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Prepublished online as a Blood First Edition Paper on October 17, 2002; DOI 10.1182/blood-2002-04-1055.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Institute for Immunology and Transfusion
Medicine, Ernst-Moritz-Arndt-University Greifswald, Greifswald,
Germany; the Institute for Experimental Haematology and
Transfusion Medicine, University Bonn, Bonn, Germany; and the
Department of Orthopedic Surgery, Sahlgrenska University
Hospital/ÖSTRA, Göteborg University,
Sweden.
Recombinant hirudin has been found to be immunogenic in patients
treated with lepirudin following heparin-induced
thrombocytopenia (HIT). We assessed the incidence of
immunoglobulin G (IgG) antihirudin antibodies by
enzyme-linked immunosorbent assay in 112 patients enrolled in a
dose-finding study with desirudin. Patients received desirudin
subcutaneously following orthopedic hip surgery at 10 mg twice
a day (n = 17), 15 mg twice a day (n = 75), and 20 mg twice a day
(n = 20). Of 112 patients, 11 (9.8%) developed antihirudin antibodies independently of the dose. The rate of immunization did not
differ from that observed in HIT patients treated with lepirudin
(P = .113). Plasma concentrations of desirudin did not differ between antihirudin antibody-positive and -negative patients. Antihirudin antibodies had no impact on incidences of deep vein thrombosis and/or pulmonary embolism, allergic reactions, and hemorrhage. However, the total number of immunized patients observed was low and so infrequent (but severe) effects of antihirudin antibodies cannot be excluded.
(Blood. 2003;101:2617-2619) There are 2 recombinant hirudins approved
in the European Union: desirudin (Revasc; Aventis, Frankfurt, Germany)
and lepirudin (Refludan; Schering, Berlin, Germany). Desirudin
is approved for thrombosis prophylaxis following orthopedic
hip-replacement surgery and lepirudin for anticoagulation in patients
with heparin-induced thrombocytopenia (HIT) and thromboembolic
complications. Both compounds differ from each other in their
N-termini: desirudin possesses a valine-valine and lepirudin a
leucine-threonine structure. Otherwise the desirudin and
lepirudin are very similar, with molecular weights of 6.96 kDa and 6.98 kDa, respectively.
Rhesus monkeys did not show a specific immune response following
intravenous boluses of desirudin.1 In contrast, balb/c mice showed a 100% antihirudin antibody response after immunization by
albumin-conjugated hirudin or hirudin fragments.2,3 Also, dogs generated antihirudin antibodies following immunization with desirudin.4
In healthy volunteers, a few allergic reactions were observed following
intravenous as well as subcutaneous applications of desirudin.5 Allergic reactions occurred in 3 volunteers,
in another 4 the skin test became positive. In one volunteer
antihirudin antibodies were detected.
In none of the major clinical studies of patients with acute
coronary syndromes was an immune reaction toward 1 of the 2 hirudins reported.6-10 However, these patients were usually treated
for 2 to 3 days only, which is too short of a period for a B-cell response.
In 1996, we reported that a high proportion of patients receiving
lepirudin for HIT developed antihirudin antibodies,11,12 a
finding confirmed by others.13,14 During a mean duration of treatment of 14.4 days, 44% of 198 patients treated with lepirudin developed antihirudin antibodies of the immunoglobulin G (IgG) class, which occurred as early as day 4 (peak at days 8-9). Of 87 patients with detectable antihirudin antibodies, 61 (70.1%) developed
during treatment, 23 (26.4%) occurred after cessation of lepirudin,
and 3 (3.5%) became positive during a second treatment course with
lepirudin only.12
In another study with lepirudin given to patients with HIT (up to
26 days), up to 74% of patients developed antihirudin antibodies of
the IgG, IgA, and/or IgM class.13,14 None of these
patients developed allergic reactions.
Desirudin is approved for thrombosis prophylaxis following orthopedic
hip-replacement surgery based on 3 prospective
trials.15-17 These patients usually require treatment for
7 to 10 days, which is within the time frame for induction of
antihirudin antibodies. The purpose of the present study was to assess
whether desirudin given subcutaneously in prophylactic dose in patients
following orthopedic hip-replacement surgery is immunogenic, and to
compare the outcomes of patients testing positive and negative for
antihirudin antibodies.
Patients
Hirudins
Laboratory assays Antihirudin antibody enzyme-linked immunosorbent assay (ELISA) was performed as described.12 Microtiter wells (Covalink, Nunc, Germany) coated with desirudin or lepirudin were incubated with patient plasma (1:50 for 60 minutes at 37°C), and IgG binding assessed by anti-human IgG-peroxidase labeled antibodies. Sera of HIT patients with known antihirudin antibodies served as positive controls, plasma of healthy blood donors served as negative controls. The assay was performed by personnel blinded for the dose of desirudin given to the patients.Concentrations of free desirudin in patient plasma samples were assessed by a chromogenic assay using the thrombin substrate S2238, as described previously.18 Incidences of deep vein thrombosis and pulmonary embolism, allergic reactions, and hemorrhages were compared by Fisher exact test (2-tailed). Occurrence of antihirudin antibodies over time was calculated by Kaplan-Meier statistics. Plasma concentrations of antihirudin antibody-positive and -negative patients were compared by Wilcoxon rank-sum test.
Samples from 112 patients were studied of 20 patients who received a dosage of 20 mg desirudin subcutaneously twice a day, of 75 patients who received a dosage of 15 mg subcutaneously twice a day, and of 17 patients who received a dosage of 10 mg subcutaneously twice a day. In 11 (9.8%) of 112 patients, antihirudin antibodies were
detectable. Results were identical whether desirudin or lepirudin was
used in the ELISA, indicating a 100% cross-reactivity. Seroconversion rates were similar among the 3 dosing groups: 2 (10%) of
20 patients received 20 mg desirudin twice a day, 6 (8.0%) of 75 patients received 15 mg twice a day, and 3 (17.6%) of 17 patients
received 10 mg twice a day. Compared with the occurrence of antihirudin antibodies in HIT patients treated with lepirudin, no differences were
observed (P = .113, log-rank test, Figure
1).
Desirudin plasma concentrations of antihirudin antibody-positive patients did not differ from those of antihirudin antibody-negative patients within the 3 dosing groups: (1) 10 mg, 0.1 µg/mL versus 0.094 µg/mL, respectively (P = 1.0); (2) 15 mg, 0.17 µg/mL versus 0.28 µg/mL, respectively (P = .33) and (3) 20 mg, 0.1 µg/mL versus 0.17 µg/mL, respectively (P = .63). Clinical end points could be evaluated in 109 patients. Outcomes of antihirudin antibody-positive patients did not differ compared with antihirudin antibody-negative patients: deep vein thrombosis and/or pulmonary embolism, 2 (18.2%) of 11 vs 11 (10.9%) of 101, P = .61; allergic reactions, 0 of 11 vs 1 of 101, P = 1.0; hemorrhage, 1 (9.1%) of 11 vs 6 (5.9%) of 101, P = .53.
The present study shows that subcutaneous desirudin is immunogenic in patients after major orthopedic surgery. At day 8 of treatment, approximately 10% of patients are immunized. Numerically this rate is much lower than the rate observed in patients with HIT receiving lepirudin.12-14 However, using Kaplan-Meier calculation, the risk of immunization per time exposed seems to be identical in both patient populations (Figure 1). Thus the total rate of immunization depends on treatment duration and time point of blood sampling. The 2 hirudins differ in the structure of the N-terminal part. However, antibodies of patients treated with desirudin showed a 100% cross-reactivity to lepirudin and vice versa, and therefore must recognize structures common to both hirudins with the same immunogenicity. Also, different routes of application (intravenous versus subcutaneous) are of no relevance for immunization; in the present study, patients received hirudin only subcutaneously, whereas lepirudin was given only intravenously in HIT patients. As the dose was higher in lepirudin-treated patients (approximately 250 mg per day in a 70-kg patient) compared with the present study, immunization is not dose related, at least if 10 mg twice a day is given. The result is surprising as the 2 patient groups are very different. We suspected HIT patients to be more likely to form antihirudin antibodies, because they have already formed antibodies toward platelet factor 4/heparin complexes. This indicates that HIT patients do not have an increased tendency to form antibodies to other drugs. Antihirudin antibody desirudin complexes cannot be filtered by the glomeruli. However, neither a difference in desirudin plasma levels, nor in bleeding complications, could be observed between antihirudin antibody-positive and -negative patients. Also we found no evidence for inhibitory antibodies, which have been described in HIT patients receiving lepirudin.12,19 Because of the small numbers of immunized patients, this observation must be considered preliminary. In conclusion, desirudin given in prophylactic dose in patients after orthopedic hip-replacement surgery is immunogenic. The overall risk of immunization seems to be the same as in HIT patients receiving lepirudin in therapeutic dose. As the treatment and observation period was shorter in the present study, the percentage of immunized patients was lower. The overall number of immunized patients, however, is still small, and thus less frequent severe adverse effects might have been overlooked until now. Patients under treatment should be carefully observed for adverse events.
Submitted April 5, 2002; accepted August 14, 2002.
Prepublished online as Blood First Edition Paper, October 17, 2002; DOI 10.1182/blood-2002-04-1055.
Supported by Deutsche Forschungsgemeinschaft DFG Gr1096/2-3a and by a grant from Novartis Pharma and by the German Federal Ministry for Education and Research (NBL3 program, reference 01 ZZ0103).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Andreas Greinacher, Institut für Immunologie und Transfusionsmedizin, Ernst-Moritz-Arndt-Universität, Sauerbruchstr/Diagnostikzentrum, 17487 Greifswald, Germany; e-mail: greinach{at}uni-greifswald.de.
1. Bichler J, Gemmerli R, Fritz H. Studies for revealing a possible sensitization to hirudin after repeated intravenous injections in baboons. Thromb Res. 1991;61:39-51[Medline] [Order article via Infotrieve]. 2. Schlaeppi JM, Vekemans S, Rink H, Chang JY. Preparation of monoclonal antibodies to hirudin and hirudin peptides. Eur J Biochem. 1990;188:463-470[Medline] [Order article via Infotrieve]. 3. Schlaeppi JM. Preparation of monoclonal antibodies to the thrombin/hirudin complex. Thromb Res. 1991;62:459-470[CrossRef][Medline] [Order article via Infotrieve]. 4. Gygax D, Botta L, Ehrat M, et al. Immunoassays in monitoring biotechnological drugs. Ther Drug Monit. 1996;18:405-409[CrossRef][Medline] [Order article via Infotrieve]. 5. Close P, Bichler J, Kerry R, et al. Weak allergenicity of recombinant hirudin CGP 39393 (Revasc) in immunocompetent volunteers: The European Hirudin in Thrombosis Group (HIT Group). Coron Art Dis. 1994;5:943-949[Medline] [Order article via Infotrieve].
6.
Zoldhelyi P, Webster MW, Fuster V, et al.
Recombinant hirudin in patients with chronic, stable coronary artery disease: safety, half-life, and effect on coagulation parameters.
Circulation.
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Topol EJ, Fuster V, Harrington RA, et al.
Recombinant hirudin for unstable angina pectoris: a multicenter randomized angiographic trial.
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Circulation.
1997;96:769-777 10. Organization to Assess Strategies for Ischemic Syndromes (OASIS-2) Investigators. Effects of recombinant hirudin (lepirudin) compared with heparin on death, myocardial infarction, refractory angina, and revascularization procedures in patients with acute myocardial ischaemia without ST elevation: a randomized trial. Lancet. 1999;353:429-438[CrossRef][Medline] [Order article via Infotrieve]. 11. Eichler P, Greinacher A. Anti-hirudin antibodies induced by recombinant hirudin in the treatment of patients with heparin-induced thrombocytopenia (HIT). Ann Hematol. 1996;72(suppl 1):21.
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1999;100:1528-1532 14. Huhle G, Hoffmann U, Song X, Wang LC, Heene DL, Harenberg J. Immunologic response to recombinant hirudin in HIT type II patients during long-term treatment. Br J Haematol. 1999;106:195-201[CrossRef][Medline] [Order article via Infotrieve]. 15. Eriksson BI, Ekman S, Kaelebo P, Zachrisson B, Bach D, Close P. Prevention of deep-vein thrombosis after total hip replacement: direct thrombin inhibition with recombinant hirudin, CGP 39393. Lancet. 1996;347:635-639[CrossRef][Medline] [Order article via Infotrieve].
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1997;79:326-333 18. Pötzsch B, Madlener K, Seelig C, Riess CF, Greinacher A, Müller-Berghaus G. Monitoring of r-hirudin anticoagulation during cardiopulmonary bypass: assessment of the whole blood ecarin clotting time. Thromb Hemost. 1997;77:920-925[Medline] [Order article via Infotrieve]. 19. Huhle G, Liebe V, Hudek R, Heene DL. Anti-r-hirudin antibodies reveal clinical relevance through direct functional inactivation of r-hirudin or prolongation of r-hirudin's plasma halflife. Thromb Haemost. 2001;85:936-938[Medline] [Order article via Infotrieve].
© 2003 by The American Society of Hematology.
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Abstract
Introduction
30°C or below.
