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Voclosporin is a potent immunosuppressive agent currently approved for treating active lupus nephritis. Based on its potential antiviral activity, it has also been investigated as immunosuppressive agent in an investigator-initiated study in SARS-CoV2 positive kidney transplant recipients. As with many immunosuppressive agents, optimizing dosing regimens to achieve therapeutic efficacy while minimizing toxicity remains a critical challenge in clinical practice. To prevent organ rejection as well as infections, the prescribed immunosuppression needs to be well balanced. Dried blood spot (DBS) sampling has enabled development of remote voclosporin therapeutic drug monitoring. Here, we report on the development and analytical validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for quantification of voclosporin in dried blood spots. Method development was based on previously developed assays for the quantification of tacrolimus, everolimus, sirolimus, cyclosporin, mycophenolic acid, creatinine and iohexol in DBS and voclosporin in whole blood using LC-MS/MS. HemaXis™ volumetric blood spot devices were used for sample collection. The sample purification was based on the extraction of voclosporin from the DBS samples. Stable isotopically labeled voclosporin-D4 was used as an internal standard prior to sample purification. Bland Altman and Passing bablok analysis were performed for cross validation between whole blood and DBS samples. The method was successfully validated following the current ICH M10 guidelines. The dynamic range for the analyte was 10-600 µg/L with an excellent mean coefficient of correlation of 0.9978. The within run and between run precision and accuracy were both within the acceptance criteria. The cross-validation against the whole blood method shows that the quantified voclosporin results are promising. This developed dried blood spot LC-MS/MS method was successfully validated and provides an easy, efficient workflow for therapeutic drug monitoring in kidney transplant patients or remote pharmacokinetic studies in lupus nephritis patients treated with voclosporin., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Y.K.O. Teng reports financial support and equipment, drugs, or supplies were provided by Aurinia Pharmaceuticals Inc. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The recommended immunosuppressive treatment after kidney transplantation consists of tacrolimus, mycophenolate mofetil, and low-dose corticosteroids. Drug concentrations are monitored using therapeutic drug monitoring (TDM), which does not necessarily correlate with pharmacodynamic activity. To find the balance between optimal efficacy and minimal toxicity, it might be more informative to monitor patients' immunological status rather than drug concentrations. We selected a panel of T-cell-based immune assays, which were used for immunomonitoring of 14 stable kidney transplantation patients. Whole blood was incubated with a T-cell stimulus, after which T-cell proliferation, T-cell activation marker expression and cytokine production were measured to study residual immune activity in vitro (before drug intake; drug added to the incubation) and ex vivo (after drug intake). T-cell proliferation was completely suppressed in all patients over the full day, while IL-2, IFN-γ, CD71, and CD154 showed fluctuations over the day with a strong inhibition (75%-25%) at 2 h post-dose. The level of inhibition was variable between patients and could not be related to pharmacokinetic parameters or the presence of regulatory or senescence immune cells. Moreover, the level of inhibition did not correlate with the in vitro tacrolimus drug effect as studied by incubating pre-dose blood samples with additional tacrolimus. Overall, IL-2, IFN-γ, CD71, and CD154 seem to be good markers to monitor residual immune activity of transplantation patients. To evaluate the correlation between these pharmacodynamic biomarkers and clinical outcome, prospective observational studies are needed., (© 2024 The Author(s). Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)