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Direct Oral Anticoagulants

Direct Oral Anticoagulants

6/19/18

Direct Oral

A Comprehensive History and Current Developments

BCSLS Telehealth Seminar, Vancouver, BC June 26, 2018

Terence M. Litavec B.Sc., MLT(CSMLS), SHCM(ASCP)HCM,MLTCM,HTLCM(ASCP)QIHCCM

Acknowledgements: 2017-2018 KGH Student Interns British Columbia College of New Institute of Caldonia, Technology, Burnaby Prince George  Stefani Guidi, MLT  Soraya Hadjirul, MLT  Henry Lu, MLT  Neelam Lilly, MLT  Jenna Zhang, MLT  Alix Savoy, MLT  Dawny Tabilangan, MLT

Overview and Objectives (A Work In Progress)  Provide background information about the classification and function of DOAC’s

 Outline the various subtypes of DOAC’s listing their similarities and differences

 List the brief histories and the most current advances in the field of DOAC drug development

 Discuss the abnormalities in routine screening coagulation assays for patients taking DOAC’s

 Describe the laboratory assays used to measure the level of DOAC in plasma samples

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Direct Oral Anticoagulants (DOAC)  A new class of drugs to treat and prevent related to: (ACS), (AF), Cerebrovascular Accident (CVA), (MI), Joint Replacement, etc.

 Can be given as an immediate treatment for an acute crisis, or as a long-term regiment

 Can be given as alternatives to traditional “clot- busting” in patients who have developed sensitivities to (HIT antibody formation) or (Coumadin-related limb gangrene due to inhibition)

 Do not require scheduled monitoring, but they typically cause abnormalities in coag screening assays (PT, aPTT)

Alternate Names For DOAC’s  NOAC = Novel Oral Anticoagulants, label initially applied to (Praxada) when it was introduced to the US market in 2010  Since 2010, additional medications with similar mechanisms of action have been developed which render the description “novel” no longer applicable  Novel - (adj.) of a new kind, different from anything seen or known before, having no precedent; (syn.) new, original, first-ever, unique, uncommon, unheard-of  NOAC = Non-vitamin K Oral Anticoagulants, was introduced by the American College of Chest Physicians (Feb. 2016) to keep the same acronym, but it has been criticized as being too vague and cumbersome  NOAC can be mistaken as “NO AntiCoagulants”

Alternate Names For DOAC’s

 TSOAC = Target-Specific Oral Anticoagulant

 ODI = Oral Direct Inhibitor

 SODA = Specific Oral Direct Anticoagulant

“DOAC seems to be a very reasonable term moving forward that should be embraced by clinicians to describe these new oral anticoagulants and oral anticoagulants with similar direct mechanisms that haven’t yet been released.”

Source: Kane, S., NOAC, DOAC, or TSOAC: What Should We Call Novel Oral Anticoagulants? Sept.19, 2016 www.pharmacytimes.com

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United States Food and Drug Administration (USFDA) Drug Development Process  After a drug is manufactured and tested using animal models, human testing is carried out in 3 Clinical Trial Phase Studies (Source: www.fda.gov)  Phase I – Safety and Dosage on a group of healthy volunteers – Several Months  Phase II – Efficacy and Side Effects on a group of patients with a disease or condition – Up to 2 Years  Phase III – Efficacy and Monitoring of Adverse Reactions on a group of volunteers with a disease or condition – 1 to 4 Years – 25-30% of all drugs tested will be approved for further review

Biological Half-Life Definition: The time required for the body to eliminate half of an administered dose of any substance by regular physiological processes

The plasma drug level falls at a logarithmic rate that is dependent upon factors such as: the nature and composition of the drug, accumulation of the drug in tissues, activity of the drug metabolites, and interactions between the drug and its target receptor within the body

Examples:

 Oxaliplatin (Colorectal Cancer medication) = 14 minutes

 Bedaquiline (Tuberculosis medication) = 5½ months

Drug Levels Displaying 1-Hour Half-Life www.slideshare.net

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Subcategories of DOAC’s

 Direct Inhibitors – Bind to one or more active sites or exosites on both free and -bound thrombin (Factor IIa) thereby preventing the conversion of soluble (Factor I) into a fibrin monomer; also display an anti- effect by reducing the thrombin- mediated activation of

 Factor Xa Inhibitors – Bind directly to both free and clot- bound Factor Xa resulting in an interruption of both the intrinsic and extrinsic pathways of the cascade sequence without requiring cofactors such as ATIII

 (Sources: Nisio, M., et al. N. Engl. J. Med. 2005;353:1028-1040, Samama, M. Thromb. Res. 2011 Jun;127(6):497-504)

Direct Thrombin Inhibitors

 Univalent – Bind only to the active site of thrombin: Dabigatran, , , Efegatran, Melagatran ()

 Bivalent – Bind to both the active site and exosite 1 of thrombin: and derivatives , Desirudin,

 Allosteric Inhibitors – New class of medications currently under investigation which inhibit thrombin using a variety of approaches: Ichorcumab (parenteral thrombin inhibitor), BMS-986177 (oral FXIa inhibitor), BAY- 1213790 Xisomab and Aximab (parenteral FXIa inhibitors), several FXIIa monoclonal antibodies are under investigation

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Inogatran - Parenteral

 Entered Phase I Clinical Trials in 1995

 Half-life = 1 hour, aPTT and TCT were doubled

 Discontinued by AstraZeneca during Phase II Clinical Trials due to a lack of efficacy compared to heparin for preventing ischemic events in patients with unstable coronary artery disease (CAD)

 (Sources: Teger-Nilsson, A. et al. J. Amer. Coll. Cardiol. Feb 1995, Volume 25, Issue 2, Supp. 1, p 117A-118A; TRIM-Thrombin Inhibition in Myocardial Ischaemia Study Group. Euro. Heart J. 1997(18):1416-1425)

Efegatran - Parenteral  Tested in a Phase II Clinical Trial from May 1995 to June 1996 in cardiovascular patients with , and MI patients undergoing thrombolytic therapy

 Half-life in humans not published, caused a dose- dependent prolongation of aPTT and TCT

 Analysis of Phase II data demonstrated that Efegatran “exhibited equivalent efficacy” to Heparin and was discontinued by Eli Lilly Pharmaceuticals

 (Sources: The PRIME Investigators, Am. Heart J. 2002;143:95-105; Shuman, R. and Gesellchen, P. Integration of Pharmaceutical Discovery and Development Ch. 4 pp. 57-80, Springer, Boston, MA. ISBN – 978-0-306-47384-5)

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Argatroban (Acova) - Parenteral  Licensed in 2000 and USFDA approved in 2002 for use in percutaneous coronary interventions (angioplasty) in patients who have HIT or are at risk for developing HIT

 Half-life = 50 minutes, metabolized by the liver

 Monitored using the aPTT, therapeutic range is 1.5 to 3x the initial baseline value, but can not exceed 100 sec

 In healthy patients (without HIT), anticoagulation was 4x faster with Argatroban than with UFH and produced more predictable dose-related increases of the activated clotting time (ACT) and the aPTT

 (Sources: Bambrah, R., et al. Ther. Adv. Chronic Dis. 2013 Nov;4(6):302-304, Swan, S., et al. Pharmacotherapy 2000 July;20(7):756-770)

Melagatran (Ximelagatran)

 Ximelagatran was the first oral direct thrombin inhibitor developed and approved for Clinical Trials in Dec. 2003

 Ximelagatran is a pro-drug which is converted by the liver to the active agent Melagatran

 During clinical trials, the results were comparable to Warfarin and LMWH for VTE prophylaxis

 However, 6%-12% of patients developed elevated liver enzymes subsequent to severe hepatotoxicity leading to its withdrawal by AstraZeneca in 2006

 (Sources: Eriksson, B. I. Thromb. Haemost. 2002 Feb;87(2):231-7. Brighton, T. A. Med. J. Aust. 2004 Oct 18;181(8):432-7)

Dabigatran (Praxada)  USFDA approved in October 2010 for treatment and prophylaxis of DVT and PE as well as prophylaxis of VTE and stroke in patients with atrial fibrillation

 Half-life = 12-17 hours, eliminated by the kidney (80%)

 Recommended as an alternative to Warfarin in 2011 with no need for frequent monitoring due to its stable hematologic response and short half-life

 In the therapeutic range (27-411 ng/mL), the PT, aPTT, and ACT were not affected, the TCT and dilute TCT displayed good linear correlation with lower doses only

 (Sources: Ganetsky, M., et al. J. Med. Toxicol. 2011 Dec;7(4):281-287, Hawes, E., et al. J. Thromb. Haemost. 2013 Aug;11(8):1493-1502)

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Idarucizumab (Praxabind)

 A monoclonal antibody fragment developed by Boehringer Ingelheim Pharmaceuticals for the reversal of Dabigatran (Praxada)  Displays a binding affinity with Dabigatran that is 350x stronger than Dabigatran’s binding affinity with thrombin ensuring rapid and complete reversal of Dabigatran within minutes after IV infusion  USFDA approved in 2015  (Source: Pollack, C., et al. N. Engl. J. Med. 2015 Aug;373:511-520)

Hirudin and Derivatives  Hirudin is produced by the salivary glands of the European Medicinal Leech Hirudo medicinalis, first isolated and studied in 1884 by John Berry Haycraft

 Native (Unmodified) Hirudin is not recommended for acute coronary syndrome due to significantly higher incidence of bleeding compared to Heparin

 Native Hirudin is not given as a medication, but it has been evaluated as a universal anticoagulant in collection tubes for hematology, clinical chemistry, and blood bank testing (Source: Menssen, HD, et al. Clin. Chem. Lab Med. 2001 Dec;39(12):1267-1277)

 All Hirudin derivatives must be given parenterally by IV or subcutaneous bolus injection

Lepirudin (Refludan)

 Hirudin derivative which is derived from yeast cells using recombinant biotechnology

 Forms a non-covalent, irreversible complex with thrombin inhibiting its activity

 Was approved for patients with thrombosis due to HIT, but was discontinued in 2012 due to formation of anti- Lepirudin antibodies in 44-74% of HIT patients that displayed cross-reactivity with desirudin and bivalirudin

 Lepirudin served as a useful model for the development of other Hirudin-derived medications

 (Source: Eichler, P., et al. Blood 15 Jan 2004, V.103 N.2)

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Desirudin (Iprivask or Revasc)  Hirudin derivative which is derived from yeast cells using recombinant biotechnology  Forms a non-covalent, irreversible complex with thrombin inhibiting its activity  Approved for treatment in Europe for DVT treatment and prophylaxis  Currently in phase III FDA clinical trials in the US where it has been shown to be more effective than UF and LMWH in preventing DVT’s in patients undergoing elective hip replacement surgery  (Source: Lee, C. and Ansell, J. Br. J. Clin. Pharmacol. 2011 Oct; 72(4): 581-592)

Bivalirudin (Angiomax or Angiox)

 Modified 20-amino acid synthetic analog of Hirudin

 USFDA approved in December 2000 for HIT patients, patients with stable angina, unstable angina undergoing percutaneous transluminal coronary angioplasty (PTCA), ST-Elevated Myocardial Infarction (STEMI) and Non-STEMI

 Has only been studied and approved in patients receiving concurrently

 Reversible by Recombinant Factor VII concentrate

 (Source: Young, G., et al. Blood Coagul. 2007 Sep;18(6):547-553)

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Allosteric Inhibition

 Definition: the regulation of an enzyme by binding an effector molecule at an allosteric site which is different from the enzyme’s active site

 Occurs when the binding of one functional group or allosteric site decreases the affinity for substrate at other allosteric sites (in this case, causing disruptions in the cascade sequence before thrombin formation)

 New targets for anticoagulation include: FXIa, FXIIa, protein disulfide isomerase, and polyphosphatases

 (Source: Gómez-Outes, A. et al. Discovery methods of coagulation-inhibiting drugs. Expert Opinion on Drug Discovery, Volume 12, 2017, Issue 12)

Ichorcumab  Monoclonal antibody against exosite 1 of thrombin, developed by Janssen Pharmaceuticals

 In Greek mythology, ichor was the ethereal fluid in the blood of the gods that conveyed their immortality

 Development arose from a discovery in August 2008 of a patient with an abnormal aPTT that formed a rare IgA paraprotein that reacted with thrombin by stabilizing her cerebral hemorrhage, but prevented the clotting from expanding into the intravascular spaces

 Phase I clinical trials began in September 2017

 (Source: Grainger, David. Ichorcumab: the blood of the gods? June 16, 2013 www.fiercebiotech.com)

BMS-262084 and BMS-986177

 Both developed by Bristol-Myers Squibb as parenteral, small-molecule, direct, irreversible FXIa inhibitors

 BMS-262084 prolonged the aPTT, but did not affect the PT, TT, or platelet function testing in rabbits; began Phase I Clinical Trials in July 2014

 BMS-986177 began Phase I Clinical Trials in Nov. 2016 to test its effectiveness on end-stage renal disease patients undergoing hemodialysis with UFH treatments

 (Sources: Wong, P .et al. J. Thromb. . 2011 Aug;32(2):129-137, www.clinicaltrials.gov)

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BAY-1213790

 An Anti-FXIa antibody developed by Bayer Pharmaceuticals

 Shows strong anti-thrombotic efficacy without increasing the risk of bleeding in rabbits

 Began Phase I Clinical Trials on Sept. 21, 2017 to compare its safety and efficacy to existing therapies (Enoxaparin or ) in patients undergoing total knee arthroplasty

 (Sources: Buchmueller, A. ISTH Academy. July 10, 2017;186857, www.clinicaltrials.gov)

Xisomab and Aximab

 Both are monoclonal Anti-FXIa antibodies developed by Aronora Biopharmaceuticals in conjunction with Oregon Health Sciences University

 Aximab underwent initial testing in 2009, it inhibited plasma FXI by 99%, reduced platelet activity, and prevented fibrin formation in baboons; application for Phase I Clinical Trials has been submitted

 Xisomab was approved for Phase I Clinical Trials in August 2017 in healthy adult subjects

 (Sources: Tucker, E, et al. Blood. 2009 Jan 22;113(4): 936-44, Rojas-Burke, J. OHSU News. September 7, 2017 www.news.ohsu.edu, www.clinicaltrials.gov)

Several Factor XIIa Antibodies Are Currently Being Investigated  Recombinant antibody clone 3F7 has been tested along with Heparin and was shown to block thrombus formation in mice and rabbits without increasing bleeding from wounds or impairing the overall hemostatic capacity (Source: Larsson, M., et al. Sci. Transl. Med. 2014 Feb 5;6(222):222ra17)

 Recombinant antibody clone 15H8 reduced fibrin formation in experimental baboons and prolonged the aPTT in both baboon and human plasmas (Source: Matafonov, A., et al. Blood. 2013-04- 499111)

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Factor Xa Inhibitors

Discontinued Currently in Use  Razaxaban, Letaxaban

, Eribaxaban Apixaban  Not DOAC’s   Enoxaparin  /Idrabiotaparinux

Diagram of Factor Xa Inhibition

Source: Yeh, C., Fredenburgh, J., Weitz, J. Oral Factor Xa Inhibitors, Circulation Research 2012;111:1069- 1078

Razaxaban - Discontinued

 In 2002, it was the first FXa inhibitor to provide clinical validation of the effectiveness of FXa inhibitors

 In combination with aspirin and/or (Plavix), Razaxaban showed improved activity without enhanced bleeding in rabbits

 It was discontinued in 2005 due to Apixaban being developed by the same drug company (Bristol-Myers Squibb) and displaying a more favorable safety profile

 (Sources: Wong, P. et al. J. Thromb. Thrombolysis 2011 May;31(4):478-492, and 2007 Aug;24(1):43-51)

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Darexaban (Tanexaban) - Discontinued  Half-life = 14-18 hours, metabolized mainly by the liver

 In clinical trials, patients with acute coronary syndrome displayed a 2- to 4-fold increase in major bleeding events with no decrease in subsequent thrombotic events compared to patients receiving placebo

 Discontinued by Astellas Pharma in 2011 before additional clinical trials for VTE prophylaxis began citing “difficulty finding a suitable partner” for further study and “intensified competition for this product”

 (Source: Grogan, K. Aug. 31, 2011, Feb. 16, 2011, Sept. 29, 2011, www.pharmatimes.com)

Discontinued Factor Xa Inhibitors

 Eribaxaban – Discontinued by Pfizer in 2010 after Phase II Clinical Trials in favor of cooperation with Bristol-Myers Squibb for Apixaban development

 Letaxaban – Discontinued by Takeda in 2011 during Phase II Clinical Trials due to adverse bleeding events

 Otamixaban – Discontinued by Sanofi in 2013 after Phase III Clinical Trials due to increased bleeding and failure to prevent ischemic events in acute coronary syndrome patients

 (Sources: Ahrens, I. June 23, 2012, www.discoverymedicine.com; Hughes, S. Sept. 1, 2013, www.medscape.com)

Rivaroxaban (Xarelto)  Initially developed by Bayer, USFDA approved in 2011

 Half-life = 5-9 hours, excreted by the liver (67%)

 Compared to LMWH, Rivaroxaban displayed a lower risk of VTE, but no difference in the risk of bleeding in patients discharged from total hip or knee arthroplasty

 Compared to Warfarin (Coumadin), Rivaroxaban was “non-inferior” for the prevention of stroke or systemic embolism with no significant difference in major bleeding, but intracranial and fatal bleeding occurred less frequently in AF patients (ROCKET AF Clinical Trial)

 (Sources: Lazo-Langner, A., et al. J. Thromb. Haemost. 2014 Oct:12(10):1626-1635; Patel, M., et al. N. Engl. J. Med. Sept. 8, 2011;365:883-891)

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COMPASS Clinical Trial  Cardiovascular OutcoMes for People Using Anticoagulation StrategieS; sponsored by Bayer, Janssen Research and Development, and Hamilton Health Sciences Corporation, affl. McMaster University, Ontario

 Began Feb. 28, 2013, Completed July 21, 2017

 27395 CAD and/or PAD patients in 602 medical centers in 33 countries were given either 5mg Rivaroxaban, 100mg Aspirin, or 2.5mg Rivaroxaban plus 100mg Aspirin

 Patients who received low-dose Rivaroxaban plus Aspirin had fewer recurrent cardiovascular events than either medication alone, but had increased bleeding events

 (Sources and Table: Eikelboom, J., et al. N Engl. J Med. Oct. 5, 2017;377:1319-1330; www.clinicaltrials.gov)

Apixaban (Eliquis)  Developed in a joint venture by Pfizer and Bristol-Myers Squibb, approved in Europe in 2012 and by the USFDA in 2014 for treatment and prophylaxis of DVT and PE

 Half-life = 9-14 hours, excreted by the bile (75%) and the kidney (25%)

 Causes only minimal prolongation of the PT and aPTT, Apixaban levels are measured using the Anti-Xa assay

 Performs with highly comparable stroke/VTE prevention results to Rivaroxaban and Dabigatran with a slightly lower risk of bleeding in patients with atrial fibrillation

 (Sources: Cohen, A., et al. PLoS ONE10(12) Dec. 2015 Noseworthy, P. et al. Chest 2016 Dec;150(6):1302-1312)

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Rivaroxaban vs. Apixaban vs. Dabigatran vs. Coumadin

 In 76354 patients with Atrial Fibrillation, the effects of DOAC’s were directly compared to traditional Coumadin (Warfarin) therapy:  Apixaban – lower risk of both stroke and major bleeding, n=15390  Dabigatran – similar risk of stroke but lower risk of major bleeding, n=28614  Rivaroxaban – similar risk of both stroke and major bleeding, n=32350

 (Source: Yao, X., et al., JAHA June 2016;5:e03725)

Betrixaban (Bevyxxa)

 Received USFDA approval June 23, 2017 for VTE prophylaxis in hospitalized, acutely ill at-risk patients

 Half-life = 20-27 hours, excreted by the bile

 Clinical trial data showed comparable results between Betrixaban and LMWH along with a slightly better “net clinical benefit” in patients with reduced mobility and specific risk factors for VTE

 (Sources: Horn, J. and Hansten P. Betrixaban: A Factor Xa Inhibitor. Oct. 25, 2017. www.pharmacytimes.com, Cohen, A., et al. N. Engl. J. Med. 2016;375:534-544)

Edoxaban (Savaysa, Lixiana)  Approved in 2011 in Japan and by the USFDA in 2015

 Half-life = 10-14 hours, excreted by kidney (50%) and bile (<50%)

 In clinical trials in patients with symptomatic VTE, Edoxaban was “non-inferior” to dose adjusted Warfarin (INR 2.0-3.0) for prevention of recurrent VTE and also had a significantly lower risk of bleeding events

 (Source: Bounameaux, H., Camm, A.J. Drugs 2014 Jul;74(11):1209-1231. Erratum in Drugs 2014 Aug;74(12):1455)

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Enoxaparin Is NOT a DOAC  Enoxaparin is a Low Molecular Weight Heparin approved for use in 1993, also known as Lovenox and Clexane

 All LMWH’s bind to ATIII for indirect inactivation of FXa

 All LMWH’s are given parenterally by IV or SQ injection and are measured using the chromogenic Anti-Xa assay with medication-specific standards for calibration

 In a recent clinical trial, Rivaroxaban caused fewer asymptomatic deep venous and muscular venous thromboses, but slightly more ecchymoses and wound complications compared to Enoxaparin in patients undergoing primary total knee replacement

 (Source: Xie, J., et al. Med. Sci. Monit. 2017;23:704-11)

Fondaparinux (Arixtra) Is NOT a DOAC  Approved by the USFDA in 2001, it is composed of the same pentapeptide active site as Heparin

 Half-life = 17-21 hours, excreted by the kindey

 Binds to ATIII for indirect Factor Xa inhibition

 Given to patients with established HIT since it has no affinity to thrombin or PF4

 Has been shown to prolong the PT by about 1 sec, and the aPTT by 4-5 sec, but give no significant changes in the Fibrinogen, ATIII, or TT assays

 (Source: Smogorzewska, A. et al. Arch. Pathol. Lab Med. 2008 Nov;130(11):1605-1611)

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Idraparinux and Idrabiotaparinux Are NOT DOAC’s  Idraparinux is a discontinued polymethylated synthetic pentasaccharide similar to Fondaparinux

 Idrabiotaparinux is Idraparinux modified with a biotin conjugate which enables rapid deactivation by avidin

 Both bind to ATIII for indirect Factor Xa inhibition

 Idraparinux has a half-life of 80 hours and has caused severe bleeding when given for VTE prophylaxis

 Idrabiotaparinux is currently undergoing clinical trials

 (Source: Harenberg, J. Expert Rev. Clin. Pharmacol. 2010 Jan;3(1):9-16)

Factor Xa Inhibitor Reversal Agents Under Investigation  PseudoXa – derived from Australian Brown Snake venom

 Andexanet alfa – a synthetic, truncated form of enzymatically inactive Factor Xa reverses the inhibitory activity of Rivaroxaban and other Factor Xa Inhibitors

 Aripazine – a synthetic small molecule which appears to have broad-spectrum reversal activity of Dabigatran, Rivaroxaban, Apixaban, and Edoxaban in rat models

 Activated charcoal – reduces absorption in cases of prolonged bleeding, accidental ingestion, or overdoses

 (Sources: Ward, C., et al. Thromb. J. Dec. 2013;11:27, Southey, F., Sept. 20, 2017 in-pharmatechnologist.com)

Newly Approved Factor Xa Inhibitor Reversal Agent – Andexanet Alfa (Andexxa)  According to a statement by Portola Pharmaceuticals, on May 3, 2018, the USFDA granted accelerated approval to Andexanet Alfa (Andexxa) as an Anti-Xa “breakthrough therapy” which must still undergo trials

 It has been approved for Rivaroxaban and Apixaban, but it has NOT been approved for Edoxaban and Betrixaban

 It remains under review by the European Medicines Agency with a final decision expected in early 2019

 (Sources: Lou, N. May 4, 2018 www.medpage.com; Neale, T. May 4, 2018 www.tctmd.com)

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Monitoring vs. Measurement  Therapeutic Drug Monitoring (TDM) – the clinical laboratory measurement of a chemical parameter that, with appropriate medical interpretation, will directly influence drug prescribing procedures

 Useful in drugs that meet the following criteria:  Difficulty in interpreting clinical evidence of therapeutic or toxic effects  A good relationship between the plasma drug concentration and the therapeutic and/or toxic effect  A low therapeutic : toxic ratio  Does not metabolize to important active metabolites

 (Source: Kang, J., and Lee, M. Korean J. Intern. Med. 2009 Mar; 24(1):1-10)

Monitoring vs. Measurement  Coumadin (Warfarin) - Half-life = 20-60 hours with its effect lasting 2-5 days in duration

 Coumadin is the most commonly prescribed anticoagulant for treatment and prevention of VTE, PE, SE, AF, and for heart valve replacement

 Since the duration of Coumadin administration can last for the patient’s lifetime, patient education of drug interactions and regular monitoring of the PT/INR is required to prevent overdose and other adverse events

 Therapeutic INR range = 2.0-3.0 but it can be as high as 4.0 in patients with mechanical heart valves and LA

 (Source: Horton, J. and Bushwick, B. Am. Fam. Physician 1999 Feb. 1;59(3):635-646)

Monitoring vs. Measurement

 UFH – Half-life = 30-90 minutes, SQ administration is optimal, highly dosage-dependent, bioavailability ranges from 30% at low doses to 70% at higher doses

 UFH is one of the oldest drugs still in widespread clinical use, may be used for treatment and prevention of VTE, PE, SE, AF, heart valve replacement, and to prevent coagulation during surgery as the patient’s blood passes through an extracorporeal circuit (ECMO)

 Due to the unpredictable anticoagulant response and side effects of UFH, close monitoring of the patient’s aPTT and/or drug level by Anti-Xa is required

 (Source: Tahir, R. US Pharm. 2007:32(7):HS26-HS36)

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Monitoring vs. Measurement

 For DOAC’s, scheduled monitoring is not recommended due to short half-life of each drug, but measurements of plasma concentration are useful in the following cases:  Suspected Drug Failure (Incidents of thrombosis or major/life-threatening bleeding while on DOAC’s)  Urgent/emergent surgery or invasive procedure while on DOAC’s  Patients displaying any weight extreme, renal insufficiency, or hepatic dysfunction  Peak effect is 2-4 hours after administration

Effects of DOAC’s on Routine Coag Screening Assays

 In a Dabigatran study, the aPTT and TT from 75 plasma samples was measured using 4 different aPTT reagents

 The aPTT values were used to establish therapeutic ranges (90-180 ng/mL) with “modest correlation” but “became less reliable at higher Dabigatran levels”

 The TCT value was >300 sec with a plasma Dabigatran level of 60 ng/mL and “is too sensitive to quantify Dabigatran levels, but a normal TCT value suggests minimal or no plasma Dabigatran”

 (Source: Hapgood, G., et al. Thromb. Haemost. 2013 Aug;110(2):308-15)

Effects of DOAC’s on Routine Coag Screening Assays  In several Rivaroxaban studies, the PT/INR results using different reagents showed “modest and variable” correlation to plasma levels of Rivaroxaban with minimal responsiveness of the PT assays to Apixaban

 Rivaroxaban showed a non-linear dose-dependent increase in aPTT results with minimal sensitivity and significant variability between different reagents

 Rivaroxaban and Apixaban have no effect on TCT results

 (Sources: Siegal, D. and Konkle, B. ASH Education Book Dec. 5, 2014 v.2014(1):334-6; Ciurus, T., et al. Polish J. Thoracic and Cardiov. Surg. 2015 June;12(2):111-8)

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Effects of DOAC’s on Routine Coag Screening Assays  Dabigatran, Argatroban, Rivaroxaban, Apixaban, and Fondaparinux have been tested for their effects on point-of-care (POC) and platelet function assays

 Each medication was spiked into normal blood samples at therapeutic and supratherapeutic concentrations

 All samples were run using thromboelastography (TEG), activated clotting time (ACT) and platelet aggregometry

 Therapeutic and supratherapeutic drug concentration samples showed dose-dependent prolongation of clotting times with a decrease in platelet aggregation

 (Source: Eller, T., et al. Clin. Chem. Lab Med. 2014 June;52(6):835-844)

Effects of DOAC’s on Lupus Anticoagulant (LA) Assays  In addition to high and low phospholipid aPTT reagents, Dabigatran, Rivaroxaban, and Apixaban have been shown to interfere with the reagents in the Dilute Russell Viper Venom Time (DRVVT) assays which are used in a panel to confirm the presence of antiphospholipid antibodies in LA patients

 “It is crucial to take into account the finding that even samples containing DOAC concentrations below the limit of detection of the drugs may lead to false-positive DRVVT measurements.”

 (Source: Flieder, T., et al. Thrombosis Research 2018 May;165:101-106)

Effects of DOAC’s on Routine and More Specialized Coag Assays  DOAC’s interfere with the functional assays which are run as part of thrombophilia panels for patients with hereditary, congenital, or acquired thrombotic disorders

III (ATIII) activity assays based on anti-FIIa inhibition are affected by Dabigatran, those based on anti-FXa inhibition are affected by Rivaroxaban, et al.

 Clot-based assays for Protein C and Protein S and Activated Protein C Resistance are affected by all DOAC’s, chromogenic assays are not affected by DOAC’s

 (Source for above, next page, and following table: Favaloro, E., Lippi, G. Blood Transfus. 2017;15:491-494)

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Effects of DOAC’s on Routine and More Specialized Coag Assays  “The risk of falsely diagnosing, or alternatively falsely excluding, a hereditary thrombophilia, such as Activated Protein C Resistance/Factor V Leiden, or Protein C, Protein S, or Antithrombin III deficiency, most likely exceeds, by several orders of magnitude, the likelihood of detecting true cases of hereditary thrombophilia as previously reported during the era of Vitamin K antagonists and , but now further accentuated by the existence and use of the DOAC.”

 “… we and others continue to advise against testing patients for thrombophilia markers, including Activated Protein C Resistance, while the patients are on anticoagulant therapy, of which Dabigatran is now one of many anticoagulants affecting hemostasis testing.”

 = Increase in clotting time  = Decrease in clotting time or fibrinogen level O = No effect on assay results O/ or O/ = Variable effect on assay result, dependent upon reagent manufacturer or assay methodology (clot-based vs. chromogenic)

Dilute Thrombin Time Assay For Direct Thrombin Inhibitors  The patient plasma is diluted with frozen Patient Normal Plasma reagent in a 4:1 ratio of PNP:sample

 A Thrombin Clotting Time assay is run on the mixture

 A normal value (14-19 sec) indicates a medication level that does not exceed the therapeutic range

 This assay is only capable of detecting an overdose of direct thrombin inhibitor meds; levels within and below the therapeutic range will give normal results

 It is a tool for physicians to rapidly detect overdoses and/or adjust patient dosages based upon the result

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HEMOCLOT Direct Thrombin Inhibitors Clotting Assay  A modification of the Dilute Thrombin Time assay for the quantitative measurement of Dabigatran

 Calibration standards and QC solutions are used to determine the concentration of Dabigatran in citrated plasma samples within a range of 50 - 500 ng/mL

 Developed by HYPHEN Biomed in France, this assay has been shown to display high sensitivity and suitability for measuring plasma Dabigatran levels in patients with both therapeutic and very high doses of Dabigatran

 (Sources: Stangier, J., et al. Blood. 2011 118:2307; Samoš, M., et al. J. Thromb. Thrombolysis. 2015 Jan;39(1):95-100)

Confirmatory Assay For Dabigatran Measurement  NMS Labs, Willow Grove, PA, www.nmslabs.com

 Direct Measurement of Dabigatran (Therapeutic Drug Monitoring Assay) by High Performance Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS)

 Red Top Serum or EDTA Plasma Required (No Gel Tubes)

 Lower limit of assay linearity = 10 ng/mL

 Peak plasma concentrations of Dabigatran following 3x daily dosing for 7 days:  50 mg (150 mg/day) = 42 ng/mL  100 mg (300 mg/day) = 130 ng/mL

Chromogenic Anti-Xa Assay For Factor Xa Inhibitors  The chromogenic Anti-Xa assay is used to measure the levels of all Factor Xa Inhibitor medications  Reagents are: a known concentration of Factor Xa and a chromogenic substrate (para-nitroanaline) - (Figure on Next Page: Smith, L. Coagulation Assays Part 2 www.slideshare.net, modified by Terence Litavec)  The medication in the patient’s plasma binds to the reagent Factor Xa, thereby inactivating it  The unbound Factor Xa reagent catalyzes the chromogenic substrate yielding an end product with an intensity indirectly proportionate to the inhibitor level  Calibration standards and QC test solutions are required for each medication to provide accurate measurement of patient plasma levels using this Anti-Xa method

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Anti-Factor Xa Assay Diagram pNA = Paranitroanaline Chromophore ------Plasma Rivaroxaban

------[Rivaroxaban-Xa]

Anti-Xa Assay Case Study  A 9-year-old obese child with a history of renal failure, HIT, and thrombosis was given Fondaparinux

 The need for appropriate dosing along with accurate plasma concentration measurements presented significant challenges in a lab which only had calibrators for UFH and LMWH for its anti-Xa assay

 Since sending the samples for Fondaparinux levels to a reference lab caused a 24 hour delay, a decision was made to run the assay with the LMWH calibration curve

 The anti-Xa values for Fondaparinux, LMWH and UFH are not equivalent, separate calibration curves are required

 (Source for above and next graph: Johnson, P., et al. J. Pediatr. Pharmacol. Ther. 2013 Oct-Dec;18(4):318-319)

Comparison of Calibration Curves

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DOAC Conclusions Direct Thrombin Inhibitors (DTI’s) Most commonly prescribed:

 Dabigatran (Praxada) – Half-life = 12-17 hours (may be prolonged in patients with renal insufficiency), Reversal Agent = Idarucizumab (Praxabind)

 Argatroban (Acova) – Half-life = 50 minutes (may be prolonged in patients with hepatic insufficiency), Can be reversed using broad-spectrum reversal agents like Arizapine and activated charcoal

 Levels are measured using the HEMOCLOT Assay, or overdose is detected and levels can be roughly measured using the Dilute Thrombin Time Assay

DOAC Conclusions Factor Xa Inhibitors (-xaban) Most commonly prescribed:

 Rivaroxaban (Xarelto) – Half-life = 5-9 hours (may be prolonged in patients with hepatic insufficiency)

 Apixaban (Eliquis) – Half-life = 9-14 hours (may be prolonged in patients with hepatic/renal insufficiency)

 Edoxaban (Savaysa, Lixiana) and Betrixaban (Bevyxxa) may become more prominently used in the future

 Plasma levels are measured using the Anti-Xa assay with different calibration curves needed for each medication

 Rivaroxaban and Apixaban can be reversed with Andexxa Edoxaban and Beritrxaban need broad-spectrum agents

DOAC Conclusions Abnormalities in Routine Assays  Direct Thrombin Inhibitors (DTI’s) will cause prolongation of PT, aPTT, TCT results, with no affect Fibrinogen results and no reactivity with the Anti-Xa assay giving a negative result  Factor Xa inhibitors will cause prolongation of PT and aPTT results, with no affect on TCT and Fibrinogen results, but positive reactivity with the Anti-Xa assay giving a positive result value ** Interpret ALL coagulation tests with caution for patients on DOAC’s **

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Final Thoughts - Colman, et al. p3 “Thrombosis may occur if the hemostatic stimulus is unregulated, either because the capacity of inhibitory pathways is impaired or, more commonly, because the capacity of the natural anticoagulant mechanism is overwhelmed by the intensity of the stimulus. Thrombosis may be regarded as an accident of nature that has not had time to adapt through the lengthy process of evolution to the advances of modern medicine, which allow patients to survive the hemostatic challenges of major surgery and trauma but leave them vulnerable to .”

Additional References 1. Colman, et al., Hemostasis and Thrombosis, Basic Principles and Clinical Practice, 5th Ed. Lippincott, Williams and Wilkins 2006 ISBN 978-0-7817-4996-1 2. Gulati, G. et al., Case Studies in Hematology and Coagulation. ASCP Press, 2012 ISBN 978-0-89189-585-5 3. McKenzie, S. et al., Textbook of Hematology, 2nd Edition. Williams and Wilkins, 1996 ISBN 0-683-18016-9 4. Monagle, P. et al., Haemostasis Methods and Protocols. Humana Press, 2013 ISBN 978-1-62703-338-1 5. Rodak, B. et al., Hematology: Clinical Principles and Applications, 3rd Edition. Saunders, 2007 ISBN 978-1- 4160-3006-5 6. Rodgers, G and Alday, A., Hemostasis Casebook, Lab Diagnosis and Management. ASCP Press, 2013 ISBN 978-089189-6173

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