinical and early clinical studies were expanded upon in large, randomized phase III studies, which demonstrated the clinical efficacy and safety of rivaroxaban in a broad spectrum of patients. This article provides an overview of the discovery and development of rivaroxaban, describing the pharmacodynamic profile established in preclinical studies and the optimal translation to clinical studies in healthy subjects and patient populations.

Rivaroxaban and other novel oral anticoagulants: pharmacokinetics in healthy subjects, specific patient populations and relevance of coagulation monitoring

Unlike traditional anticoagulants, the more recently developed agents rivaroxaban, dabigatran and apixaban target specific factors in the coagulation cascade to attenuate thrombosis. Rivaroxaban and apixaban directly inhibit Factor Xa, whereas dabigatran directly inhibits thrombin. All three drugs exhibit predictable pharmacokinetic and pharmacodynamic characteristics that allow for fixed oral doses in a variety of settings. The population pharmacokinetics of rivaroxaban, and also dabigatran, have been evaluated in a series of models using patient data from phase II and III clinical studies. These models point towards a consistent pharmacokinetic and pharmacodynamic profile, even when extreme demographic factors are taken into account, meaning that doses rarely need to be adjusted. The exception is in certain patients with renal impairment, for whom pharmacokinetic modelling provided the rationale for reduced doses as part of some regimens. Although not routinely required, the ability to measure plasma concentrations of these agents could be advantageous in emergency situations, such as overdose. Specific pharmacokinetic and pharmacodynamic characteristics must be taken into account when selecting an appropriate assay for monitoring. The anti-Factor Xa chromogenic assays now available are likely to provide the most appropriate means of determining plasma concentrations of rivaroxaban and apixaban, and specific assays for dabigatran are in development.