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BIOLOGICS

Biomarkers and Coagulation Tests for Assessing the Biosimilarity of a Generic Low-Molecular-Weight Heparin: Results of a Study in Healthy Subjects With Enoxaparin

Karina Kuczka, MD, Sebastian Harder, MD, Bettina Picard-Willems, BSc, André Warnke, BSc, Frank Donath, MD, Pietro Bianchini, PhD, Bruna Parma, PhD and Henning Blume, PhD

From pharmazentrum frankfurt/ZAFES, Institute for Clinical Pharmacology, University Hospital Frankfurt, Germany (Dr Kuczka, Dr Harder, Ms Picard-Willems); SocraTec R&D GmbH, Oberursel, Germany (Mr Warnke, Dr Donath, Dr Blume); and OPOCRIN S.p.A. Modena, Italy (Dr Bianchini [deceased], Dr Parma).

Address for reprints: Sebastian Harder, MD, Institute for Clinical Pharmacology at the pharmazentrum frankfurt, University Hospital, Frankfurt/Main, Theodor Stern Kai 7, D-60590 Frankfurt am Main, Germany; e-mail: harder{at}em.uni-frankfurt.de.


   ABSTRACT
TOP
 ABSTRACT
Study Design
 Assay Methodology
 Sample Size
 Pharmacodynamic Analyses
 Statistics
 RESULTS
 DISCUSSION
 REFERENCES
 
Low-molecular-weight heparins (LMWHs) differ considerably in their influence on clotting tests and release of tissue factor pathway inhibitor (TFPI). Biosimilarity therefore becomes an issue when generic forms of LMWHs are developed. So far, no bioequivalence study with a generic LMWH has been reported. A generic enoxaparin (test) was compared with the originator (reference) in 20 volunteers after single-dose subcutaneous administration (40 mg enoxaparin sodium, 4000 IU/mL anti–factor Xa (anti-FXa; activity). Target variables were anti-FXa and anti-FIIa activity, activated partial thromboplastin time (aPTT), prothrombinase-induced clotting time (PiCT), and TFPI over 24 hours. The statistical evaluation of the anti-FXa activity profile demonstrated bioequivalence of test and reference with confidence intervals of area under the plasma concentration-time curve (AUC0-tlast) (93%-99%) and Amax (88%-95%). Confidence intervals of AUC0-tlast (89%-102%) and Amax (90%-103%) of anti-FIIa activity also fulfill bioequivalence criteria. The 90% confidence interval for the maximum concentration of TFPI ranged from 90% to 113%. The claim of similarity was also supported by aPTT and PiCT profiles. Bioequivalence with the originator enoxaparin could be demonstrated by ex vivo inhibition of FXa and FIIa activity, by coagulation tests (aPTT and PiCT), and by in vivo release of TFPI. Whether such data also prove biosimilarity of the generic enoxaparin needs to be determined.

Key Words: Biosimilarity • enoxaparin • factor Xa • tissue factor pathway inhibitor

Because the primary aim of a generic drug is to reduce cost without compromising patient care, a generic drug is required to be chemically and biologically equivalent to the pioneer drug. Approval of a generic variant usually requires evidence for pharmacokinetic bioequivalence with the originator product, unless a trial in patients with clinical endpoints is performed.1 So far, the approval requirement for a generic variant by using a pharmacokinetic bioequivalence is applicable to only small molecules. Provided that a chemical assay of the active ingredient is feasible, the conduct of such studies, the pharmacokinetic target variables, and the statistical approach are subject to thorough regulatory guidelines.2 Currently, a large number of low-molecular-weight heparins (LMWHs) are marketed as brand products, where each of these LMWHs has a characteristic molecular weight profile and biological activity in terms of anti–factor Xa (FXa) and anti-FIIa potency.3,4 In contrast to many other drugs, heparins cannot be reliably detected in vivo as a chemical entity; therefore, pharmacodynamic surrogates are usually employed to describe their pharmacokinetic properties and their bioavailability. These pharmacodynamic surrogates include anti-FXa activity, anti-FIIa activity, and several clotting tests (eg, activated partial thromboplastin time [aPTT] or Heptest). Currently, there are no guidelines enforced on the conduct and target parameters of a bioequivalence study with a generic LMWH. Recently, several companies have filed for regulatory approval of a generic version of a branded product in the United States and in Europe. So far, no data have been published on the pharmacodynamic profile of a generic variant of an LMWH. It has been claimed that because LMWHs represent complex natural mucopolysaccharide drugs that have undergone chemical and enzymatic modifications, in vitro data regarding molecular weight and the anti-FXa:anti-FIIa ratio alone do not allow conclusion of generic equivalency to the originator drug.5,6 In this article, we report the conduct of a bioequivalence study with a generic version of enoxaparin. The pharmacodynamic parameters of interest were anti-FIIa activity and anti-FXa activity in plasma. These parameters will be influenced directly by the amount of LMWH present in the plasma sample and therefore do not necessarily reflect the biological activity in the organism. Thus, we additionally analyze the profile of tissue factor pathway inhibitor (TFPI), which is released by the endothelium under LMWH therapy and which should serve as an indirect parameter of the in vivo activity of the LMWH.7-9


   Study Design
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 ABSTRACT
 Study Design
Assay Methodology
 Sample Size
 Pharmacodynamic Analyses
 Statistics
 RESULTS
 DISCUSSION
 REFERENCES
 
This was a double-blind, single-dose, randomized, 2-period, 2-sequence, crossover study under fasting conditions. The study consisted of 2 treatment phases of 36 hours each. After approval by the Ethics Committee of the Physicians Chamber of Thuringia (Germany) and after the subjects provided informed consent, 20 healthy subjects (10 men, 10 women, aged 27-37 years, weighing 66-90 kg) were randomly assigned to 1 of the 2 treatment sequences (test-reference or reference-test). The test preparation consisted of 1 syringe with 40 mg of enoxaparin (4000 IU anti-FXa, manufactured by OPOCRIN S.p.A., Italy); the reference preparation was 1 syringe with 40 mg of enoxaparin (4000 IU anti-FXa, manufactured by Sanofi-Aventis, France [Lovenox]). The study medication was injected subcutaneously. A 6-day washout phase was kept between treatments. During both treatment periods, blood samples were taken at the following time points: 1 predose sample and then 0.5, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 10, 12, 16, 20, 24, and 36 hours postdose. Citrated blood was drawn (via a 1.2-gauge venous cannula). Blood samples for pharmacodynamic analysis were centrifuged and stored at –25°C until analyzed.

The primary endpoint for the assessment of the bioequivalence of test versus reference was area under the plasma concentration-time curve (AUC0-tlast) and Amax of anti-FXa activity. Secondary objectives were the comparative characterization of several other pharmacodynamic effects: anti-FIIa activity, aPTT, prothrombinase induced clotting time (PiCT), and analysis of total TFPI. All analyses except TFPI were prespecified in the protocol.


   Assay Methodology
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 ABSTRACT
 Study Design
 Assay Methodology
Sample Size
 Pharmacodynamic Analyses
 Statistics
 RESULTS
 DISCUSSION
 REFERENCES
 
The analysis of anti-FXa and anti-FIIa activity was performed on an ACL coagulation analyzer (6000/7000 series; Instrumentation Laboratories, Munich, Germany). The test principle is to measure changes in the absorption following the cleavage of a chromogenic substrate by the coagulation factors IIa and Xa, respectively. The cleaved substrate is generated by free FXa or FIIa, which depends on the amount of heparin-antithrombin complex (which inactivates FXa and FIIa) in the sample. The activity of FXa and FIIa is proportional to the absorption (dA), which is the signal generated by the ACL system. FXa and FIIa activity were calibrated against a standard of known anti-FXa or anti-FIIa activity (second international standard for LMWH, provided by the NIBSC, Potters Bar, UK). Factor Xa activity was measured by a chromogenic assay, employing bovine factor Xa (68 nkat), and quantification was by the chromogenic substrate S2765. The dA was measured for 35 seconds at 405 nm. The accuracy of the assay was between 95.7% and 109.8%, and the precision ranged from 0.8% (at 0.70 IU/mL anti-FXa activity) to 5.0% at the lower limit of quantitation (LLOQ; 0.05 IU/mL anti-FXa activity). Factor IIa activity was measured by a chromogenic assay, employing bovine factor IIa (92 nkat), and quantification was by the chromogenic substrate S2238. The dA was measured for 120 seconds at 405 nm. The accuracy was between 93.8% and 110.4%, and the precision ranged from 1.9% (at 0.104 IU/mL anti-FIIa activity) to 12.3% at the LLOQ (0.015 IU/mL anti-FIIa activity). All reagents were provided by Instrumentation Laboratories. The methods were validated according to recommendations given in the FDA Guidance for Industry "Bioanalytical Method Validation" (May 2001).

The aPTT was measured with the aPTT-SP liquid reagent (Instrumentation Laboratories). The normal range on the ACL system is 28 to 33 seconds.

PiCT is a plasma clotting assay based on the activation of coagulation using a combination of a defined amount of FXa, phospholipids (mimicking the platelet membrane or other negatively charged surfaces), and an enzyme that specifically activates factor V (FV; FV activator from the Russel's viper venom). All reagents used in this test were provided by Pentapharm Ltd (Basel, Switzerland; Pefakit PiCT). The results are given in seconds (predose normal range on the ACL system: 24-26 seconds).

The IMUBIND Total Tissue Factor Pathway Inhibitor enzyme-linked immunosorbent assay (ELISA) kit is an enzyme-linked sandwich immunoassay for the quantization of TFPI in plasma as well as in cell culture supernatants (American Diagnostica, Stamford, Connecticut). This ELISA detects both intact and truncated forms of TFPI as well as complexes with tissue factor (TF) and factor VIIa (TF/VIIa/TFPI). The lower limit of detection for this assay is 0.36 ng TFPI/mL sample (undiluted).


   Sample Size
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 ABSTRACT
 Study Design
 Assay Methodology
 Sample Size
Pharmacodynamic Analyses
 Statistics
 RESULTS
 DISCUSSION
 REFERENCES
 
Data obtained from a multiple-dose study with enoxaparin sodium in healthy volunteers10 have shown an intraindividual variance of 16.6% (overall maximum value for the parameters AUC0-tlast and Amax for anti-FXa and anti-FIIa activity). However, because this study was performed in a single-dose design, a higher intraindividual variance of 20% was assumed. Thus, assuming a type I error of {alpha} = .05, a statistical power of 1 – β = .80, the equivalence acceptance intervals of 80% to 125%, and a mean ratio of test versus reference (µT/µR) between 0.95 and 1.05, a sample size of N = 20 has been calculated to be appropriate for this study.


   Pharmacodynamic Analyses
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 ABSTRACT
 Study Design
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 Pharmacodynamic Analyses
Statistics
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 DISCUSSION
 REFERENCES
 
Anti-FXa and anti-FIIa activity. This is the AUC0-tlast area under the anti-FXa or FIIa activity versus time curve from dosing time to the last measurement time point with an activity value greater than the LLOQ, calculated by linear/log trapezoidal method, which uses the linear trapezoidal rule up to Amax and log trapezoidal rule for the remainder of the curve. Amax, maximum of anti-FXa and anti-FIIa activity in plasma, and tmax were directly taken from measured activity values.

Tissue factor pathway inhibitor. Pharmacokinetic calculations were based on plasma concentrations that exceeded the LLOQ, after the baseline TFPI level measured in the predose sample was subtracted. For this purpose, the LLOQ was defined as any value resulting from this subtraction that is ≤0 ng/mL. This procedure is unavoidable because TFPI is physiologically present in human blood. The AUC0-tlast was calculated after baseline subtraction. Maximum TFPI levels and time to reach maximum activity (tmax) were directly taken from measured TFPI profiles.


   Statistics
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 ABSTRACT
 Study Design
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 Pharmacodynamic Analyses
 Statistics
RESULTS
 DISCUSSION
 REFERENCES
 
The statistical analysis for anti-FXa and anti-FIIa activity and TFPI was carried out on the basis of a multiplicative model for all AUC and Amax values while tmax was evaluated on the basis of an additive model. Analyses of variances were performed for AUC0-tlast and Amax values, including the factors formulation, period, sequence, and volunteer. Intrasubject variability were estimated and period, subject, and sequence effects were determined on a significance level of .05 (type I error probability). Parametric point and interval estimates of the test-reference ratio were calculated for AUC0-tlast and Amax values. Relative bioavailability of test versus reference was assessed by the ratios of geometric means (point estimates). Ninety percent confidence intervals served as interval estimates and were determined by parametric analysis (2 one-sided t tests). With regard to tmax, means and the difference between the means were given. Tmax values for test and reference were additionally compared by a 2-sample rank test according to Mann-Whitney.

aPTT and PiCT maximum clotting time prolongation was calculated relative to the baseline value measured prior to administration. Mean values of aPTTmax and PiCTmax of test and reference were evaluated by paired Student t test. Corresponding median values were compared by 2-sample rank test according to Mann-Whitney.


   RESULTS
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 ABSTRACT
 Study Design
 Assay Methodology
 Sample Size
 Pharmacodynamic Analyses
 Statistics
 RESULTS
DISCUSSION
 REFERENCES
 
The mean curves of anti-FXa activity after administration of the test and the reference product were similar with regard to course and shape (Figure 1A). The extent of anti-FXa activity represented by the geometric mean AUC0-tlast values was slightly lower for test (2.57 h·IU/mL) compared with reference (2.69 h·IU/mL), as the maximum anti-FXa activity, represented by the geometric mean Amax values, was lower after administration of the test product (0.38 IU/mL) compared with reference (0.42 IU/mL; Table I). Confidence intervals of AUC0-tlast (93%-99%) and Amax (88%-95%) fulfilled the acceptance criterion of 80% to 125% (Table II).


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  		Table I Mean Pharmacodynamic Variables of Anti-FXa Activity After Subcutaneous Single-Dose Administration of Either a Test or a Reference Formulation of 40 mg of Enoxaparin Sodium (Corresponding to 4000 IU Anti-FXa) Under Fasted Conditions to 20 Volunteers

 


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  		Table II Parametric Point Estimates and 90% Confidence Intervals Determined for AUC0-tlast and Amax of Anti-Xa Activity; Anti-FIIa Activity and TFPI Release, Comparison of Test Versus Reference, N = 20

 
Mean curves of anti-FIIa activity reached their maximum mean anti-IIa activity of approximately 0.06 IU/mL for test and for reference at about 3 hours (Figure 1B). The extent of anti-FIIa activity represented by geometric mean AUC0-tlast values was almost similar after administration of test (0.28 h·IU/mL) compared with reference (0.30 h·IU/mL), as was the maximum anti-FIIa activity (0.06 IU/mL; Table III). Confidence intervals of AUC0-tlast (89%-102%) and Amax (90%-103%) fulfilled the acceptance criterion of 80% to 125% (Table II).


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  		Table III Mean Pharmacodynamic Variables of Anti-FIIa Activity After Subcutaneous Single-Dose Administration of Either a Test or a Reference Formulation of 40 mg of Enoxaparin Sodium (Corresponding to 4000 IU Anti-FXa) Under Fasted Conditions to 20 Volunteers

 
The concentration time curves for TFPI release indicated a similar profile after application of test and reference product (Figure 1C). The predose baseline of total TFPI was 31 ± 8 ng/mL. In general, test and reference preparation led to a maximal increase of total TFPI levels by approximately 30% to 50% of the baseline values. The geometric mean AUC0-tlast value for TFPI (baseline subtracted) was 95 h·ng/mL after the test formulation and 92 h·ng/mL after the reference product. The maximum concentration (Cmax) of total TFPI (baseline subtracted) observed after the test formulation was 13.0 ng/mL; after the reference product, the Cmax averaged 12.9 ng/mL (Table IV). The confidence interval of Cmax (90%-113%) fulfilled the acceptance criterion of 80% to 125%, but the confidence interval of AUC0-tlast (75%-140%) exceeded these limits at both edges.


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  		Table IV Mean Pharmacodynamic Variables of Baseline-Corrected TFPI After Subcutaneous Single-Dose Administration of Either a Test or a Reference Formulation of 40 mg of Enoxaparin Sodium (Corresponding to 4000 IU Anti-FXa) Under Fasted Conditions to 20 Volunteers

 
Both mean aPTT and PiCT versus time curves show a nearly indistinguishable prolongation after administration of the test and the reference product (Figure 2). Both mean and median {Delta}aPTTmax and {Delta}PiCTmax prove an analogous clotting time prolongation after test and reference administration ({Delta}aPTTmax mean: 9 seconds vs 8 seconds, median: 9 seconds vs 9 seconds; {Delta}PiCTmax mean: 26 seconds vs 26 seconds, median: 18 seconds vs 16 seconds).


   DISCUSSION
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 ABSTRACT
 Study Design
 Assay Methodology
 Sample Size
 Pharmacodynamic Analyses
 Statistics
 RESULTS
 DISCUSSION
REFERENCES
 
According to our knowledge, this is the first report of a bioequivalence trial with a generic version of an LMWH claiming biosimilarity with an originator. Other studies have compared LMWHs with different chemical properties.3,8,11 Because of the biological nature of an LMWH, which is a mixture of different polysaccharide chains extracted from porcine mucosa, bioequivalence cannot be assessed by direct measurement of enoxaparin concentrations. Furthermore, LMWHs act indirectly, as they bind to antithrombin, which in turn then inhibits FXa and thrombin (FIIa). The different domains of the polysaccharide chains have different binding properties to antithrombin. Anti-FXa activity alone does not distinguish between the concentrations of the pharmacologically more active C-domain from the less active A domain, and therefore, FIIa activity must be taken into account.12

Bioassays determining the anti-FXa and anti-FIIa activities served as surrogates for the pharmacokinetic properties of the LMWH and were established and validated in this study according to the FDA Guidance for Industry "Bioanalytical Method Validation." Indeed, mean curves of anti-FXa and anti-FIIa activities after administration of the test and the reference product are similar, and the parametric statistical evaluation of the primary pharmacodynamic variables of anti-FXa activities clearly demonstrate the bioequivalence of test and reference, with confidence intervals of AUC0-tlast (93%-99%) and Amax (88%-95%) being completely inside the preset acceptance criterion of 80% to 125%. The tight confidence intervals are the result of a low intraindividual variability. Confidence intervals obtained for anti-FIIa activities also support the bioequivalence conclusion. Given the fact that the antithrombotic activity of heparins primarily relies on their anti-FXa and anti-FIIa potency,4,12 application of the same confidence intervals as requested for small-molecule generic drugs seems justified.


Figure 1
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  		Figure 1. (A) Mean anti-FXa activity versus time curves (arithmetic means with SD, concentrations above the LLOQ) after fasted subcutaneous administration of single doses of test (V) and reference (S) to 20 volunteers (40 mg of enoxaparin sodium per treatment). (B) Mean anti-FIIa activity versus time curves (arithmetic means with SD, concentrations above the LLOQ) after fasted subcutaneous administration of single doses of test (V) and reference (S) to 20 volunteers (40 mg of enoxaparin sodium per treatment). (C) Mean baseline corrected TFPI plasma concentration versus time curves (arithmetic means with SD) after fasted subcutaneous administration of single doses of test (V) and reference (S) to 20 volunteers (40 mg of enoxaparin sodium per treatment).

 


Figure 2
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  		Figure 2. (A) Mean aPTT versus time curves (arithmetic means with SD) after fasted subcutaneous administration of single doses of test (V) and reference (S) to 20 volunteers (40 mg of enoxaparin sodium per treatment). (B) Mean PiCT versus time curves (arithmetic means with SD) after fasted subcutaneous administration of single doses of test (V) and reference (S) to 20 volunteers (40 mg of enoxaparin sodium per treatment).

 
Comparison with published data shows that anti-FXa (as well as anti-FIIa) activities determined in the current study are generally in accordance to data reported in the literature from healthy volunteer studies.3,10,11 Moreover, this bioequivalence assessment is strongly supported by statistical evaluation of secondary pharmacodynamic parameters aPTT and PiCT, which do not indicate statistically significant differences between both products. The PiCT is a clotting assay based on the activation of coagulation using a combination of a defined amount of FXa, phospholipids, and an enzyme that specifically activates FV (FV activator isolated from Russell's viper venom) in the analytical sample.13 PiCT is approved for determining the anticoagulant activity of indirect, antithrombin-dependent inhibitors such as unfractionated heparin, LMWH, danaparoid, and pentasaccharide and is also sensitive (in a modified form) to new direct FXa inhibitors.14,15 Because the assay provides all the components of the prothrombinase complex, the clotting endpoint becomes solely dependent on the activity of FXa (as supplemented by the assay) and FIIa (as generated from prothrombin in the sample). Therefore, the similarity of the PiCT profiles might also indicate the similarity of the combined anti-FXa and anti-FIIa activity under an LMWH.

The TFPI is a multivalent serine proteinase inhibitor that plays a central role in the extrinsic pathway of blood coagulation and is mainly expressed by endothelial cells.5,16 Heparin and LMWHs mobilize TFPI from the vascular endothelium into the blood circulation. TFPI strongly inhibits the activity of the procoagulant tissue factor TF, and it is assumed that this effect provides an important contribution to the clinical effects of heparins.5,7,9 It has already been shown that differences exist between LMWHs regarding TFPI release. In one study,8 the pharmacokinetic profiles of bemiparin (3500 IU anti-FXa) and tinzaparin (4500 IU anti-FXa) administered subcutaneously to 12 healthy male volunteers were compared. Bemiparin exerts a significantly more rapid, more potent, and more prolonged anti-Xa activity than tinzaparin, but the plasma level increase for free and total TFPI was significantly lower with bemiparin than with tinzaparin. Although the baseline total TFPI levels in our study averaged 31 ng/mL and were thus lower than the approximate of 60 ng/mL described in other reports,7,8 the increase (relative to baseline) seen after 4000 IU of test and reference preparation was comparable to results observed with another LMWH at this dose level.8 Analysis of bioequivalence showed that the Cmax of total TFPI was similar, and the confidence intervals for the test-reference ratio fell within generally accepted limits for bioequivalence. However, the AUC0-t for baseline subtracted total TFPI showed a wide interindividual variation; therefore, the confidence intervals were too wide to allow a valid conclusion for bioequivalence. The CV analysis of variance for AUC0-t was 62.5%, and a post hoc power analysis yielded a sample size requirement of 88 subjects to prove existing bioequivalence with this parameter.

It is acknowledged that despite proof of bioequivalence with several pharmacodynamic surrogates, the claim of biosimilarity is tempered by several limitations of the current study design. The tight confidence intervals for anti-FXa and anti-FIIa activity indicate a low intraindividual variation of the plasma concentrations of the LMWH. The anti-FXa assay, however, is an in vitro test with plasma sample drawn from the subjects. This also applies to aPTT and PiCT because the coagulation is initiated in the test tube with the subject's plasma specimen. Only TFPI indicates the biological activity in the vascular endothelium of the subject who has received the study drug, but this parameter shows a much wider intraindividual variation, requiring a larger sample size if confirmatory tests are planned (see above). Furthermore, TFPI exhibits a circadian rhythm in clinically healthy men, with highest levels in the morning (+11% compared with levels at noon), at the beginning of the time of daily activity, and a constant decline over the day.16 Considering chronogenicity of TFPI, one might suggest a placebo arm in future bioequivalence studies that interferes with current statistical approaches on bioequivalence (2-way crossover design). Therefore, we would suggest TFPI as only a secondary viable target in future trials. One might further suggest tissue plasminogen activator (tPA; giving rise to fibrinolytic activity), which is also released by heparins,17,18 as an additional biological marker, but it needs to be noted that tPA is enhanced only under larger, therapeutic doses of heparin.17 Immunogenicity adds another layer to the safety assessment required for generic LMWH.19 A possible immunogenic effect seen with LMWHs is the occurrence of heparin-induced thrombocytopenia (type II). However, because heparin-induced thrombocytopenia type II is reported at a rate of 0.1% to 1% of patients treated with LMWHs,20 it will occur in large clinical studies but is not expected to be seen in a single-dose phase 1 study. Furthermore, occurrence of immunogenecity during the bioequivalence evaluation in the current crossover design (with a 1-week washout phase between administrations) would not allow for discrimination between the test and reference product because antiplatelet factor 4/heparin antibodies can occur about 10 days after the first LMWH administration.21

In conclusion, bioequivalence of the generic enoxaparin formulation tested and the originator product could be demonstrated on the basis of commonly accepted bioequivalence margins for the primary pharmacodynamic parameter anti-FXa activity. Additional biomarker analysis and coagulation tests support this finding. Whether data of this kind are sufficient to prove biosimilarity or which type of additional clinical study is needed should be subject to a regulatory approach. Most recently, a guideline on similar biological medicinal products containing LMWHs has been drafted by the European Medicines Agency.22 In this draft, anti-FXa activity is suggested as a primary target variable, and anti-FIIa and TFPI are suggested as secondary variables. However, in addition, a clinical study (preferably in the field of thromboprophylaxis in high-risk patients) is suggested, using a noninferiority approach.

DOI: 10.1177/0091270008322911

Financial disclosure: This study was supported by OPOCRIN S.p.A. All the documentation about the traceability of the test preparation is available at OPOCRIN S.p.A., via Pacinotti, 341043 Corlo di Formigine, (Modena) Italy.


   REFERENCES
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 Study Design
 Assay Methodology
 Sample Size
 Pharmacodynamic Analyses
 Statistics
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 REFERENCES
 

1. Directive 2001/83/EC of the European Parliament and of the Council of 6 November 2001 on the community code relating to medicinal products for human use. Official Journal L. 311;28/11/2001: 67-128.

2. Prugnaud JL. Similarity of biotechnology derived medicinal products: specific problems and new regulatory framework. Br J Clin Pharmacol. 2008;65: 619-620.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

3. Collignon F, Frydman A, Caplain H, et al. Comparison of the pharmacokinetic profiles of three low molecular mass heparins—dalteparin, enoxaparin and nadoparin—administered subcutaneously in healthy volunteers. Thromb Haemost. 1995;73: 630-640.[Web of Science][Medline] [Order article via Infotrieve]

4. White RH, Ginsberg JS. Low-molecular-weight heparins: are they all the same? Br J Haematol. 2003;121: 12-20.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

5. Alban S, Gastpar R. Plasma levels of total and free tissue factor pathway inhibitor (TFPI) as individual pharmacological parameters of various heparins. Thromb Haemost. 2001;85: 824-829.[Web of Science][Medline] [Order article via Infotrieve]

6. Maddineni J, Walenga JM, Jeske WP, et al. Product individuality of commercially available low-molecular-weight heparins and their generic versions: therapeutic implications. Clin Appl Thromb Hemost. 2006;12: 267-276.[Abstract/Free Full Text]

7. Gori AM, Pepe G, Attanasio M, et al. Tissue factor reduction and tissue factor pathway inhibitor release after heparin administration. Thromb Haemost. 1999;81: 589-593.[Web of Science][Medline] [Order article via Infotrieve]

8. Depasse F, González de Suso MJ, Lagoutte I, Fontcuberta J, Borrell M, Samama MM. Comparative study of the pharmacokinetic profiles of two LMWHs—bemiparin (3500 IU, anti-Xa) and tinzaparin (4500 IU, anti-Xa)—administered subcutaneously to healthy male volunteers. Thromb Res. 2003;109: 109-117.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

9. Gouin-Thibault I, Pautas E, Depasse F, Andreux JP, Siguret V. Heparin-releasable TFPI is not depleted after repeated injections of tinzaparin at therapeutic dose for up to 30 days. J Thromb Haemost. 2003;1: 2694-2695.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

10. Frydman AM, Lara L, Le Roux Y, Woler M. Chauliac F, Samama MM. The antithrombotic activity and pharmacokinetics of enoxaparin, a low-molecular weight heparin, in humans given single subcutaneous doses of 20 to 80 mg. J Clin Pharmacol. 1988;28: 609-618.[Abstract]

11. Eriksson BI, Söderberg K, Widlund L, Wandeli B, Tengborn L, Risberg B. A comparative study of three low-molecular weight heparins (LMWH) and unfractionated heparin (UH) in healthy volunteers. Thromb Haemost. 1995;73: 398-401.[Web of Science][Medline] [Order article via Infotrieve]

12. Hemker HC, Al Dieri R, Béguin S. Laboratory monitoring of low-molecular-weight heparin therapy—part II. Monitoring LMWH therapy? For the moment a non-question. J Thromb Haemost. 2005;3: 571-573.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

13. Calatzis A, Spannagl M, Gempeler-Messina P, Kolde HJ, Schramm W, Haas S. The prothombinase-induced clotting test (PiCT®): a new technique for the monitoring of anticoagulants. Haemostasis. 2000;30: 172-174.[CrossRef][Web of Science]

14. Graff J, Hentig Nv, Misselwitz F, et al. Effects of the oral, direct factor Xa-inhibitor rivaroxaban (BAY 59-7939) on platelet-induced thrombin generation and prothrombinase activity. J Clin Pharm. 2007;47: 1398-1407.[CrossRef]

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