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Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody (mAb) by Neutralizing TNF Using an In Vitro Bioanalytical Method
Published on: September 16, 2017
A screen for apoptotic synergism between clinical relevant nephrotoxicant and the cytokine TNF-α
Giulia Benedetti1, Sreenivasa Ramaiahgaris, Bram Herpers
1Division of Toxicology, Leiden Academic Center for Drug Research, Leiden University, The Netherlands.
Abstract:
Nephrotoxicity remains one of the main reasons for post-market drug withdrawal. Tumour necrosis factor α (TNF-α) secretion has been shown to underlie the nephrotoxicity induced by some of these drugs. Yet, there is currently no reliable and sensitive in vitro assay available to screen for nephrotoxicants of which toxicity largely depends on TNF-α secretion. Therefore, we developed and applied a sensitive fluorescence-based in vitro assay for TNF-α-mediated nephrotoxicity screening using mouse immortalized proximal tubular epithelial cells (IM-PTECs). Our assay allows rapid evaluation of TNF-α-mediated toxicant-induced apoptosis and necrosis using fixed endpoint and live cell measurements. To evaluate our assay, sixteen nephrotoxicants and two control non-nephrotoxicants were used. Out of the sixteen nephrotoxicants, eight induced cell death, of which five induced apoptosis as well as necrosis. Moreover, TNF-α significantly enhanced apoptotic cell death induced by cisplatin, cyclosporine A, tacrolimus and azidothymidine. These nephrotoxicants are known to induce inflammation in vivo which has been linked to an enhancement of nephrotoxicity for cisplatin, cyclosporine A and tacrolimus, confirming the functionality of our assay. Overall, our assay allows rapid and sensitive measurement of apoptosis and necrosis induced by a combination of nephrotoxicants and inflammatory components such as TNF-α and can be used as an alternative assay for nephrotoxicity prediction in vitro.
Insights
A new in vitro assay effectively screens for drug-induced nephrotoxicity by measuring TNF-α-mediated apoptosis and necrosis in kidney cells, aiding in early drug safety evaluation.
Area of Science:
- * Pharmacology and Toxicology
- * Renal Cell Biology
- * Drug Safety Assessment
Background:
- * Nephrotoxicity is a primary cause for post-market drug withdrawal.
- * Tumor necrosis factor-alpha (TNF-α) secretion contributes to drug-induced kidney damage.
- * Current in vitro assays lack sensitivity for screening TNF-α-dependent nephrotoxicants.
Purpose of the Study:
- * To develop and validate a sensitive fluorescence-based in vitro assay for screening TNF-α-mediated nephrotoxicity.
- * To evaluate the assay's capability in detecting toxicant-induced apoptosis and necrosis.
- * To confirm the assay's functionality using known nephrotoxicants and inflammatory mediators.
Main Methods:
- * Utilized mouse immortalized proximal tubular epithelial cells (IM-PTECs).
- * Employed a fluorescence-based assay for fixed endpoint and live cell measurements.
- * Tested sixteen nephrotoxicants and two non-nephrotoxicants, assessing TNF-α's role.
Main Results:
- * The assay detected cell death (apoptosis and necrosis) induced by eight out of sixteen nephrotoxicants.
- * TNF-α significantly enhanced cisplatin-, cyclosporine A-, tacrolimus-, and azidothymidine-induced apoptosis.
- * Results correlated with known in vivo inflammatory effects of these nephrotoxicants.
Conclusions:
- * The developed assay provides rapid and sensitive measurement of TNF-α-mediated nephrotoxicity.
- * It effectively quantifies both apoptosis and necrosis in kidney cells.
- * This assay serves as a valuable in vitro alternative for predicting drug-induced nephrotoxicity.
