Effects of Second-Generation Calcineurin Inhibitor, Voclosporin, on Calcineurin Isoform Activity and Nephrotoxicity in Comparison to Cyclosporine and Tacrolimus

Abstract ID 90301 Poster Board 341 INTRODUCTION: Calcineurin inhibitors (CNI) are a class of immunosuppressive drugs that have revolutionized autoimmune medicine. These drugs function by inhibiting the activity of calcineurin (CN), a key enzyme involved in T cell activation and proliferation, in turn suppressing immune response. Cyclosporine (CsA) and tacrolimus (Tac) are examples of CNIs used to treat many immunological disorders. However, long-term use of CsA or Tac induced toxicity and nephritis. Voclosporin (VCS), a structural analog of CsA, was developed as an alternative to CsA for treating lupus nephritis. Previous studies have suggested VCS possessed a higher stability than its predecessor with improved potency on CN inhibition. However, the mechanism by which VCS blocks CN activity is poorly understood. Evidence from the literature indicates that alpha and beta isoforms of CN catalytic subunit A provide differential functionality in embryonic development and CsA-induced nephrotoxicity. OBJECTIVES: The objectives of this study were to assess 1) the degree of VCS cytotoxicity in comparison to existing treatment options of CsA and Tac, 2) the difference in the effect of CN inhibition by VCS compared to CsA and Tac, and 3) the specificity of which isoform of CN is affected by CN activity, nephrotoxicity and fibrosis. METHODS: Human Embryonic Kidney HEK293 cells were used as the cell model. CN alpha and beta isoform knocked-out (KO) HEK293 cell lines were developed using CRISPR gene editing. Using presto blue cell viability assay, a dose-dependent cytotoxicity assay was performed with CsA, Tac, and VCS-treated HEK293 cells. CN inhibition studies were performed in wild-type and isoform-specific KO HEK293 cells by measuring cellular CN phosphatase activity after drug treatment. Nephrotoxicity, fibrosis, and CN downstream inhibition assays were performed using RT-PCR and western blot. RESULTS: VCS, CsA, and Tac inhibited the growth of HEK293 cells in both dose- and time-dependent manners. After 48-hr incubation, VCS was less cytotoxic to HEK293 cells than CsA at 20 and 40 μM (p < 0.05), and its IC50 value (42.3 μM) was much higher than that of CsA (21.6 μM). RT-PCR arrays showed less pro-nephrotic gene upregulation when treated with VCS than CsA (p < 0.05). In addition, our data demonstrated that VCS was more effective at inhibiting CN phosphatase activity in HEK293 cells than CsA and Tac at the same dosage of 20 μM. Inhibition of CN activity was retained in the alpha isoform KO but was mostly absent in the beta isoform KO HEK293 cells. Significant fold change in anti-fibrotic marker Bone Morphogenetic Protein-7 and nephrotoxic marker Activating Transcription Factor 3 was observed when treated with VCS compared to CsA. Downstream targets of CN activity were downregulated in the beta isoform KO, not alpha isoform KO, HEK293 cells. CONCLUSIONS: These data suggest that VCS may be a more effective and safer alternative to CsA for immunosuppressive therapy regarding nephritis and fibrosis. This study uncovers some of the mechanisms of CN inhibition by elucidating that these classes of drugs target CN A beta isoform and which pathways are affected by drug treatment. Further studies are needed to fully evaluate the clinical significance of these findings and the drug's long-term safety. DISCLOSURE T. Dang, J. Tong, J.Nish, R. Saede, A. Staskiewicz, H. Ma, S. Rayalam, S. Taval, M. George-Weinstein, X. Wang, None. T. Collins, R. Huizinga, Aurinia.