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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.

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.

Rivaroxaban is a direct factor Xa inhibitor, which can be monitored by anti-factor Xa chromogenic assays. This ex vivo study evaluated different assays for accurate determination of rivaroxaban levels. Eighty plasma samples from patients receiving rivaroxaban (Xarelto) 10 mg once daily and 20 plasma samples from healthy volunteers were investigated using one anti-factor Xa assay with the addition of exogenous antithrombin and two assays without the addition of antithrombin. Two different lyophilised rivaroxaban calibration sets were used for each assay (low concentration set: 0, 14.5, 59.6 and 97.1 ng/ml; high concentration set: 0, 48.3, 101.3, 194.2 and 433.3 ng/ml). Using a blinded study design, the rivaroxaban concentrations determined by the assays were compared with concentrations measured by HPLC-MS/MS. All assays showed a linear relationship between the rivaroxaban concentrations measured by HPLC-MS/MS and the optical density of the anti-FXa assays. However, the assay with the addition of exogenous antithrombin detected falsely high concentrations of rivaroxaban even in plasma samples from controls who had not taken rivaroxaban (intercept values using the high calibrator set and the low calibrator set: +26.49 ng/ml and +13.71 ng/ml, respectively). Plasma samples, initially determined by the high calibrator setting and containing rivaroxaban concentrations <25 ng/ml, had to be re-run using the low calibrator setting for precise measurement. In conclusion, anti-factor Xa chromogenic assays that use rivaroxaban calibrators at different concentration levels can be used to measure accurately a wide range of rivaroxaban concentrations ex vivo. Assays including exogenous antithrombin are unsuitable for measurement of rivaroxaban.

Rivaroxaban is one of the novel oral direct factor Xa inhibitors, which is effective in preventing thromboembolic complications at fixed doses (i.e., once daily), without the need for dose adjustment according to laboratory monitoring. Nearly 60% of rivaroxaban is cleared from circulation by glomerular filtration, 30% of which is excreted as active drug. Therefore, as renal elimination plays a pivotal role in the metabolism of this drug, impairment of renal function may be important during anticoagulation with rivaroxaban over long periods of time. The assessment of the anticoagulant effect/concentration of rivaroxaban may thus be useful in special patient populations such as in the elderly and eldest, during acute diseases with concurrent dehydration, before surgery, during bleeding or thrombotic episodes, or to verify adherence to therapy. Rivaroxaban prolongs prothrombin time in a dose-dependent, linear fashion. Activated partial thromboplastin time (APTT) is also prolonged, but in an exponential manner. Substantial differences in test results might be generated by different thromboplastin and APTT reagents. One-step prothrombin-induced clotting time assay is sensitive to low concentrations of rivaroxaban. Chromogenic substrate assays specific for factor Xa are also sensitive to rivaroxaban. Several initiatives are currently ongoing to standardize the various methods to determine rivaroxaban in human plasma samples, some of which will be summarized in this article along with the dose-dependent effects of rivaroxaban on relevant coagulation parameters. Therefore, although rivaroxaban prolongs all coagulation assays used to assess the anticoagulant effects of most anticoagulants, the most specific assay cannot be identified at present. Moreover, clinical trials are needed to determine the relationship of assay results with bleeding or thrombotic complications., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Rivaroxaban is an oral, direct factor Xa inhibitor. Routine coagulation monitoring is not required, but a quantitative determination of rivaroxaban concentrations might be useful in some clinical circumstances. This multicentre study assessed the suitability of the anti-factor Xa chromogenic assay for the measurement of rivaroxaban plasma concentrations (ng/ml) using rivaroxaban calibrators and controls, and the inter-laboratory precision of the measurement. Twenty-four centres in Europe and North America were provided with sets of rivaroxaban calibrators (0, 41, 209 and 422 ng/ml) and a set of rivaroxaban pooled human plasma controls (20, 199 and 662 ng/ml; the concentrations were unknown to the participating laboratories). The evaluation was carried out over 10 days by each laboratory using local anti-factor Xa reagents as well as the centrally provided reagent, a modified STA® Rotachrom® assay. A calibration curve was produced each day, and the day-to-day precision was evaluated by testing three human plasma controls. When using the local anti-factor Xa reagents, the mean rivaroxaban concentrations (measured/actual values) were: 17/20, 205/199 and 668/662 ng/ml, and the coefficient of variance (CV) was 37.0%, 13.7% and 14.1%, respectively. When the modified STA Rotachrom method was used, the measured/actual values were: 18/20, 199/199 and 656/662 ng/ml, and the CV was 19.1%, 10.9% and 10.0%, respectively. The results suggest that, by using rivaroxaban calibrators and controls, the anti-factor Xa chromogenic method is suitable for measuring a wide range of rivaroxaban plasma concentrations (20-660 ng/ml), which covers the expected rivaroxaban plasma levels after therapeutic doses.

Apixaban and other factor Xa (FXa) inhibitors are in late-stage clinical development for prevention and treatment of thromboembolic diseases. Although routine monitoring will not be required, in certain situations assessment of drug level may be helpful. This study evaluated the suitability of commercially available prothrombin time/international normalised ratio (PT/INR) and anti-FXa activity assays to measure FXa inhibitors in plasma. Twelve PT (ISI 0.89-1.88) and three anti-Xa assays were evaluated in vitro using human plasma spiked with four FXa inhibitors (0-2,000 ng/ml). Assay variability and correlation with drug plasma exposure were evaluated in patients with venous thromboembolism (VTE) treated with apixaban. All FXa inhibitors prolonged PT; however, assay sensitivity was dependent on thromboplastin reagents used and FXa inhibitors tested. To achieve a doubling of PT, the concentration of each FXa inhibitor varied 2.6- to 8-fold between thromboplastin reagents. The rank order of a FXa inhibitor's effect on PT ratio varied across thromboplastin reagents. Conversion to INR increased variability. Different anti-Xa assays showed different dynamic ranges for each FXa inhibitor; however, their rank order was consistent. For apixaban, the dynamic range of <7.8-240 ng/ml, and inter- and intra-assay precision of <6% coefficient of variation by Rotachrom assay appeared suitable for the anticipated apixaban plasma concentrations with 2.5 and 5 mg bid clinical doses. The stronger correlation between apixaban plasma concentration and anti-Xa activity (r2 = 0.88-0.89) compared with PT/INR (r2 = 0.36) in patients undergoing VTE treatment suggested that anti-Xa activity was the better indicator of apixaban plasma concentrations.

Although there is no need for routine coagulation monitoring with rivaroxaban--an oral, direct factor Xa inhibitor--a haemostasis assay might be valuable to measure its pharmacodynamic effects. This study aimed to find assays, among those commercially available, to measure rivaroxaban pharmacodynamics. Several global conventional clotting tests, as well as clotting or chromogenic assays to measure anti-factor Xa activity, were studied. A thrombin generation test using calibrated automated thrombogram was also done. Tests were performed with the indirect factor Xa inhibitor fondaparinux for comparison. A concentration-dependent prolongation of prothrombin time (PT), dilute PT, and activated partial thromboplastin time was observed with rivaroxaban. The results varied depending on the reagents. This variability cannot be standardised with the international normalised ratio system commonly used for vitamin K antagonists. Using a standard calibration curve, PT test results can be expressed in plasma concentrations of rivaroxaban rather than PT seconds or ratio. Standard methods for HepTest and two-step prothrombinase-induced clotting time (PiCT) resulted in a paradoxical response, with low concentrations of rivaroxaban reducing clotting times. This was not observed with shorter incubation times, or when antithrombin-deficient (immunodepleted) plasma was used. The chromogenic tests found a dose-dependent relationship between anti-factor Xa activity and rivaroxaban concentration. Modified specific factor Xa chromogenic assays are being further investigated. One-step PiCT and HepTest with shortened incubation times, as well as the widely available PT assay (using a rivaroxaban calibrator) could be useful to monitor the pharmacodynamic effects of rivaroxaban accurately. Finally, all clotting and chromogenic assays showed a concentration-dependent effect induced by rivaroxaban.

Current anticoagulant therapies for the prevention and treatment of thromboembolic disorders have many drawbacks: vitamin K antagonists interact with food and drugs and require frequent laboratory monitoring, and heparins require parenteral administration. Oral rivaroxaban (BAY 597939) is a new, highly selective and potent direct factor-Xa (FXa) inhibitor with a predictable pharmacodynamic and pharmacokinetic profile and could therefore be an attractive antithrombotic drug. It was the objective of this study to investigate the antithrombotic efficacy of oral rivaroxaban in two rabbit models of experimental venous thrombosis. In the venous stasis (prevention) model, animals were randomized to receive oral rivaroxaban 0.3, 1.0, 3.0 or 10.0 mg/kg or vehicle control. Thrombosis was induced by jugular vein stasis and injection of thromboplastin into the ear vein. In the venous thrombosis (treatment) model, intravenous (1.0 and 3.0 mg/kg) and oral (3.0 mg/kg) rivaroxaban was compared with intravenous nadroparin (40 U bolus and 20 U/h), fondaparinux (42 microg/kg) and vehicle control. Thrombus growth was assessed by measuring the accretion of radiolabelled fibrinogen into preformed clots in the jugular veins. Bleeding was assessed using an ear bleeding model. In the prevention model, rivaroxaban reduced thrombus formation dose-dependently (calculated ED(50) 1.3 mg/kg). In the treatment model, oral rivaroxaban (3.0 mg/kg) reduced thrombus growth to a similar extent to intravenous rivaroxaban (1.0 mg/kg), nadroparin and fondaparinux. Oral rivaroxaban did not prolong bleeding time. In conclusion, the orally available selective, direct FXa inhibitor rivaroxaban is effective in the prevention and treatment of venous thrombosis in two well-established models of experimental thrombosis.

Plasma concentrations of viloxazine were determined in twenty depressed inpatients during 4 weeks of treatment with progressively increasing dosage. Viloxazine plasma levels varied markedly during the day, due to the short half-life of the drug. Plasma levels rose to peak values after 7-10 days of treatment and then decreased, perhaps due to enzymatic induction by viloxazine. A negative linear correlation was found between the plasma concentration of viloxazine and its clinical effect, with the best clinical improvement in patients whose plasma concentration was 20-500 ng/ml at 7 a.m. Performance in psychomotor tests (visual reaction time and ring of Pierron was improved in many patients after treatment and was correlated with the plasma viloxazine level and the Hamilton Rating score. Assessment of viloxazine effects of electroencephalogram showed a decrease in EEG amplitude in the eight clinically improved patients.