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Immunogenicity of Low Molecular Weight Heparins and Their Biosimilars J.M. Walenga, D. Hoppensteadt, J. Cunanan, C. Adiguzel, O. Iqbal, W.P. Jeske, M. Prechel, and M. Bakhos Cardiovascular Institute, Loyola University Medical Center, Maywood, IL 60153 USA INTRODUCTION Heparin and low molecular weight heparins (LMWHs) used for the treatment of thrombotic and cardiovascular disorders are heterogeneous mixtures of glycosaminoglycans (GAGs). The chemical properties of a LMWH → translate into its biological properties Micro-chemical differences in the heparin molecules affect the biological activities of GAGs. Chemical properties make each LMWH unique → which translates into biological differences between LMWHs INTRODUCTION The chemical entities of LMWHs readily bind to numerous plasma proteins. GAGs bound to proteins can promote immunogenic responses. If compositional variations exist between branded LMWHs, or between an innovator and biosimilar LMWH, then interactions with endogenous proteins such as platelet factor 4 (PF4) would differ. The result would be a differential immunogenic response between drugs. INTRODUCTION Heparin-induced thrombocytopenia (HIT) is the most common of the known immunogenic responses to heparin. + PF4 Heparin Heparin-PF4 Complex INTRODUCTION Although symptomatic HIT infrequently occurs in patients treated with LMWHs, anti-heparin/PF4 antibodies (HIT antibodies) do develop in up to 25% of treated patients. There are reports that HIT antibodies that do not generate a symptomatic HIT response may still play a role in the clinical outcome of patients and also contribute to variations in the heparin therapeutic response. The US Food and Drug Administration has required that comparative data on the immunogenic potential of all biosimilar versions of LMWHs in comparison to the innovator product be provided. STUDY OBJECTIVE Since the molecular profile and composition of branded (and perhaps biosimilar) LMWHs varies, it was hypothesized that the immunogenic potential of each LMWH differs. METHODS - The generation of HIT antibodies and their subtypes were evaluated in blood samples obtained from clinical studies of different LMWHs. - The interaction of branded and biosimilar LMWHs with HIT antibodies was determined in functional platelet activation assays. - The ability for LMWHs to mobilize PF4 and serotonin from platelets, and interact with these substances, was determined. MATERIAL 1. Branded LMWHs included: • enoxaparin (Sanofi-aventis) • dalteparin (Pfizer) • nadroparin (GSK) • reviparin (Abbott) • parnaparin (Alpha Wasserman) • tinzaparin (Leo) 2. Heparin was obtained from Gentium 3. Biosimilar versions of enoxaparin included: • Cutenox • Clenox • Dripanina • Dilutol • Fibrinox • Lupenox 4. Biosimilar version of dalteparin was Daltehep MATERIAL 1. Frozen citrated plasma samples from clinical trial subjects and frozen serum samples from clinically diagnosed HIT patients (and healthy subjects) were used. 2. The GTI ELISA kit (Waukesha, WI) was used to measure the HIT antibodies and their subtypes. 3. The interaction of LMWHs with HIT antibodies was studied using platelet function assays: HIPA aggregation with platelet rich plasma and 14C-Serotonin Release Assay (SRA). 4. PF4 and serotonin released from platelets [whole blood incubated with drug at 37°C for 30 min with stirring] were measured by ELISAs (PF4, Stago, Parsippany, NJ; serotonin, Alpco, Salem, NH). 5. Routine anti-FXa and anti-FIIa chromogenic assays were used on the ACL (Beckman-Coulter); PF4 was from Hyphen BioMed (Neuville-sur-Oise, France). Comparison of the Immunoglobulin Subtypes in Normals, Clinically Diagnosed HIT Patients, and ELISA Positive Non-HIT Patients ELISA IgG, A, M 1.4 IgA IgG 1.2 IgM 1.0 O.D. 0.8 0.6 0.4 0.2 0.0 Normal Sera HIT Patients (n=110) (n=111) ELISA (+) HIT (-) LMWH group (n=110) Prevalence of Anti-Heparin/PF4 Antibodies in Various Patient Groups Treated with Different LMWHs LMWH (Clinical Trial) Dosage Patient Group Prevalence Reviparin (ECHOS) 4,200 AXa U o.d. Neurologic patients 12% Enoxaparin (ONCENOX) 1 mg/kg o.d. Cancer patients 15% Certoparin (PARAT) 1 mg/kg o.d. Acute coronary syndrome 18% Heparin 7,500 U b.i.d. DVT 24% Antibody Sub-typing of HIT Antibody Positive Samples from Various Branded LMWH Treated Groups IgA 1.0 IgG IgM 0.8 O.D. 0.6 0.4 0.2 0.0 Reviparin Enoxaparin 4200 AXa U O.D. 1 mg/kg O.D. Certoparin 1 mg/kg O.D. Heparin 7500 AXa U B.I.D. 1. 2. 3. 4. Saline Dalteparin Enoxaparin Nadroparin 5. Reviparin 6. Parnaparin 7. Tinzaparin 8. Heparin In Vitro Cross-Reactivity with HIT Antibodies in the Platelet Aggregation Assay 50 25 0 1 2 3 4 5 6 7 8 Branded LMWHs (1 μg/mL) 75 % Aggregation % Aggregation 75 1. 2. 3. 4. Saline Clenox Cutenox Dripanina 5. Dilutol 6. Lupenox 7. Daltehep 50 25 0 1 2 3 4 5 6 Biosimilar Versions of Enoxaparin and Dalteparin (1 μg/mL) 7 In Vitro Cross-Reactivity of Biosimilar Versions of Enoxaparin with HIT Antibodies in the SRA 100 Enoxaparin % Serotonin R elease 80 Dilutol Fibrinox 60 40 20 0 0 0.01 0.1 1 -20 Concentration (ug/mL) 10 100 Serotonin Release (ng/mL) Differential Serotonin Release in Whole Blood / Platelet Activation Heparin 70 60 50 40 30 20 10 0 Enoxaparin Tinzaparin Reviparin Parnaparin Pentasaccharide Danaparoid All agents were tested at 10 ug/mL. Saline PF4 Release (ng/mL) Differential PF4 Release in Whole Blood / Platelet Activation Heparin 120 100 80 60 40 20 0 Enoxaparin Tinzaparin Reviparin Parnaparin Pentasaccharide All agents were tested at 10 ug/mL. Danaparoid Saline PF4 Neutralization of the Anti-FXa Activity % Neutralization 100 Heparin 80 Enoxaparin 60 Tinzaparin 40 Reviparin Parnaparin 20 Pentasaccharide 0 Danaparoid All agents were tested at 5 ug/mL. PF4 Neutralization of the Anti-FIIa Activity % Neutralization 100 Heparin 80 Enoxaparin 60 Tinzaparin 40 Reviparin Parnaparin 20 Danaparoid 0 All agents were tested at 5 ug/mL. SUMMARY 1. The prevalence of HIT antibodies varied from 12-18% in patients treated with different branded LMWHs. 2. The antibody sub-typing showed a higher proportion of IgG antibodies in patients with symptomatic HIT. 3. The proportion of IgG to IgA and IgM HIT antibodies generated in LMWH treated patients differed between the branded products. Certoparin produced a higher proportion of IgG, whereas enoxaparin and reviparin produced a lower proportion. 4. In the in vitro platelet aggregation HIT assay, each of the branded innovator LMWHs produced a response to HIT antibodies, but the response differed between LMWHs in terms of onset of platelet aggregation, slope, and time to achieve maximum aggregation. SUMMARY 5. In the platelet aggregation HIT assay and SRA, each biosimilar version of enoxaparin showed a response to HIT antibodies, but differences were observed between biosimilars, as well as in comparison to the branded product. 6. In the platelet aggregation HIT assay, the biosimilar version of dalteparin showed a response to HIT antibodies that was similar to the response of the branded product. 7. Differences were found in the ability of each LMWH to release PF4 and serotonin from platelets. 8. Differences were found in the ability of each LMWH to interact with PF4 as shown by the neutralization of both the anti-FXa and antithrombin activities of the LMWH. CONCLUSIONS • The branded LMWHs exhibit differential capacities to generate HIT antibodies. • Differences in the interaction with HIT antibodies are evident for each of the branded LMWHs and also for the various enoxaparin biosimilars. • Differences are evident in the interaction of each LMWH with platelets and PF4. • These studies suggest that compositional differences of each LMWH (branded or biosimilar) are determinants of the immunogenic effects of these drug. • Impurities and contaminants that adulterate these drugs can add an additional cause of immunogenicity. PROPOSED RECOMMENDATIONS • Because each LMWH is characterized by its own immunogenic capacity, the immunogenic profile should be used to define the bioequivalence of LMWHs; sub-typing of antibodies generated would provide useful information. • In vitro cross-reactivity studies alone would be insufficient to characterize the immunogenic profile of a LMWH due to the complex and polycomponent nature of these drugs. • Well-designed, population balanced, clinical trials that include patients particularly at risk for developing HIT (critically ill, septic, multiple pathologies) are desirable.