Analytical Specifications RIVAROXABAN

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1 Page 1 of 9 ANALYTE NAME AND STRUCTURE - RIVAROXABAN SYNONYMS Xarelto CATEGORY Anticoagulant TEST CODE PURPOSE Therapeutic Drug Monitoring GENERAL RELEVANCY BACKGROUND Xarelto (rivaroxaban) is an orally administered anticoagulant approved for a number of uses: (1) to reduce the risk of stroke and systemic embolism in patients with nonvalvular atrial fibrillation, (2) for the treatment/reduction of risk for deep vein thrombosis and pulmonary embolism, and (3) for the prophylaxis of thrombotic events for patients undergoing knee or hip replacement surgery [1]. Rivoraxaban acts as a Factor Xa inhibitor. The only orally administered anticoagulants previously available were vitamin K antagonists (VKAs), such as warfarin, which require frequent monitoring, have a narrow therapeutic index, and display pharmacogenetic variation. Xarelto offers an improved efficacy and safety profile. The prescribed dosage for Xarelto is 15 mg or 20 mg tablets with food or 10 mg tablets without food [1]. MECHANISM OF ACTION

2 Page 2 of 9 Rivoraxaban directly binds to the active site of Factor Xa, inhibiting its action and then decreasing thrombin generation and thrombus development [1]. Factor Xa is a key component of the prothrombinase complex that catalyzes the conversion of prothrombin to thrombin in the coagulation cascade that is responsible for fibrin clot formation. Rivoraxaban also indirectly inhibits platelet aggregation induced by thrombin. This process is reversible and highly selective. *AT stands for antithrombin, a circulating plasma protein that inhibits serum protease clotting enzymes. Escobar CE, et al., Introduction to hemostasis. In: Harmening DM, ed. Clinical Hematology and Fundamentals of Hemostasis. 4th ed. Philadelphia, PA: FA Davis Company; 2002: ADVERSE EFFECTS Rivoraxaban increases the risk of bleeding and can cause serious bleeding events [1]. Other adverse reactions include anemia, nausea, pain, and dyspepsia. Rivoraxaban is not recommended for patients with severely impaired renal function or undergoing dialysis due to increased risk for bleeding [1]. Significant drug interactions

3 Page 3 of 9 can occur when used in combination with rifampin, ketoconazole, and other compounds that inhibit para-glycoprotein (P-gp) and cytochrome P450 3A4 [1]. Rivaroxaban should also not be administered with other anticoagulants. PHARMACOKINETICS t 1/2 : ~ h [2] The bioavailability of rivaroxaban is dose and food dependent. At a 10 mg dose, the bioavailability is % and is not affected by food. At 20 mg, the bioavailability of rivaroxaban is 66% in the fasted state; however, food increases the bioavailability and peak plasma concentration [1]. Absorption of rivaroxaban is rapid after oral administration, with the maximum concentration of the drug being reached within 2-4 hours [2]. Plasma protein binding is approximately 92-95% and the mean volume of distribution of the drug at steady state is 50 L [1]. Approximately 51% of an orally administered dose is recovered in urine and feces as metabolites [1]. METABOLISM Unchanged rivaroxaban was identified as the main compound in human plasma after oral administration of the drug and all metabolites were found to be inactive [3]. Metabolism of rivaroxaban occurs primarily via the cytochrome P450 (CYP) enzymes CYP3A4/3A5 and CYP2J2 [1]. Metabolism of rivaroxaban occurs via two major pathways: oxidative degradation of the 6-membered heterocyclic ring and hydrolysis of the different amide bonds [3]. A few minor metabolites were identified; shown as M1, M4, M5, M7, and M8/M9 in the figure below which is a depiction of the predicted metabolism of rivaroxaban.

4 Page 4 of 9 [3] THERAPEUTIC AND TOXIC CONCENTRATIONS Mean peak plasma concentrations of rivaroxaban following a single oral administration of 5, 10, or 20 mg oral doses are as follows [2]: 5 mg (n=8): 67.2 ng/ml [range, 53.2 to 81.2] 10 mg (n=8): 143 ng/ml [range, 110 to 175] 20 mg (n=8): 204 ng/ml [range, 179 to 229] Mean peak plasma concentrations of rivaroxaban at steady state following a multi-dose administration of 5, 10, or 20 mg oral doses every 12 hours are as follows [2]: 5 mg (n=8): 115 ng/ml [range, 91.1 to 139] 10 mg (n=8): 215 ng/ml [range, 178 to 252] 20 mg (n=8): 415 ng/ml [range, 355 to 474] TECHNICAL CONSIDERATIONS

5 Page 5 of 9 ANALYTES TO BE DETERMINED Rivoraxaban PROPER SPECIMEN TYPES Serum and/or plasma RECOMMENDED COLLECTION TUBES Serum/Plasma Plastic container (preservative-free) SP Special Handling - Serum: Collect sample in Red top tube Plasma: Collect sample in Lavender top tube (EDTA) or Pink top tube. Promptly centrifuge and separate Serum or Plasma into a plastic screw capped vial using approved guidelines. CALIBRATION RANGE For the purposes of therapeutic monitoring, a suggested calibration range is 0.5 to 500 ng/ml. MAXIMUM ACCEPTABLE ERROR The interpretation of concentrations in biological specimens is facilitated by analytical methods with <20% total error. INTERFERING SUBSTANCES Clopidogrel, ketoconazole, erythromycin, warfarin, ranitidine, midazolam, rifampicin, atorvastatin, acetylsalicylic acid, and naproxen may be co-administered with rivoraxaban. Dabigatran and apixaban may be considered to have similar structures to rivoraxaban. MISCELLANEOUS

6 Page 6 of 9 Rivaroxaban was stable in all different matrices and under different conditions [4]. However, stability should be evaluated through normal NMS Labs validation procedures. REFERENCE COMMENTS CLINICAL Mean peak plasma concentrations of rivaroxaban following a single oral administration of 5, 10, or 20 mg oral doses are as follows: 5 mg: 67.2 ng/ml [range, 53.2 to 81.2] 10 mg: 143 ng/ml [range, 110 to 175] 20 mg: 204 ng/ml [range, 179 to 229] Mean peak plasma concentrations of rivaroxaban at steady state following a multi-dose administration of 5, 10, or 20 mg oral doses every 12 hours are as follows: 5 mg: 115 ng/ml [range, 91.1 to 139] 10 mg: 215 ng/ml [range, 178 to 252] 20 mg: 415 ng/ml [range, 355 to 474] Elevated Flag: over 500 ng/ml Source: Zhao X et al. Safety, pharmacokinetics and pharmacodynamics of single/multiple doses of the oral, direct Factor Xa inhibitor rivaroxaban in healthy Chinese subjects. British Journal of Clinical Pharmacology. 2009;68(1): FORENSIC Rivaroxaban is an orally administered anticoagulant approved to reduce the risk of thrombotic events. The prescribed dosage for Xarelto is 15 mg or 20 mg tablets with food or 10 mg tablets without food. Mean peak plasma concentrations of rivaroxaban following a single oral administration of 5, 10, or 20 mg oral doses are as follows: 5 mg: 67.2 ng/ml [range, 53.2 to 81.2] 10 mg: 143 ng/ml [range, 110 to 175] 20 mg: 204 ng/ml [range, 179 to 229]

7 Page 7 of 9 Mean peak plasma concentrations of rivaroxaban at steady state following a multi-dose administration of 5, 10, or 20 mg oral doses every 12 hours are as follows: 5 mg: 115 ng/ml [range, 91.1 to 139] 10 mg: 215 ng/ml [range, 178 to 252] 20 mg: 415 ng/ml [range, 355 to 474] Rivaroxaban may cause bleeding, anemia, nausea, pain, and dyspepsia. Significant drug interactions can occur when used in combination with rifampin, ketoconazole, and other compounds that inhibit metabolism. Source: Zhao X et al. Safety, pharmacokinetics and pharmacodynamics of single/multiple doses of the oral, direct Factor Xa inhibitor rivaroxaban in healthy Chinese subjects. British Journal of Clinical Pharmacology. 2009;68(1): Xarelto prescribing information. Titusville, NJ: Janssen Pharmaceuticals, 2012.

8 Page 8 of 9 REFERENCES 1. Xarelto prescribing information. Titusville, NJ: Janssen Pharmaceuticals, Zhao X, Sun P, Zhou Y, Liu Y, Zhang H, Mueck W, et al. Safety, pharmacokinetics and pharmacodynamics of single/multiple doses of the oral, direct Factor Xa inhibitor rivaroxaban in healthy Chinese subjects. British Journal of Clinical Pharmacology. 2009;68(1): Weinz C, Schwarz T, Kubitza D, Mueck W, Lang D. Metabolism and excretion of Rivaroxaban, an oral, direct factor Xa inhibitor, in rats, dogs, and humans. 2009;37(5); Rhode G. Determination of rivaroxaban a novel, oral, direct Factor Xa inhibitor in human plasma by high-performance liquid chromatography-tandem mass spectrometry. Journal of Chromatography B. 2008; 872:43-50.

9 Page 9 of 9 SIGNATURES: DMP 3/20/2013 Specification Writer Date EJB 3/21/2013 Toxicology Technical Leader Date Laboratory Director Date

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