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Evaluation of Immunostimulatory Potential of Branded and US-Generic Enoxaparins in an In Vitro Human Immune System Model.

Luna E, Agrawal P, Mehta R, Vernhes C, Viskov C, Amiral J, Warren WL, Drake DR - Clin. Appl. Thromb. Hemost. (2014)

Bottom Line: Low-molecular-weight heparins (LMWHs) have several positive therapeutic effects and can also form immunostimulatory complexes with plasma proteins, such as platelet factor 4 (PF4).Production of tissue factor pathway inhibitor (TFPI), a physiologic heparin-induced inhibitor of tissue factor-induced coagulation that was used as a functional readout of biological activity of enoxaparins in these assays, was heightened in the presence of branded enoxaparin complexes, but its levels were variable in cultures treated with complexes containing US-generic enoxaparins.Analytical analyses suggest that the heightened immunostimulatory potential of some of the US-generic enoxaparin product lots could be tied to their capacity to form ultra-large and/or more stable complexes with PF4 than the other LMWHs included in this study.

View Article: PubMed Central - PubMed

Affiliation: Sanofi Pasteur, VaxDesign Campus, Orlando, FL, USA.

No MeSH data available.


Related in: MedlinePlus

Brand and generic enoxaparins have different capacities to induce TFPI secretion in the MIMIC® PTE construct. A, MIMIC® PTE cultures were treated with heparins alone (Lot 1) for 48 hours, the culture supernatants were then harvested and examined for TFPI secretion by ELISA. The data show the means ± SD of the percentage change over the control (no treatment condition) for 5 donors. B, Culture supernatants from the blinded study cultures shown in this figure and in Figure 3 were harvested at 48 hours posttreatment and examined for TFPI by ELISA. The data show the means ± SD of the percentage change over the control (no treatment condition) for 10 donors included in the blinded study. Ag indicates antigen; ELISA, enzyme-linked immunosorbent assay; MIMIC® PTE, Modular IMmune In vitro Construct system-peripheral tissue equivalent; PF4, platelet factor 4; SD, standard deviation; TFPI, tissue factor pathway inhibitor; UFH, unfractionated heparin.
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fig4-1076029614562037: Brand and generic enoxaparins have different capacities to induce TFPI secretion in the MIMIC® PTE construct. A, MIMIC® PTE cultures were treated with heparins alone (Lot 1) for 48 hours, the culture supernatants were then harvested and examined for TFPI secretion by ELISA. The data show the means ± SD of the percentage change over the control (no treatment condition) for 5 donors. B, Culture supernatants from the blinded study cultures shown in this figure and in Figure 3 were harvested at 48 hours posttreatment and examined for TFPI by ELISA. The data show the means ± SD of the percentage change over the control (no treatment condition) for 10 donors included in the blinded study. Ag indicates antigen; ELISA, enzyme-linked immunosorbent assay; MIMIC® PTE, Modular IMmune In vitro Construct system-peripheral tissue equivalent; PF4, platelet factor 4; SD, standard deviation; TFPI, tissue factor pathway inhibitor; UFH, unfractionated heparin.

Mentions: Tissue factor pathway inhibitor plays a crucial role in regulating the coagulation cascade and is one of the critical pharmacodynamic markers of heparin function.18,26 As TFPI is constitutively synthesized by vascular endothelial cells,27 and endothelial cells serve as a principal component of the MIMIC® PTE construct, we questioned whether it could be used as a marker of heparin function in this study. As a proof-of-concept experiment, cultures harvested from MIMIC® PTE assays treated with noncomplexed UFH and variant enoxaparins showed 2- to 3-fold increases in free TFPI accumulation over the baseline control condition (Figure 4A). Subsequently, the effect of PF4–heparin complexes on TFPI production in MIMIC® PTE assays was examined using the same culture supernatants profiled in Figure 3. When compared against the phenotype results, it is notable that TFPI production was inversely correlated with APC activation, such that strongly immunostimulatory complexes triggered significant decreases in TFPI production below the baseline level observed in control condition. For example, the highly immunostimulatory PF4–UFH complexes reduced TFPI production by approximately 50% and both lots of PF4–branded enoxaparin complexes, which were only weakly immunostimulatory, increased TFPI production ∼3- to 4-fold above the baseline level (Figure 4B). On the other hand, complexes formed with the generic enoxaparins (Sandoz and Amphastar) generated variable TFPI responses (Figure 4B) that were inversely matched to the APC phenotype data shown in Figure 3.


Evaluation of Immunostimulatory Potential of Branded and US-Generic Enoxaparins in an In Vitro Human Immune System Model.

Luna E, Agrawal P, Mehta R, Vernhes C, Viskov C, Amiral J, Warren WL, Drake DR - Clin. Appl. Thromb. Hemost. (2014)

Brand and generic enoxaparins have different capacities to induce TFPI secretion in the MIMIC® PTE construct. A, MIMIC® PTE cultures were treated with heparins alone (Lot 1) for 48 hours, the culture supernatants were then harvested and examined for TFPI secretion by ELISA. The data show the means ± SD of the percentage change over the control (no treatment condition) for 5 donors. B, Culture supernatants from the blinded study cultures shown in this figure and in Figure 3 were harvested at 48 hours posttreatment and examined for TFPI by ELISA. The data show the means ± SD of the percentage change over the control (no treatment condition) for 10 donors included in the blinded study. Ag indicates antigen; ELISA, enzyme-linked immunosorbent assay; MIMIC® PTE, Modular IMmune In vitro Construct system-peripheral tissue equivalent; PF4, platelet factor 4; SD, standard deviation; TFPI, tissue factor pathway inhibitor; UFH, unfractionated heparin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2 - License 3
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getmorefigures.php?uid=PMC4401814&req=5

fig4-1076029614562037: Brand and generic enoxaparins have different capacities to induce TFPI secretion in the MIMIC® PTE construct. A, MIMIC® PTE cultures were treated with heparins alone (Lot 1) for 48 hours, the culture supernatants were then harvested and examined for TFPI secretion by ELISA. The data show the means ± SD of the percentage change over the control (no treatment condition) for 5 donors. B, Culture supernatants from the blinded study cultures shown in this figure and in Figure 3 were harvested at 48 hours posttreatment and examined for TFPI by ELISA. The data show the means ± SD of the percentage change over the control (no treatment condition) for 10 donors included in the blinded study. Ag indicates antigen; ELISA, enzyme-linked immunosorbent assay; MIMIC® PTE, Modular IMmune In vitro Construct system-peripheral tissue equivalent; PF4, platelet factor 4; SD, standard deviation; TFPI, tissue factor pathway inhibitor; UFH, unfractionated heparin.
Mentions: Tissue factor pathway inhibitor plays a crucial role in regulating the coagulation cascade and is one of the critical pharmacodynamic markers of heparin function.18,26 As TFPI is constitutively synthesized by vascular endothelial cells,27 and endothelial cells serve as a principal component of the MIMIC® PTE construct, we questioned whether it could be used as a marker of heparin function in this study. As a proof-of-concept experiment, cultures harvested from MIMIC® PTE assays treated with noncomplexed UFH and variant enoxaparins showed 2- to 3-fold increases in free TFPI accumulation over the baseline control condition (Figure 4A). Subsequently, the effect of PF4–heparin complexes on TFPI production in MIMIC® PTE assays was examined using the same culture supernatants profiled in Figure 3. When compared against the phenotype results, it is notable that TFPI production was inversely correlated with APC activation, such that strongly immunostimulatory complexes triggered significant decreases in TFPI production below the baseline level observed in control condition. For example, the highly immunostimulatory PF4–UFH complexes reduced TFPI production by approximately 50% and both lots of PF4–branded enoxaparin complexes, which were only weakly immunostimulatory, increased TFPI production ∼3- to 4-fold above the baseline level (Figure 4B). On the other hand, complexes formed with the generic enoxaparins (Sandoz and Amphastar) generated variable TFPI responses (Figure 4B) that were inversely matched to the APC phenotype data shown in Figure 3.

Bottom Line: Low-molecular-weight heparins (LMWHs) have several positive therapeutic effects and can also form immunostimulatory complexes with plasma proteins, such as platelet factor 4 (PF4).Production of tissue factor pathway inhibitor (TFPI), a physiologic heparin-induced inhibitor of tissue factor-induced coagulation that was used as a functional readout of biological activity of enoxaparins in these assays, was heightened in the presence of branded enoxaparin complexes, but its levels were variable in cultures treated with complexes containing US-generic enoxaparins.Analytical analyses suggest that the heightened immunostimulatory potential of some of the US-generic enoxaparin product lots could be tied to their capacity to form ultra-large and/or more stable complexes with PF4 than the other LMWHs included in this study.

View Article: PubMed Central - PubMed

Affiliation: Sanofi Pasteur, VaxDesign Campus, Orlando, FL, USA.

No MeSH data available.


Related in: MedlinePlus