Dicetones-1,4 thiosubstituees

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Data from: Evaluation of the innate immunostimulatory potential of originator and non-originator copies of insulin glargine in an in vitro human immune model

Background: The manufacture of insulin analogs requires sophisticated production procedures which can lead to differences in the structure, purity, and/or other physiochemical properties of resultant products that can affect their biologic activity. Here, we sought to compare originator and non-originator copies of insulin glargine for innate immune activity and mechanisms leading to differences in these response profiles in an in vitro model of human immunity. Methods: An endothelial/dendritic cell-based innate immune model was used to study antigen-presenting cell activation, cytokine secretion, and insulin receptor signalling pathways induced by originator and non-originator insulin glargine products. Mechanistic studies included signalling pathway blockade with specific inhibitors, analysis of the products in a Toll-like receptor reporter cell line assay, and insulin removal from the products by immunopurification. Findings: All insulin glargine products elicited at least a minor innate immune response comparable to human insulin, but some lots of a non-originator copy product induced the elevated secretion of the cytokines, IL-8 and IL-6. In studies aimed at addressing the mechanisms leading to differential cytokine production by these products, we found (1) the inflammatory response was not mediated by bacterial contaminants, (2) the innate response was driven by the insulin receptor through the MAPK pathway, and (3) the removal of insulin significantly reduced their capacity to induce innate activity. No evidence of product aggregates was detected, though the presence of some high molecular weight proteins argues for the presence of insulin dimers or others contaminants in these products. Conclusion: The data presented here suggests some non-originator insulin glargine product lots drive heightened in vitro human innate activity and provides preliminary evidence that changes in their biochemical composition (dimers, impurities) might be responsible for their greater immunostimulatory potential

repository data for Plos One publication 2018

repository data for Plos One publication 201

Evaluation of the innate immunostimulatory potential of originator and non-originator copies of insulin glargine in an <i>in vitro</i> human immune model

<div><p>Background</p><p>The manufacture of insulin analogs requires sophisticated production procedures which can lead to differences in the structure, purity, and/or other physiochemical properties of resultant products that can affect their biologic activity. Here, we sought to compare originator and non-originator copies of insulin glargine for innate immune activity and mechanisms leading to differences in these response profiles in an in vitro model of human immunity.</p><p>Methods</p><p>An endothelial/dendritic cell-based innate immune model was used to study antigen-presenting cell activation, cytokine secretion, and insulin receptor signalling pathways induced by originator and non-originator insulin glargine products. Mechanistic studies included signalling pathway blockade with specific inhibitors, analysis of the products in a Toll-like receptor reporter cell line assay, and natural insulin removal from the products by immunopurification.</p><p>Findings</p><p>All insulin glargine products elicited at least a minor innate immune response comparable to natural human insulin, but some lots of a non-originator copy product induced the elevated secretion of the cytokines, IL-8 and IL-6. In studies aimed at addressing the mechanisms leading to differential cytokine production by these products, we found (1) the inflammatory response was not mediated by bacterial contaminants, (2) the innate response was driven by the native insulin receptor through the MAPK pathway, and (3) the removal of insulin glargine significantly reduced their capacity to induce innate activity. No evidence of product aggregates was detected, though the presence of some high molecular weight proteins argues for the presence of insulin glargine dimers or others contaminants in these products.</p><p>Conclusion</p><p>The data presented here suggests some non-originator insulin glargine product lots drive heightened in vitro human innate activity and provides preliminary evidence that changes in the biochemical composition of non-originator insulin glargine products (dimers, impurities) might be responsible for their greater immunostimulatory potential.</p></div

Insulin glargine (Glaritus) lots 32 and 96 induce heightened innate activity in the MIMIC^® PTE.

<p>MIMIC^® PTE cultures were treated with different batches of insulin glargines at 30 nM (5 U/ml) for 48 hours. Thereafter, the culture supernatant were collected and evaluated for the secretion of different cytokines by multiplex assay. The plotted values represent mean ± SEM (pg/ml) of IL-8 secretion (<b>A</b>) and IL-6 secretion (<b>B</b>) for 12–14 donors. (<b>C</b>) Graphical representation of Pearson correlation analysis using confidence intervals (CI) of IL-8 and IL-6 secretion induced by all insulin glargine products. **, p<0.01; ****, p<0.001; -, No treatment; L+R, LPS+R848; Bas, Basalin; B, Bonglixan; HI, Human Insulin (Insuman). Lot numbers represent the last two digits of the lot numbers shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197478#pone.0197478.t001" target="_blank">Table 1</a>.</p

Statistical analysis of IL-8 and IL-6 secretion: Glaritus versus Lantus.

<p>Statistical analysis of IL-8 and IL-6 secretion: Glaritus versus Lantus.</p

The innate response induced by insulin glargine in MIMIC^® PTE assays is largely driven by insulin signaling through the IRA/IRB and MEK pathways.

<p>MIMIC^® PTE cultures were incubated with the indicated treatments immediately following PBMC application. (S961 is the insulin receptor AB antagonistic peptide.) 1 hour later, 30 nM (5 U/ml) insulin glargines were added to the wells and incubated for 48 hours. Thereafter, the culture supernatants was collected and analyzed for IL-8 secretion by multiplex assay. Data represented as mean ± SEM of 8–12 healthy donors. Lot numbers represent the last two digits of the lot numbers shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197478#pone.0197478.t001" target="_blank">Table 1</a>.</p

Insulin glargines employed in this study.

<p>Insulin glargines employed in this study.</p

Glaritus lots 32 and 96 show differential by-product profiles by SEC-HPLC analysis.

<p>(<b>A</b>) 500 ng insulin glargines were analyzed by 3–12% gradient Bis-Tris Native-PAGE under non-reducing conditions followed by silver staining. Molecular markers were used as size standards. Images were taken with a Kodak GL 1500 Imaging system and the insulin glargine band was observed at 6–10 kDa. (<b>B</b>) Representative SEC-HPLC analysis of Glaritus lots 32 and 96 compared with the originator lot 06. Arrows indicate the presence of distinct peaks observed between the originator and non-originator insulin glargines.</p