It must be recognized that biological macromolecules can express MULTIPLE functions, thus defining the process or more specifically chemical reaction is NECESSARY to avoid unnecessary ambiguity in both discussion and in measurement of function.
Structures are shown because they provide the basis from which measurement methods can be developed, and functional properties related to structure of the heparins.
Pharmacopoeial definition of LMWH: Avg. MW <8,000 Da Anti-Xa potency >+70 IU/mg aXa/aIIa ratio of at least 1/5
Enoxaparin (Lovenox, Sanofi-Aventis, Paris, France) Dalteparin (Pfizer, New York, USA) Cutenox (Gland Pharma, Hyderabad, India) Dripanina (Ariston, Sao Paulo, Brazil) Dilutol (Lazar, Buenos Aires, Argentina) Clenox (Pharmayect, Barranquilla, Colombia) Lupenox (Lupin, Mumbai, India) Daltehep (Gland Pharma, Hyderabad, India)
Overall, anti-Xa activity alone is not sufficient to predict the safety and efficacy profile of a LMWH Anti-Xa activity is mediated by the AT-III binding The proportion of octasaccharides and their AT-III binding sites is process dependent AT-III affinity as well as the half-life of the octasaccharides may strongly differ although overall anti-Xa activity might be similar Enoxaparin: Shows characteristic structural fingerprints as well as specific ATIII-binding sequences A complete characterization of about 40 different ATIII-binding sites has been performed (hexa, octa, and decasaccharides).
The 1,6-anhydro ring structure is involved in: Anticoagulant activity of oligosaccharides fractions Inflammation Smooth muscle cell proliferation Angiogenesis Pharmacokinetics Safety profile
Low-molecular-weight heparins (LMWHs) have significantly different pharmacokinetic patterns when their respective anti-Xa activities and plasma half-lives are compared. These data compare the ex vivo potency in healthy subjects of LMWHs administered subcutaneously at doses recommended for prophylaxis of deep-vein thrombosis. • Collignon et al. Thromb Haemost 1995;73(4):2-12 When normalized to the same injection dose (1,000 IU anti-Xa), the plasma anti-Xa activity generated by: enoxaparin is 1.48 times greater (p < 0.001) than nadroparin and 2.28 times greater (p < 0.001) than dalteparin nadroparin is 1.54 times greater (p < 0.001) than that of dalteparin. • Eriksson et al. Thromb Haemost 1995;73(3):398-401 Comparison between dalteparin (5,000 IU anti-Xa), enoxaparin (4,000 IU anti-Xa), and tinzaparin (50 IU anti-Xa), n = 12 The anti-Xa half-life of enoxaparin (4.28 ± 1.06 h) was significantly longer compared with dalteparin (2.31 ± 0.6 h, p < 0.05) or tinzaparin (2.97 ± 1.01 h, p < 0.05). “ Thus, enoxaparin, nadroparin and dalteparin clearly differ from each other and produce pharmacodynamic effects on the coagulation cascade which vary differently with time.” 1 Reference: 1. Collignon F, et al. Thromb Haemost 1995;73:2-12
Combination therapy for cerebrovascular and cardiovascular indications. Results from LMWH clinical trials confirm that LMWHs are not interchangeable. “ Properties associated with one LMWH cannot be extrapolated to a different LMWH. For this reason, the findings of clinical trials apply only to the particular LMWH evaluated and should not be generalized to the LMWHs at large” - Ref. Hyers, Chest 1998
Heparin and Low Molecular Weight Heparins Safety of Heparin Biosimilars Jeanine M Walenga, PhD & Craig M Jackson, PhD Presentation developed on behalf of the
SUBTOPICS: Chemical Background – Context for sections to follow Mechanism of heparin anticoagulant activity (in vitro ) Heparins and Low Molecular Weight Heparins (Products) Heparin Actions in vivo – Animal Models and Patients Heparin Adulteration – Unrecognized “Side Effects” Unfractionated heparins Low molecular weight heparins Safety Concerns – Low Molecular Weight Heparin Biosimilars
BLOOD COAGULATION SYSTEM CONTEXT FOR HEPARIN ANTICOAGULANT ACTION Contact Activation ( in vitro ) TFPI VII Va TFPI VIIIi Fibrin Gel Fibrinogen PS APC & Xa IIa TF TF IIa Xa PS & START – Extrinsic Pathway PC TM APC IXa TF VIIa X V i IIa vWF VIII IIa IIa XI XIa V II IX IX TF VIIa GAG IIa VIIa Inh Xa Inh IIa Inh Xa -2 M IIa -2 M START – Intrinsic Pathway VIIIa APC Inh APC Inh
SOURCES OF HEPARIN: Animal Sources: Pigs , Cattle, Sheep Tissue Sources: Intestinal mucosa , lung Geographic Origins: China, North America, South America SOURCES OF LOW MOLECULAR WEIGHT HEPARINS: Heparins listed above – processed by chemical or enzymatic degradation to provide lower molecular weight forms of heparins Low molecular weight heparins are produced by several different processes that generate chemically distinct products. HEPARIN SALTS: Na + , Ca 2+ (Outside USA), Li + (diagnostic test use)
Heparin, Chemical Background (Unfractionated heparin as isolated and purified) Glycosaminoglycan – polysaccharide polymer consisting of two types of monosaccharide: a uronic acid residue and a glucosamine residue that together provide the disaccharide building blocks for the heparin polymer Unfractionated heparin is heterogeneous - molecules of different molecular weights are present – one type of heterogeneity in heparin GENERAL PROPERTIES OF UNFRACTIONATED HEPARIN Linear polysaccharide – MW 5,000 – 50,000, MW 8,000 – 18,000 Repeating disaccharide – 1,4-linked glucosamine & uronic acid Sulfated polysaccharide – 2.2 – 2.8 sulfo groups per disaccharide Predominant uronic acid – iduronic acid (75-99%), > 99% not 2-O sulfated Glucosamine – N-sulfated (80-99%), N-acetylated (1-20%) Glucosamine – 6-O sulfated (>75%); 3-O sulfated (< 10%)
CHEMICAL STRUCTURE OF HEPARIN Disaccharide unit of heparin consists of one glucuronic or one iduronic acid and one glucosamine residue. These residues are substituted with sulfate or acetyl groups. … a disaccharide unit Sulfur (sulfate with oxygens ) Nitrogen (amino, substituted with acetyl or sulfonyl) Carbon (uronic acid or glucosamine backbone residues) Oxygen (carboxylate on uronic acids, OH on monosaccharide rings)
Synthetic pentasaccharide Functionally important central glucosamine residue 3-O sulfate: High affinity pentasaccharide is present in 20-60% of the molecules High Affinity Heparin - Binding to Antithrombin A KEY SPECIFIC HEPARIN STRUCTURAL FEATURE Presence or absence of the high affinity pentasaccharide is a second type of heterogeneity characteristic of heparin O CO 2 CO 2 O O O O O OH OH OH O HNSO 3 OSO 3 O OH HNSO 3 O OH HNAc OSO3 CH 2 OSO 3 CH 2 OSO 3 CH 2 OSO 3 O OH
Heterogeneity of Heparin (Variable locations of the high-affinity pentasaccharide ) Location of the high affinity pentasaccharide in the heparin chains is a third type of heterogeneity characteristic of heparin
HEPARIN ANTICOAGULANT ACTIVITY GENERAL MECHANISM ( in vitro ) – Catalysis of the inactivation of coagulation proteases by the inhibitors: ANTITHROMBIN , heparin cofactor II and Protein C inhibitor CATALYST – INCREASES the rate (speed) of a chemical reaction WITHOUT being consumed – one heparin molecule can increase the rate of inactivation of many protease molecules by an equal number of inhibitor molecules. After each reaction, the heparin molecule is free to participate in another inactivation reaction. The rate increases caused by heparin catalytic action are EXTREMELY large – as much as one MILLION times! (Time equivalents of one MILLION time increase – one minute is required for the same extent of reaction that would otherwise occur in TWO (2) YEARS !) Actually the effect is much less – one MILLION is for the highest mol wt heparin and each heparin molecule contains the high affinity pentasaccharide. WHY – heterogeneity and binding of heparin in blood to many plasma proteins NOT ONLY ANTITHROMBIN
CATALYSIS OF PROTEASE (FACTOR Xa) INACTIVATION BY ANTITHROMBIN – PENTASACCHARIDE This is the in vitro BIOLOGICAL ACTIVITY of high affinity heparin INREASE IN RATE – 200 times faster than in the absence of heparin Molecular weight dependence is relatively small – ANTI Xa ACTIVITY Pentasaccharide – actual shape when bound to antithrombin XaAT + Xa AT- H Xa…AT H H +
IIa AT- H IIaAT + + IIa … AT H CATALYSIS OF PROTEINASE THROMBIN INACTIVATION BY ANTITHROMBIN – HIGH MOL WT HEPARIN This is the in vitro BIOLOGICAL ACTIVITY of high affinity heparin INREASE IN RATE – 20,000 times faster than in the absence of heparin Molecular weight dependence is LARGE – ANTI IIa (anti-THROMBIN) ACTIVITY
RELATIVE INCREASES IN RATE RELATIVE TO ANTITHROMBIN Inactivation Rate Constants, k 2 /K d = Value (M 1 s 1 ) Data are for pH 7.4, ionic strength ~ 0.15 Temperature ~ 25 o C They are a composite of several investigators published results Average number of monosaccharide residues; pentasaccharide = 5 Heparin Mol Wt 1768( 5 ) 6000 - 7000 8000( 26 ) 15000 21000( 70 ) No Heparin Xa 6.2 x 10 5 1.2 x 10 6 1.8 x 10 6 9 x 10 6 3.4 x 10 3 IIa 1.4 x 10 4 1.4 x 10 7 2.3 x 10 7 1.8 x 10 8 6.9 x 10 3 ~ 3X ~ 10,000 X ~ 200X ~ 2X
OTHER TARGET PROTEASES Factor IXa, Factor VIIa – antithrombin, high affinity heparin Thrombin – Heparin Cofactor II, Protein C Inhibitor – NO requirement for the high affinity pentasaccharide sequence , efficiency depends on glycosaminoglycan charge density Activated Protein C – Protein C Inhibitor NO requirement for the high affinity pentasaccharide sequence Protease inactivation in plasma where all inhibitors are present is thus the sum of the inactivation by each inhibitor and catalysis by the all the glycosaminoglycans added in a “heparin” or heparin “biosimilar” product. Measurements are complicated by protease trapping by the non-serpin inhibitor, -2 macroglobulin.
DIFFERENCES AMONG PROPERTIES LOW MOLECULAR WEIGHT HEPARINS Chemical entity differences at the reducing end of the oligosaccharide chains of the cleaved heparin chains is a fourth type of heterogeneity characteristic of low molecular weight heparins. Molecular weight differences : average mol wts, mol wt distribution of molecules in the different heparin preparations High affinity heparin content: percentage of molecules that contain the high Affinity pentasaccharide sequence, locations of the pentasaccharide in the heparin chains Bioavailability : fraction of administered heparin measureable in plasma Chain end chemical entities : O O S O 3 N a O H N H S O 3 N a O H O O S O 3 N a O H N H S O 3 N a O H O O S O 3 N a O H C H 2 O H O O 3 N a O O S O 3 N a O H C H 2 O H O O 3 N a O O O H NHSO 3 Na O 1,6-anhydro ring Tinzaparin Enoxaaparin Dalteparin
Mulloy, et al. (1997) Thromb Haemost 77: 668-674 MOL WT DISTRIBUTIONS VARIOUS Low Mol Wt HEPARINS (Smaller molecules have longer retention times) UFH Enoxaparin Fragmin
MEASUREMENT OF HEPARIN ACTIVITIES ANTI-Xa AND ANTI-IIa (thrombin) activities Procedures measure the loss in Xa activity (anti-Xa) or IIa activity (anti-IIa) when the individual protease is incubated with antithrombin and the various heparins or low molecular weight heparins – “pure components” Reference materials and either a conventional bioassay methods are used to calibrate the measurement of the activity assays. Potency – activity per unit mass (units/mg) is established by these activity measurements. Units and methods are established by either the USP or EP Pharmacopeias. The USP method for potency assignment used prior to 2009 was based on an assay that used sheep plasma and was subject to complications caused by interactions of heparin in plasma with proteins other than antithrombin
Recent Advances in Harmonization Heparin Activity Measurements – Unfractionated Heparins United States Pharmacopeia Monographs for heparin and heparin related products have been revised and better, more specific tests and criteria adopted. Both chemical and biological (activity) specifications are now included. European Pharmacopeia is similarly revising specifications for heparin and heparin derivatives The heparin potency unit of the USP and the International unit will be the same in the future and new reference materials have been jointly assigned the values for the activity of these materials. Assay precision for both anti IIa and anti Xa activities are improved and for several reasons the methods employed for assigning potency are more similar than ever before
BLOOD COAGULATION SYSTEM In vitro HEPARIN ASSAY USING THE aPTT, Xa and IIa TFPI VII Va TFPI Fibrin Gel Fibrinogen PS APC & Xa IIa TF TF IIa Xa PS & START – Extrinsic Pathway PC TM APC IXa TF VIIa X V i IIa vWF VIII IIa IIa XI XIa V II IX IX IIa Xa Inh VIIIa VIIIi Contact Activation START – in vitro Intrinsic Pathway TF VIIa VIIa Inh IIa Inh Xa -2 M IIa -2 M APC Inh APC Inh K XIIa HMWK Ka XII XI XIa
OTHER ACTIONS AND INTERACTIONS OF HEPARINS Reactions that occur only in vivo Bind to plasma proteins NOT related to coagulation Affinity is heparin molecular weight dependent Binding may depend on the presence of Ca 2+ (histidine-rich glycoprotein) Bind to and activate lipoprotein lipase (lipemic plasma clearing) Promote release of tissue factor pathway inhibitor (TFPI) Bind platelet factor 4 (PF4), heparin-PF4 complex is immunogenic and can lead to heparin-induced thrombocytopenia (HIT) Binding is heparin molecular weight dependent Enhance inactivation of thrombin by heparin cofactor II Ability is heparin molecular weight dependent, dermatan sulfate is ACTIVE Anti-inflammatory effects – down-regulation of cytokine production Facilitates release of proteins from endothelial cells (tPA, PAI, vWF, TFPI) Can reduce smooth muscle cell proliferation (Enoxaparin)
VARIATIONS IN THE ANTITHROMBIN AND HCII-BINDING PROPERTIES OF LOW MOLECULAR WEIGHT HEPARINS Young E, et al. Thromb Haemost. 1994;71:300-4. Linhardt RJ, et al. J Med Chem. 1990;33:1639-45. Dalteparin IC 50 = half maximal inhibitory concentration . Enoxaparin Heparin Ardeparin Tinzaparin Dalteparin High-affinity material (%) 0 10 20 30 40 50 HCII-affinity material (charge density & mol wt ) Nadroparin Enoxaparin IC 50 (µg/mL) 0 10 20 30 40 50 60 Parnaparin Ardeparin Tinzaparin High AT-affinity material (specific pentasaccharide sequence)
REDUCTION IN SMOOTH MUSCLE CELL (SMC) PROLIFERATION
Only enoxaparin contains a unique 1,6-anhydro ring at the cleavage point in 15–25% of its chains
The 1,6-anhydro structure showed a dose-dependent reduction in SMC proliferation
Courtesy of SANOFI-AVENTIS.
ID 50 (mg/kg) Lower ID 50 = more effective LMWH < 7% 1,6-anhydro Enoxaparin 15–25% 1,6-anhydro LMWH 40– 5 0% 1,6-anhydro ID 50 = half maximal inhibitory dose. Endpoint = lipopolysaccharide-induced nitrite/nitrate (NO) production Courtesy of SANOFI-AVENTIS. ANTI-INFLAMMATORY EFFECTS ASSOCIATED WITH THE 1,6 ANHYDRO RING STRUCTURE IN ENOXAPARIN 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
PHARMACOLOGICAL DIFFERENCES BETWEEN LOW MOL WT HEPARINS 1 Collignon F, et al. Thromb Haemost. 1995;73:630-40. 2 Eriksson BI, et al. Thromb Haemost. 1995;73:398-401. At approved doses for prevention of DVT, enoxaparin has a greater and more prolonged inhibitory effect on anti-Xa than other LMWHs NS = not significant. 1.0 2.0 3.0 4.0 0 5.0 Half-life (hours) NS p < 0.05 p < 0.05 Area under the curve (1,000 IU anti-Xa/mL) 0 0.4 0.8 1.2 1.6 p < 0.001 p < 0.001 p < 0.001 Collignon study 1 Eriksson study 2 Dalteparin 5,000 IU anti-Xa Nadroparin 3,075 IU anti-Xa Tinzaparin 50 IU anti-Xa/kg Enoxaparin 40 mg (4,000 IU anti-Xa)
CLINICAL DIFFERENCES BETWEEN VARIOUS LOW MOLECULAR WEIGHT HEPARINS
LMWHs are cleared primarily via renal excretion
renal insufficiency increases the half-life of the LMWH and thus the risk of bleeding complications
There is no single creatinine clearance that correlates with an increased risk of bleeding for all LMWHs
enoxaparin and nadroparin: LMWH clearance linearly related to creatinine clearance
tinzaparin: LMWH clearance not linearly related to CrCl
In patients with renal failure the amount of plasma anti-Xa activity, total body clearance, and the pattern of anti-Xa fragments recovered in urine differ between the LMWHs
Hirsh J, Raschke R. Chest. 2004;126(3 Suppl):188S-203S.
THROMBOPROPHYLACTIC DRUGS IN RENAL INSUFFICIENCY Severe renal impairment (CrCl < 30 mL/min) Moderate renal impairment (CrCl 30–50 mL/min) and Mild renal impairment (CrCl 50–80 mL/min) Should be used with caution in moderate renal impairment No information No information No dose adjustment necessary Fondaparinux 4 Tinzaparin 3 Dalteparin 2 Enoxaparin 1 “ Contraindicated” “ Should be dosed with caution” No dosage information “ Should be used with caution” No dosage information “ Adjustment of dose is recommended” and dosages are specified 1 Clexane ® –Lovenox ® prescribing information, Sanofi-Aventis. 2 Fragmin ® prescribing information, Pfizer. 3 Innohep ® prescribing information, Pharmion. 4 Arixtra ® prescribing information, GlaxoSmithKline. Prescribing information may differ between countries. CL – creatinine clearance
CLINICAL DIFFERENTIATION OF LMWHS * Acute coronary syndrome refers to unstable angina and non-ST-segment elevation myocardial infarction. GP = glycoprotein. Therapeutic use Clinical findings DVT prophylaxis Optimized dosages vary widely in terms of both gravimetric and anti-Xa IU dosages DVT treatment Optimized dosages and dosing schedules are product specific Acute coronary syndrome * Individual products do not provide similar outcomes; only one product proven superior to UFH Percutaneous coronary intervention Individual products produce varying degrees of anticoagulation Thrombotic stroke Marked variations in clinical outcomes in terms of efficacy and bleeding Combination therapy Interactions with GP IIb/IIIa inhibitors and thrombolytics are influenced by the type of LMWH
CLINICAL ADVANTAGES LOW MOLECULAR WEIGHT HEPARINS Greater bioavailability - ~ 100% versus ~30% for heparin Longer duration of action – slower clearance (but can be a problem in renal dysfunction) Less bleeding Reduced risk of heparin-induced thrombocytopenia Reduced risk of osteoporosis?
CLINICAL DISADVANTAGES LOW MOLECULAR WEIGHT HEPARINS No generally useful neutralizing agent – in contrast to protamine for neutralization of unfractionated heparin NOT all low molecular weight heparins are the same and thus an opportunity for clinician confusion exists RENAL CLEARANCE – may be contraindicated in patients with renal dysfunction
ADULTERATION OF HEPARIN In 2008 epidemiological data suggested that an unusually high frequency of adverse events and deaths were occurring in patients receiving heparin, particularly patients undergoing renal dialysis beginning in 2007. Laboratory investigations of the suspect lots of heparin indicated the presence of a heparin – like, but non heparin contaminant. The contaminant was subsequently identified a chemically over-sulfated chondroitin sulfate. Although chondroitin sulfate is a glycosaminoglycan, it differs from heparin in that galactosamine rather than glucosamine is present in the disaccharide “building block”. Over-sulfated chondroitin sulfate causes severe hypotension as the result of it’s ability to promote the cleavage of bradykinin from the kininogen present in the circulating blood. The adulterated heparin was not detected by the USP potency assay in use prior to late 2008.
BLOOD COAGULATION SYSTEM EFFECT OF OVER-SULFATED CHONDROITIN SULFATE Contact Activation BRADYKININ Kk XIIa HMWK Ka XII XI XIa Hypotension is caused by the generation of bradykinin Contact activation surface Silica Ellagic acid dispersion Sulfatide dispersion Over-sulfated chondroitin sulfate (2-dimensional surface) START – Intrinsic Pathway
WHY? Other glycosaminoglycans in production Danaparoid (mixture of heparan sulfate, dermatan sulfate and chondroitin sulfate ) Algal and marine plant glycosaminoglycans have been investigated and shown to possess anticoagulant activity Most glycosaminoglycans are LESS expensive than heparin Quality CANNOT be based on post manufacturing testing ALONE CONCERNS - ADULTERATION THAT MAY BE DIFFICULT TO DETECT
LOW MOLECULAR WEIGHT HEPARIN BIOSIMILARS CHALLENGES FOR INTERCHANGEABILITY
Heterogeneity of the starting material (opportunity for adulteration)
Process control may be difficult to achieve
In vitro activity assays ONLY measure one or a few properties
Inadequate criteria currently exist for defining interchangeability
Biological activity (how many different activities)