Pharmaceutic Principles of Anticoagulant Drugs and Their Action PDF

Pharmaceutic Principles of Anticoagulant Drugs and Their Action Robert J. Kerns, PhD Professor Department of Pharmaceutical Sciences & Experimental Therapeutics August 22, 2023 Readings: Chapter 26 Foye’s Chapter 36 Goodman and Gillman’s (14e edition) 1 Learning Objectives: From these lectures s...

Pharmaceutic Principles of Anticoagulant Drugs and Their Action Robert J. Kerns, PhD Professor Department of Pharmaceutical Sciences & Experimental Therapeutics August 22, 2023 Readings: Chapter 26 Foye’s Chapter 36 Goodman and Gillman’s (14e edition) 1 Learning Objectives: From these lectures students should know and understand: • • • • • • • • • • • • • • The principal players (II, VII, IX, Xa, prothrombin, thrombin) of the clotting factor cascade, including role of platelets, and the difference between Intrinsic and Extrinsic pathways The fundamental processes of coagulation (hemostasis) and anticoagulation in the body as well as the underlying biochemical and physiological processes with an emphasis on those where pharmaceutical agents can act to alter/inhibit coagulation. The mechanism of action for the anticoagulant agents discussed and how the structures, differences in structures and physical properties of the agents (when discussed) impact use. Correlations between which anticoagulants can or cannot be reversed with which reversal agents, as well as the structural and functional reason why. While not discussed for every agent, when discussed in class understand structure-function relationships as they relate to activity, PK/PD, metabolism, etc..) Primary formulations and routes of administration for direct and indirect thrombin inhibitors (heparins, DOACs), and vitamin K blockers (Warfarin) Fundamental PK/PD properties of unfractionated heparin (UFH), low molecular weight heparin (LMWH), warfarin and DOACs, particularly their principal routes of metabolism How generic LMWH are formulated, and the challenges that exist to showing BioEquivalence. Primary factors underlying inter-individual variation (20-fold range) in warfarin efficacy Mechanisms of drug-drug interactions with Warfarin (2C9, 3A4) Dabigatran formulation and unique PK/PD properties Mechanisms of P-gP (MDR1) mediated drug interactions Rivaroxaban PK/PD and major drug interactions 2 Edoxaban and association with renal clearance (CrCl values) I. Blood Coagulation and Hemostasis Blood needs to be fluid in the vasculature and remain fluid in the vasculature while clotting quickly when exposed to subendothelial surfaces (sites of vascular injury). Coagulation of blood (form clot) is in balance with Fibrinolysis (breakdown clot) -Thrombosis: Coagulation (blood clot) in the vasculature adhered to vascular wall -Embolus: Free floating clot -Hemorrhage: Loss of blood from vasculature -activate -aggregate -Hemophilia: often a loss of a functional coagulation factor (replacement therapy) -Thrombi (clotted blood): composed of aggregated platelets, cross-linked fibrin, and trapped red blood cells 3 Types of antithrombotic drugs in clinical use: Anticoagulants: attenuate fibrin clot formation by blocking function of coagulation factors Antiplatelet agents: inhibit platelet activation inhibit platelet aggregation Fibrinolytic agents: breakdown fibrin clots after they have formed -activate -aggregate *Antithrombotic and coagulant drugs have different criteria and/or parameters for clinical use than many other pharmacotherapies to treat diseases because too much pro- or anti- thrombotic effect can be deadly. -Reversal Agents -Monitoring for Dosing Adjustments 4 Blood Coagulation Pathways via “Fibrin Crosslinking” Activation of coagulation factors: -gamma carboxylation of glutamate -Ca++ binding Activated coagulation factors are proteases, that cleave and activate other coagulation factors Endogenous serine protease inhibitors (e.g. antithrombin, heparin cofactor II) are proteases that cleave and inactivate coagulation factors. (Phe-Pro-Arg) Fibrinolysis 5 Protease activation: roles of gamma carboxyglutamate and calcium: Gamma carboxylation of glutamate and subsequent calcium binding is required for an activated coagulation factor (protease) to cleave and activate the next coagulation factor to its active form. gamma carboxylation of glutamate is carried out by a vitamin K dependent carboxylase. Example here is conversion of prothrombin (factor II) to thrombin (factor IIa) by factor Xa cleavage of factor II at the Arg 274 peptide bond. Factor Xa converts prothrombin to thrombin…. Simple enough…. But… In the presence of factor Va, a negatively charged phospholipid surface, and Ca2+ (called the prothrombinase complex), factor Xa activates prothrombin with 109-fold greater efficiency, but only if prothrombin and factor Xa contain gamma carboxyglutamate residues to bind calcium and interact with the anionic phospholipid surface. 6 II. Natural Anticoagulant Molecules/Mechanisms: (example of serine protease inhibitors (serpins) and others) (1) Antithrombin: Plasma protein (serine protease) that inhibits coagulation enzymes (serine proteases) of the extrinsic, intrinsic, and common pathways. Heparan sulfate proteoglycans on vascular surface of endothelial cells have sulfated, polyanionic glycosaminoglycan chains that bind antithrombin and enhance the activity of antithrombin by about 1000fold, with particular effect on increasing rate of cleavage (inactivation) of thrombin and factor Xa. (2) Heparin Cofactor II (HCII) is a serpin similar to antithrombin in that dermatan sulfate and heparin sulfate expressed on surface of the vasculature bind to and activate HCII to enhance inhibition of thrombin. (3) Tissue factor pathway inhibitor is a natural anticoagulant found in the lipoprotein fraction of plasma or bound to endothelial cell surface. TFPI first binds and inhibits factor Xa, and this binary complex then inhibits factor VIIa bound to TF. By this mechanism, factor Xa regulates its own generation. (4) Others… Example of replacement therapy: Thrombate: Anticoagulatn pharmaceutical that is human antithrombin for patients with antithrombin deficiency (sterile human antithrombin (AT) in lyophilized powder form for reconstitution for intravenous injection). 7 . III. ANTICOAGULANT DRUGS 1. Heparin and Heparin Derivatives The function of heparin within the secretory granules of mast cells has been shown to involve the storage of specific granule proteases. Endogenous heparin is not detected in plasma under normal circumstances. However, exogenous heparin, from porcine intestinal mucosa, is used as an anticoagulant. The structure of a representative heparin oligosaccharide is shown below. On average, a heparin chain is composed of around 40 monosaccharide units having an average molecular weight (MW) of 12,000 to 14,000. An important structural feature of heparin is the presence of a specific pentasaccharide sequence found in 30% of heparin chains. This pentasaccharide binds to a serine protease inhibitor (serpin), antithrombin. Many of the coagulation factors including thrombin (IIa), Xa, IXa, XIa, and XIIa are serine proteases and these serine proteases are inhibited by antithrombin. Antithrombin inhibits these serine proteases (coagulation factors) by cleaving an arginine-serine peptide bond and forming a covalent 1:1 inactive complex, thus acting as an anticoagulant. Polydispersity: Variable length of saccharide chain Microheterogene ity: both glucuronic acid and Iduronic acid variable sulfonation of monosaccharides Heparin Structure: - - O 2C - O3S O O -O O 3SN H O OS O3- - O3S O - O3S O OH O OH O O NH O HO O3 S O 2C CO2 - O - OH - O3S O O -O O 3SN H O OS O3- - O O3S O OS O3- OH OH O O NH SO3 O A specific pentasaccharide seque nce within heparin binds to antithrombin III affording m uch of heparins anticoagula nt activity 8 Heparin pentasaccharide binds antithrombin; chain length determines function The significance of the antithrombin-binding pentasaccharide is two fold. First, full-length heparin chains, containing this pentasaccharide, can bind both antithrombin and thrombin. This binding event brings antithrombin and thrombin together, on heparin, affording a 1000 fold increase in the rate at which antithrombin inactivates thrombin. The net result of this is that heparin catalyses/increases the inactivation of thrombin, thus the thrombin mediated conversion of fibrinogen to fibrin is inhibited and heparin acts as an anticoagulant. This is shown pictorially on the next page in panel A. The second significant aspect of this specific pentasaccharide that binds to antithrombin is that when this pentasaccharide binds to antithrombin, antithrombin undergoes a conformational change. This conformational change in antithrombin facilitates the interaction between antithrombin and serine proteases (coagulation factors) directly, thus increasing antithrombin-mediated inhibition of these proteases (particularly important is factor Xa). This is shown pictorially on the next page in panel B. Therefore, long heparin sequences that contain the pentasaccharide are needed to optimally inhibit thrombin while only the unique pentasaccharide part of heparin is required for inhibiting other serine proteases such as Factor Xa. 9 Un-Fractionated Heparin (UFH)chemistry and pharmaceutical efficacy Unfractionated heparin is a linear chain polysaccharide with molecular weight being an average molecular weight of about 14,000. However, shorter and longer chains have different ratios of Anti-Xa vs. Anti-IIa because of the mechanisms of antithrombin binding and coagulant factor binding discussed on previous slides. Length & size of Oligosaccharide chain dictates anticoagulant mechanism and efficacy 10 Low Molecular Weight Heparin (LMWH) LMWH preparations are made by Depolymerization (i.e. Degradation) of UFH Low molecular weight heparin (LMWH) is heparin that has been depolymerized by some method so that most of the polysaccharide chains are shorter, average molecular weight around 5000. The shorter chains can no longer bind both antithrombin and thrombin on one chain and thus do not effectively promote anticoagulation via the inhibition of thrombin. The LMWH still contains the antithrombin binding pentasaccharide sequence and therefore still binds antithrombin and activates the serine protease inhibitor activity. These LMWHs, in which most saccharide chains are not long enough to efficiently catalyze the inhibition of thrombin, produce an anticoagulant effect primarily (preferentially) by activating antithrombin that then acts on (inhibits) factor Xa. Intense studies continue regarding the use of LMWH, and development of new LMWHs, in particular engineering and biotechnology approaches to make synthetic LMWH disconnect heparin-based drugs from a food supply. 11 Low Molecular Weight Heparin (LMWH) Manufacturing Processes Low molecular weight heparins are prepared by a number of chemical or enzymatic methods used to depolymerize heparin. This depolymerization affords fractions of heparin with a reduced average molecular weight (reduced chain length), but a proportion of the chains must retain intact AT-binding pentasaccharide in order to bind and activate antithrombin. Enoxaparin (Lovenox): Benzylation followed by alkaline hydrolysis. Dalteparin (Fragmin): Nitrous acid depolymerization Controlled nitrous acid depolymerization: MW = 4000-6000 -O - O3S O O O 2C 2C - A cN H O O3S O CO2 - OH NH O O O OS O3- O -O SO 3 - O3S O - O3 S HO O O HO OH - O3S O O O 3SN H O - O OS O3- OS O3- OH O O HO O3S O O NH SO3 O HNO2 , pH = 1.5 O O H OHO Reduction of aldehyde -O -O SO 3 O 2C - A cN H O O3S O OH O -O SO 3 OH HO - O 2C O HO OS O3- OH CO2 CH2 O O OS O3- O HO OH - O3S O O -O 3SN H O OS O3- O O - O3S O O HO O NH SO3 O D) Other LMWHs: Ardeparin (Normiflo); Nadroparin (Fraxiparine); Reviparin (Clivarine); Tinzaparin (Innohep). Method of chain cleavage results in different modifications; sequences/sites cleaved, new structures introduced by cleavage chemistryt, and % antithrombin binding pentasaccharides present.12 Commonly prescribed LMWH preparations • Major advantage over UFH is Fixed Dose administration, can be used at home via subcutaneous injection. • Less nonspecific binding to plasma proteins and endothelial cells along with smaller size increases bioavailability after subcutaneous administration. • More predictable anticoagulant response and less interpatient variability. Routine aPTT monitoring usually not necessary except with end-stage renal disease. • Fewer instances of heparin-induced thrombocytopenia (HIT) 13 Consequences of LMWH structural variability • • • • • • • • • • • • • • Structural differences ………….……. May impact PK/PD Molecular size ………..………………. May impact anti-Xa, anti-IIa Charge density …………………………Interaction with cells Binding to ATIII ……………………….. Antithrombotic effects Binding to HCII ………… ……………. Anticoagulant effects Ability to release TFPI ……………….. Inhibition of formed TF Interaction with proteins …………….. Decreased PK/PD Interactions with cells ……………….. Signaling effects (not understood) Ability for glycosylation ……………… Biological amplification Vascular uptake ……………………… Antithrombotic surface Endovascular uptake ……………….. Inhibition of vascular proliferation Modulation of growth factors ………... Anti-cancer, anti-apoptotic, antiangiogenic Molecular effects ……………………..Genotypic expression changes Regulatory effects…………………….Molecular and cellular effects Each LMWH is a different drug if prepared by a different method: Process makes the product 14 UFH vs LMWH Pharmacodynamics Property UFH LMWH M.W. (range in Da) 3000 – 50,000 1000 – 9000 Average M.W. 12,000 – 15,000 4000 – 5000 MoA Inactivation of factor Xa and Thrombin Preferential inactivation of Xa Routes of Admin. I.V., subcutaneous Subcutaneous only Nonspecific Binding Widespread and High Negligible Monitoring (aPTT) Tight monitoring required throughout Only required at start Dosage Adjusted based on aPTT Fixed Dosage Generics available? Yes Yes 15 Synthetic pentasaccharide Fondaparinux (Arixtra) Specific for binding and activating antithrombin Anti-Xa : Anti IIa (thrombin) activity ratio. See also Table 26.3 in text. UF Heparin - 1:1 LMWHs - 2-4:1 Fondaparinux (synthetic pentasaccharide) - Xa only 16 Thrombocytopenia: UFH vs LMWH Full length heparin (UFH) will bind to platelet factor 4 (PF4) in the body. This heparin-PF4 complex can cause a complex immunological reaction (antibodies form) leading to heparin-induced thrombocytopenia (low platelet levels) with or without thrombosis. LMWHs can, but much less frequently, cause heparininduced thrombocytopenia. Synthetic pentasaccharide, fonduparinux, forms even smaller complexes with PF4 and causes fewer events of heparininduced thrombocytopenia. 17 Heparin Overdose and Protamine Sulfate Rescue • Protamine is positively charged at physiological pH, forms ionic bonds with negatively charged polysaccharide groups on Heparin • Neutralization occurs immediately, lasts for 2 hours. • 1 mg protamine inactivates 100 U heparin. Administration by slow IV injection (< 10 mg/min) to avoid hypotension • Protamine is less effective/not effective at inactivating LMWH and does not effectively inhibit fonduparinux. 18 2. Vitamin K Antagonists: Coumarins and Indanediones. (First Oral Anticoagulant, Warfarin) The coumarin anticoagulants (including Warfarin) block the gama-carboxylation of glutamate residues in prothrombin and factors II, VII, IX, X and the endogenous anticoagulant protein C. Gama-carboxyglutamates are important because they bind Ca++ and Ca++ binding is essential for their activity and for coagulation factors interacting with platelet surface (e.g. prothrombin complex). Blockage of glutamate carboxylation results in molecules that are not active in coagulation. The mechanism of action is that the drug blocks the reduction of Vitamin K epoxide to its active hydroquinone form. Biosynthesis of active coagulation factors depends on an adequate supply of this form of Vitamin K. Therefore, Vitamin K antagonists such as warfarin decrease the carboxylation of glutamate and thus decrease the synthesis of the biologically active form of prothrombin and other coagulation factors and increase blood-clotting time. *Note: Citrate binds (sequesters) Ca++ and thus see citrate and other agents that complex Ca++ used to block coagulation in non therapeutic applications 19 Warfarin vs. Heparin Pharmacology Warfarin Heparin 20 Clotting Factors and Turnover Rates • Half-life of all Vitamin K-dependent clotting factors (ie., Warfarin-sensitive) highlighted in yellow above • The long lifetime of Factor Xa and Pro-Thrombin underlie lag-time of Warfarin anticoagulant effect (peak effect observed in ~3 days) 21 Vitamin K-Dependent clotting factor production in Liver Vitamin K-epoxide reductase complex 1 (VKORC1) Pharmacogenomics: polymorphisms in VKORC1 (target reductase enzyme) and CYP2C9 (metabolizes warfarin to inactive metabolite) alter warfarin potency and PK, respectively. 22 Warfarin chemistry, PK/PD 1. A mixture of (R) and (S) enantiomers [(S) is 3-5X more potent than (R) as anticoagulant] 2. High (>20-fold) inter-individual variation in dose requirements -due in large part to underlying genetics, polymorphisms in VKORC1 and CYP2C9 that alter warfarin potency and PK, respectively) 3. Narrow therapeutic index 4. Delayed (~3 days) onset and offset 5. Mandatory INR monitoring of coagulation 23 Warfarin Metabolism 24 Factors underlying inter-individual variation in Warfarin efficacy 25 Summary of Warfarin Interactions Outcome ↑ warfarin effect ↓ warfarin effect Promote Bleeding Mechanism of Interaction Drugs involved Displacement of Warfarin from Albumin Aspirin, other salicylates, sulfonamides Inhibition of warfarin degradation by CYPs (2C9 major, 3A4 and 1A2 minor) Acetaminophen, Amiodarone, Ciemtidine, Azole antifungals, erythromycin ↓ Clotting factor Synthesis Cephalosporins Induction of CYPs (2C9 major, 3A4 and 1A2 minor) Carbamazepine, Rifampin, Phenytoin Clotting factor synthesis Oral contraceptives, Vitamin K1 ↓ warfarin absorption (minor) cholestyramine Additive effects with Warfarin Clopidogrel, Prasugrel, (antiplatelets, other anticoagulants) aspirin, heparin 26 Vitamin K1 for Warfarin Overdose 1. Vitamin K1 (phytonadione) antagonizes action of warfarin 2. Bypasses VKORC1, and serves as alternative substrate for gamma-carboxylation of and clotting factors. 3. High doses (10 mg PO) of Vit K1 can cause prolonged resistance to warfarin Vitamin K (Phytonadione; Vit. K1) is both a reversal agent and a coagulant: -Safe in many populations (kids, pregnancy etc.) -Promotes coagulation by promoting gamma carboxylation of glutamate residues in coagulation factors. -Can be used to combat bleeding due to warfarin and other vitamin K antagonists -Short half-life (3-5 hours when given orally) -Can take up to 24h to see effect. -Green leafy vegetables: 0.05 to 1 mg per cup (from lettuces to kale) 27 3. IV dosed: Direct Inhibitors of Thrombin and Factor Xa Rivaroxaban Apixaban Edoxaban Dabigatran Hirudins Argatroban 28 Direct Inhibitors of Thrombin Hirudin and Hirudin-Like Direct Thrombin Inhibitors: Thrombin cleaves an arginine-glycine peptide bond in fibrinogen to give fibrin monomers. The 1:1 binding of hirudin to thrombin blocks the catalytic site (protease active site) in thrombin that catalyzes the cleavage of fibrinogen to fibrin. Below is a comparison of natural hirudin (found in a type of leech) and a recombinant form used clinically, (Lepirudin, Refludan, a recombinant hirudin generated using yeast cells). - - - - - - linking domai n + Active-site binding/inhibi ting domain (Proline-lysine-proline) + Active Site Binding domain 29 Direct Inhibitor of Thrombin: Bivalirudin Bivalirudin (Angiomax™) is an approved (December 2000) hirudin-like thrombin inhibitor that binds the catalytic site and the anion binding exosite of circulating and clot-bound thrombin. Bivalirudin is a 20 amino acid peptide, much smaller than hirudin, designed based on the peptide sequence of hirudin. Directly inhibits thrombin by specifically binding both to the catalytic site and to the anion-binding exosite of circulating and clot-bound thrombin. Bivalirudin inhibits thrombin in a reversible, competitive, manner and is also slowly cleaved by thrombin, with part of Bivalirudin remaining bound to, and still able to inhibit, thrombin activity. PK/PD: Dosed IV and cleared by renal and proteolytic routes. Renal function impacts half-life (20 mins to 1 hour), coagulation generally back to baseline normal an hour after administration stopped. 30 Direct Inhibitor of Thrombin: Argatroban (Acova) Argatroban (Acova): small molecule direct inhibitor of thrombin (IV dosing). -The Phe-Pro-Arg peptide sequence in fibrinogen is recognized and cleaved by thrombin. -Argatroban is a peptidomimetic of this tripeptide sequence. -Argatroban is a competitive, reversible, inhibitor of the thrombin catalytic site in both free and clot-bound thrombin FYI: Both the hirudins and argatroban directly inhibit thrombin and thus block the conversion of fibrinogen to fibrin. In addition, other thrombin effects thus blocked include blocking the thrombin activation of other coagulation factors, platelet aggregation and protein C. 31 Comparison of IV dosed Direct Inhibitor of Thrombin 32 4. Direct Oral Anticoagulants (DOACs) Dabigatran (Pradaxa®) – Direct thrombin (IIa) inhibitor - FDA approval 2010, oral capsules (very sensitive to moisture), pro-drug, major P-gp DDI Rivaroxaban (Xarelto®) – Factor Xa inhibitor - FDA approval 2011, oral tablets (15/20 mg tablets with large meal), major P-gp & 3A4 DDI Apixaban (Eliquis®) – Factor Xa inhibitor - FDA approval 2012, oral tablets (not necessary to take with meal), major P-gp & 3A4 DDI Edoxaban (Savaysa®) – Factor Xa inhibitor - FDA approval 2015, oral tablets (no need for concomitant food), Major Contraindication for CrCl <15 or >95 mL/min (unique). 33 Dabigatran Etexilate (Pradaxa) An ORALLY ACTIVE direct thrombin inhibitor Dabigatran: -Very polar and very water soluble -Poor oral absorption -Low tissue distribution Dabigatran etexilate: -Double Prodrug -More lipophilic for good oral absorption -Both prodrug groups cleaved by esterases -Capsules for oral administration 34 Dabigatran formulation- Unique Dabigatran etexilate mesylate- very low H2O solubility, only slightly soluble in EtOH Tartaric acid- packed with dabigatran to maintain acidity of capsule core, assists with drug dissolution and absorption Needs to be protected from air & moisture. Cap (shown to the right) has a gas impermeant seal with dessicant, Shelf-life of ~4 months once opened 35 REVERSAL AGENT for Dabigatran: Idarucizumab (Praxbind): - IV dosed monoclonal antibody developed to bind dabigatran and reverse/block the anticoagulant activity of dabigatran. FDA approved in 2015 - does not otherwise cause blood to clot (NOT a coagulant) - average wholesale price for 5 g dose is over $3,000.00 36 Dabigatran PK/PD Esterases in microsomes, plasma & liver PgP interactions! P-gp transporters are efflux transporters that are primarily expressed in the apical/luminal membrane of epithelia of the small intestine, hepatocytes, renal proximal tubules and other sites. 37 P-gP: Why so important with Anticoagulants? 170 kDa transmembrane glycoprotein Drug X PgP ,a.k.a. MDR1, a.k.a. ABC1 transporter; ATP-dependent efflux transporter 38 P-gP cont’d: Common drugs that inhibit P-gP (↑ DOAC effect): Amiodarone, dronedarone, clarithromycin, ciclosporin, colchicine, diltiazem, erythromycin, felodipine, lansoprazole, omeprazole, and other PPIs, nifedipine, paroxetine, sertraline, quinidine, tamoxifen and verapamil. Common drugs that induce P-gP (: ↓ DOAC effect) Rifampin, Carbamazepine, Phenytoin, St. John’s wort 39 ORALLY ACTIVE direct FXa inhibitors Edoxaban Apixaban Rivaroxaban (Xarelto) - Competitively bind and block active site of FXa - Do not inhibit thrombin and therefore no direct effects on platelets (thrombin is a platelet activator) - Very different from LMWHs, yet similar in that primary target is inhibition of FXa Betrixaban - Each agent has its own unique metabolism, PK, toxicity profile, etc… 40 REVERSAL AGENT for direct FXa Inhibitors Andexanet alfa Modified recombinant factor Xa derivative designed to have very high affinity for direct factor Xa inhibitors (e.g. Rivaroxaban (Xarelto)) Acts as a “decoy receptor” to bind and sequester the factor Xa inhibitors so that the inhibitors can no longer bind and inhibit endogenous factor Xa. 41 Rivaroxaban: Unique absorption/PK Note the mandatory food requirement for higher doses Lipophilic structure, Limited aqueous solubility 42 Differential Absorption and Metabolism for DOACs 43