Laboratory Detection of Newer Anticoagulant Drugs

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1 Laboratory Detection of Newer Anticoagulant Drugs Dorothy M. (Adcock) Funk, M.D. Colorado Coagulation, Laboratory Corporation of America Holdings Outline Newer Oral Anticoagulant Therapies A brief introduction - direct thrombin inhibitors (DTI) and direct Xa inhibitors (DXa) Laboratory detection to determine measured drug concentration Drug calibrated assays Laboratory detection to determine relative drug concentration Routine coagulation assays Effect on specialty coagulation assays Direct Oral Anticoagulant Agents (DOACs) or Target-Specific Oral Anticoagulants (TSOACs) Direct Thrombin Inhibitors Dabigatran Ximelagatran Direct Xa Inhibitors Rivaroxaban Apixaban Edoxaban Predictable pharmacodynamic and pharmacokinetic responses with little dietary effect Do not require therapeutic monitoring Dose is not adjusted based on laboratory measures Pharmaceutical manufacturers do not provide Therapeutic Ranges No published studies correlating plasma drug levels with thrombotic or hemorrhagic complications Dabigatran ng/ml Rivaroxaban ng/ml Trough Peak Trough Peak %* ** Published # Ranges are based on mass spectrometry n = mg dabigatran bid n = 29 2 mg rivaroxaban qd **Francart S, Hawes E, Deal A, et al. Thromb Haemost 214 doi /TH *Van Ryn J, Stangier J, Haertter S, et al. Thromb Haemost 21;13: Mueck W, Borris LC, Dahl OE, et al. Thromb Haemost 28;1: Although monitoring is not required, there are situations where it may be important to gauge drug level: Patients requiring emergent surgery Patients suffering potential overdose Patients with significant renal failure Patients with hemorrhagic or thrombotic complications Assessing efficacy of reversal strategies in patients with major bleeding 1

2 Laboratory Detection of DTIs and DXa Inhibitors International Society on Thrombosis and Haemostasis (ISTH) Scientific and Standardization Committee (SSC); Subcommittee on the Control of Anticoagulation, April 213 recommendation: Two types of assays should be considered #1) Assay that provides quantitative results to determine concentration of drug in serum or plasma. #2) Readily available, easily performed assay that can be used in an urgent clinical scenario to provide semiquantitative results, e.g. supratherapeutic, therapeutic or sub therapeutic anticoagulation. This assay is not to be used to determine drug concentration Baglin T, Hillarp A, Tripodi A, et al, J Thromb Haemost 213;11: Coagulation Cascade: In vitro model DXa FXII FXI FIX APTT FVIII Intrinsic pathway FV FII FXa Fibrinogen FVII PT Extrinsic pathway Thrombin FIIa TCT ECA Fibrin DTI Drug-specific assays Use a calibration curve specific for drug being measured Can be based on a variety of assays DTI: Dilute Thrombin Time (Aniara HEMOCLOT Thrombin Inhibitors), Ecarin Chromogenic Assay (Diagnostica Stago) Direct FXa: Chromogenic Anti-Xa Assay, PT Both: TFC-LC-MS/MS, Generally perform well over the on-therapy range of the drug Research or Investigational Use Only Hemoclot Thrombin Inhibitors* Aniara (HYPHEN BioMed) Quantitative measure of DTIs A modified thrombin time method Diluted test plasma is mixed with normal pooled plasma; clotting initiated by addition of a constant amount of alpha thrombin and clotting time is directly proportional to [DTI] The assay is calibrated with the specific DTI Insensitive to: Levels of coagulation factors VKA Lupus anticoagulant *RUO Assay Ecarin Chromogenic Assay* Diagnostica Stago, Inc 3 Total, Unconjugated 4. Dabigatran by LC- MS/MS Prothrombin (excess) Ecarin Meizothrombin Chromogenic Substrate X DTI pna (45)nm OD of pna is inversely proportional to [DTI] in plasma The assay is calibrated with the specific DTI Time, min Insensitive to: LLOQ (5ng/mL) 3.2e5 Levels of coagulation factors VKA Lupus anticoagulant *RUO Assay 15 Blank Defib Human Plasma e Time, min Time, min ULOQ (5ng/mL) Analyte Area / IS Area 2. "Linear" Regression ("1 / x" weighting): y =.79 x +.27 (r =.9999) Analyte Conc. / IS Conc. The assay is calibrated with the specific DTI Intra-Assay Characteristics Replicate LLOQ QC1 QC2 QC3 ULOQ Mean Std Dev Imprecision (%) Inaccuracy (%) NA NA NA

3 Anti-Xa Assays for DXa Inhibitors Anti-Xa Assay Principal Patient sample (DXa) + FXa (in excess) Residual Xa = [1/DXa] Measured with a chromogenic substrate The assay is calibrated with the specific DXa (rivaroxaban) Two different kit manufacturers were evaluated Kits supplemented with anti- Thrombin are not recommended Apixaban Treated Patients n Apixaban was administered at 5 or 1 mg bid or 2 mg qd in patients with acute VTE n Strong, linear correlation between plasma drug concentration and anti-xa activity Calibrated with apixaban Calibrated with LMWH Francart S, Hawes E, Deal A, et al. Thromb Haemost 214 doi /TH * Barrett YC. Thromb Haemost 21;14: Chromogenic Anti-Xa Assays and Direct FXa Inhibitor Drugs Using LMWH/UFH Calibrators Direct FXa inhibitor drug will be detected regardless of assay calibrator used Strong correlation with UFH and LMWH calibration curves but detection limits are too narrow. Gosselin R,Moll S, Adcock D. Unpublished Will read below detection limit when up to 3mg/mL drug is present. At peak, levels often above the highest calibrator On therapy range may extend to 3 5 IU/mL or higher Laboratory Detection of DTIs/DXa Inhibitors: Routine Assays APTT, PT and thrombin time: Responsiveness of different reagents varies significantly Tend to be too insensitive, too sensitive, or fail to show the appropriate dose response General rules: APTT more responsive to dabigatran than PT A normal APTT does not exclude on-therapy levels of dabigatran* PT more responsive to direct FXa inhibitors than APTT A normal PT does not exclude on-therapy levels of rivaroxaban** TT is exquisitely responsive to dabigatran A normal TT indicates that minimal or no dabigatran is present TT shows no response to direct Xa inhibitors **Francart S, Hawes E, Deal A, et al. Thromb Haemost 214 doi /TH Dabigatran Effect on APTT and PT Dabigatran TT Response 3 25 Thrombin Time (s) On therapy range *Hawes E, Deal A, Jeanneret, et al. J Thromb Haemost. Upper limit of normal range Dabigatran measured by LC- MS/MS ng/ml 3

4 Rivaroxaban Effect on APTT and PT On therapy range Upper limit norma lrange Laboratory Detection - DTI ISTH SCC Recommendations for Dabigatran Activated Partial Thromboplastin Time (APTT) Can be used to determine relative intensity of anticoagulation 15 mg twice daily, peak APTT is approximately 2X control Anticoagulant effect may be underestimated by the APTT when levels are supratherapeutic Each laboratory should be aware of the sensitivity of their reagent, and this can be done using commercially available controls or calibrators Prothrombin Time Relatively insensitive to dabigatran Thrombin Time (TT) A normal TT indicates that minimal or no dabigatran is present Francart S, Hawes E, Deal A, et al. Thromb Haemost 214 doi /TH Laboratory Detection - DXa ISTH SCC Recommendations for Rivaroxaban Activated Partial Thromboplastin Time (APTT) Some reagents are very insensitive, no prolongation is seen even at peak levels Prothrombin Time (PT) Can be used to determine relative intensity of anticoagulation Each laboratory should be aware of the sensitivity of their reagent, this can be done using commercially available controls or calibrators Samama MM, et al. Thromb Haemost. 21;13: Amount of rivaroxaban needed to double the PT varied from 66ng/mL to 7ng/mL depending on the reagent, with no correlation to ISI Baseli CAL PAT 2CT CAL PAT ne 2CT Dabigatra 2CT 2CT NPP Dabigatra n Rivaroxa Rivaroxa (s) n (ng/ml) ban (ng/ ban (ng/ml) ml) (ng/ml) PT Innovin Neoplastin CI APTT SynthASil PTT-A Drug concentration based on patient sample determined by MS versus drug concentration based on calibrator Gosselin R,Moll S, Adcock D. Unpublished Effect of Dabigatran on Factor Assays Factor Activity in IU/dL FVIII FIX FXI Intrinsic Factors APTT-Based The mechanism of action of direct and indirect FXa inhibitors is significantly different as direct FXa inhibitors can inhibit Xa in the prothrombinase complex which is 3, fold more efficient in converting prothrombin to thrombin than free Xa. Does a 24s PT in a patient on warfarin equate to the same level of anticoagulation when a patient has a 24s PT on rivaroxaban? Haas, S. Thromb Haemost 21; 13: Extrinsic Factors PT-Based * Adcock DM, et al. AJCP. 213;139:

5 Dabigatran and Rivaroxaban Effect on Coagulation Assays % change compared to Reference Plasma FII FV FX FVIII FIX FXI AT PC PS AT assays either IIa Laboratory Xa based PC and PS assays: clot-based APCR * Adcock DM, et al. AJCP. 213;139:12-19; ECAT Proficiency Data 212. Dabigatran Rivaroxaban Dabigatran and Rivaroxaban Effect on Coagulation Assays Drugs act as inhibitors: Incomplete correction with 1:1 plasma mix Can cause a false positive Bethesda assay False positive Lupus Anticoagulant Assays No effect on: D-dimer, VWF assays, free protein S antigen, chromogenic protein C activity, reptilase time Thank you Dot.Adcock@esoterix.com

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