Download Immunogenicity of Low Molecular Weight Heparins and Their

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.