Considerations for the Bioanalytical Part of Equivalence Studies of Biosimilar Nadroparin Calcium

According to current regulatory views, a comparative study of the pharmacodynamics (PD) of low molecular weight heparin (LMWH) products and confirmation of their equivalence require comparing three PD markers: the anti-Xa activity, the anti-IIa activity, and the tissue factor pathway inhibitor (TFPI) concentration. The aim of this study was to analyse the features specific to the bioanalytical part of an equivalence study of a nadroparin calcium biosimilar after single subcutaneous administration. Material and methods: the anti-Xa and anti-IIa activity values and TFPI content were determined in human plasma samples obtained after single subcutaneous administration of the test and the reference product in the same dose, using commercially available reagent kits and pre-validated assays. The authors calculated the main PD parameters (surrogate pharmacokinetic markers), namely the maximum activity or concentration (A_max or C_max), time to maximum activity or concentration (T_max), area under the activity–time (or concentration–time) curve (AUC ), and half-life period (T_1/2), by means of model-independent statistical moment analysis and carried out further statistical testing of the parameters. Results: the anti-Xa activity and TFPI concentration results provided for  the  possibility  of  calculating  and  comparing  the PD parameters (A_max or C_max, AUC_0-24, AUC_0-∞, Tmax, T_1/2) and estimating the confidence intervals that are necessary to confirm the bioequivalence of the studied products. The anti-IIa activity data had a characteristic pattern of slight fluctuations around one level, which prevented the calculation and comparison of PD parameters. Conclusion: the study identified specific features to consider when planning comparative PD studies of nadroparin calcium products. Firstly, it is feasible to divide samples into two test aliquots (one for anti-Xa and anti-IIa activity determination, the other for TFPI analysis) at the moment of collection in order to perform the analytical step correctly. Secondly, there is no need in full validation for the bioanalytical assays of the anti-Xa and anti-II activity and TFPI content in human plasma validated in the concentration ranges of 0.024–0.182 IU/mL, 0.0069–0.052 IU/mL and 1.56–100 ng/mL, respectively; a confirmation that the active ingredient does not interfere with the analytical procedure is adequate for the purpose. Finally, the data obtained may not allow for calculating PD parameters and comparing confidence intervals for all three markers. The listed considerations may be relevant for other LMWH products as well.

1. Hoffmann U, Harenberg J, Bauer K, Huhle G, Tolle AR, Feuring M, Christ M. Bioequivalence of subcutaneous and intravenous body-weight-independent high-dose low-molecular-weight heparin Certoparin on anti-Xa, Heptest, and tissue factor pathway inhibitor activity in volunteer. Blood Coagul Fibrinolysis. 2002;13(4):289–96. https://doi.org/10.1097/00001721-200206000-00003

2. Feng L, Shen-Tu J, Liu J, Chen J, Wu L, Huang M. Bioequivalence of generic and branded subcutaneous enoxaparin: A single-dose, randomized-sequence, open-label, two-period crossover study in healthy Chinese male subjects. Clin Ther. 2009;31(7):1559– 67. https://doi.org/10.1016/j.clinthera.2009.07.017

3. Gadiko C, Thota S, Tippabotla SK. Pharmacokinetic parameters to be evaluated for selected low molecular weight heparins in bioequivalence studies. Int J Pharm Sci Res. 2012;3(11):4065–72. https://doi.org/10.13040/IJPSR.0975-8232.3(11).4065-72

4. Gadiko C, Tippabhotla SK, Thota S, Cheerla R, Betha MR, Vobalaboina V. Bioequivalence study of two subcutaneous formulations of dalteparin: randomized, single-dose, two-sequence, two-period, cross-over study in healthy volunteers. J Drug Assess. 2013;2(1):21–9. https://doi.org/10.3109/21556660.2013.781504

5. Boubaker H, Grissa MH, Sassi M, Chakroun T, Beltaief K, Hassine M, et al. Generic and branded enoxaparin bioequivalence: A clinical and experimental study. J Bioequiv Availab. 2015;7(5):225–8. https://doi.org/10.4172/jbb.1000244

6. Martinez González J, Monreal M, Ayani Almagia IA, Llaudo Garín J, Ochoa Diaz de Monasterioguren L, Gutierro Adúriz I. Bioequivalence of a biosimilar enoxaparin sodium to Clexane® after single 100 mg subcutaneous dose: results of a randomized, double-blind, crossover study in healthy volunteers. Drug Des Devel Ther. 2018;12:575–82. https://doi.org/10.2147/DDDT.S162817

7. Zhang Y, Huo M, Zhou J, Xie S. PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel. Comput Methods Programs Biomed. 2010;99(3):306–14. https://doi.org/10.1016/j.cmpb.2010.01.007

8. Vorobiev AV, Chabrov AM, Savchenko AA, Makatsariya AD. Antithrombotic therapy in cancer patients. Akusherstvo, ginekologiya i reproduktsiya = Obstetrics, Gynecology and Reproduction. 2016;10(1):26–37 (In Russ.). https://doi.org/10.17749/2313-7347.2015.10.1.026-037

9. Bitsadze VO, Makatsariya AD. The use of low molecular weight heparins in obstetric practice. Rossiyskiy meditsinskiy zhurnal = Russian Medical Journal. 2000;8(18):772 (In Russ.)

10. Fan P, Gao Y, Zheng M, Xu T, Schoenhagen P, Jin Z. Recent progress and market analysis of anticoagulant drugs. J Thorac Dis. 2018;10(3):2011–25. https://doi.org/10.21037/jtd.2018.03.95

11. Qiu M, Huang S, Luo C, Wu Z, Liang B, Huang H, et al. Pharmacological and clinical application of heparin progress: An essential drug for modern medicine. Biomed Pharmacother. 2021;139:111561. https://doi.org/10.1016/j.biopha.2021.111561

12. Sturov NN. Clinical pharmacology of low molecular weight heparins: the most important aspects. Trudny patsient = Difficult Patient. 2006;4(11):38–42 (In Russ.)

13. Low molecular weight heparins: original drugs and their biosimilars — the choice in clinical practice. Seminar. Aterotromboz = Atherothrombosis. 2017;(2):142–51 (In Russ.)

14. Fareed J, Hoppensteadt D, Walenga J, Iqbal O, Ma Q, Jeske W, Sheikh T. Pharmacodynamic and pharmacokinetic properties of enoxaparin: implications for clinical practice. Clin Pharmacokinet. 2003;42(12):1043–57. https: //doi.org/10.2165/00003088-200342120-00003

15. Dmitriev VV, Lipay NV, Migal NV, Begun IV, Dmitriev EV. Pharmacokinetics of low molecular weight heparins in thrombosis, complicate the treatment of children with cancer. Onkogematologiya = Oncohematology. 2021;16(1):73–82 (In Russ.) https://doi.org/10.17650/1818-8346-2021-16-1-73-82

16. Rico S, Antonijoan RM, Ballester MR, Gutierro I, Ayani I, Martinez-Gonzalez J, et al. Pharmacodynamics assessment of Bemiparin after multiple prophylactic and single therapeutic doses in adult and elderly healthy volunteers and in subjects with varying degrees of renal impairment. Thromb Res. 2014;133(6):1029–38. https://doi.org/10.1016/j.thromres.2014.03.038

17. Egan G, Ensom MH. Measuring anti-factor Хa activity to monitor low-molecular-weight heparin in obesity: a critical review. Can J Hosp Pharm. 2015;68(1):33–47. https://doi.org/10.4212/cjhp.v68i1.1423

18. Nutescu EA, Burnett A, Fanikos J, Spinler S, Wittkowsky A. Pharmacology of anticoagulants used in the treatment of venous thromboembolism. J Thromb Thrombolysis. 2016;41(1):15–31. https://doi.org/10.1007/s11239-015-1314-3

19. Pannucci CJ, Fleming KI, Bertolaccini CB, Prazak AM, Huang LC, Pickron TB. Assessment of anti-factor Xa levels of patients undergoing colorectal surgery given once-daily enoxaparin prophylaxis: a clinical study examining enoxaparin pharmacokinetics. JAMA Surg. 2019;154(8):697–704. https://doi.org/10.1001/jamasurg.2019.1165

20. Cihlar R, Sramek V, Papiez A, Penka M, Suk P. Pharmacokinetic comparison of subcutaneous and intravenous nadroparin administration for thromboprophylaxis in critically ill patients on vasopressors. Pharmacology. 2020;105(1–2):73–8. https://doi.org/10.1159/000502847