Citation: Fontova, P.; van Merendonk, L.N.; Vidal-Alabró, A.; Rigo-Bonnin, R.; Cerezo, G.; van Oevelen, S.; Bestard, O.; Melilli, E.; Montero, N.; Coloma, A.; et al. The Effect of Intracellular Tacrolimus Exposure on Calcineurin Inhibition in Immediate- and Extended-Release Tacrolimus Formulations. Pharmaceutics 2023, 15, 1481. https://doi.org/10.3390/ pharmaceutics15051481 Academic Editors: Ivana Panteli´ c and Snezana Savic Received: 31 March 2023 Revised: 4 May 2023 Accepted: 10 May 2023 Published: 12 May 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). pharmaceutics Article The Effect of Intracellular Tacrolimus Exposure on Calcineurin Inhibition in Immediate- and Extended-Release Tacrolimus Formulations Pere Fontova 1,2,† , Lisanne N. van Merendonk 1,2,† , Anna Vidal-Alabró 1,2 , Raül Rigo-Bonnin 3 , Gema Cerezo 1,2 , Stefaan van Oevelen 4 , Oriol Bestard 1,2 , Edoardo Melilli 1 , Nuria Montero 1 , Ana Coloma 1 , Anna Manonelles 1 , Joan Torras 1,2 , Josep M. Cruzado 1,2 , Josep M. Grinyó 2 , Helena Colom 5, * and Nuria Lloberas 1,2, * 1 Nephrology Department, IDIBELL, Hospital Universitari de Bellvitge, 08907 Barcelona, Spain; perefontova@ub.edu (P.F.); lisannevanmerendonk@hotmail.com (L.N.v.M.); annavidal@ub.edu (A.V.-A.); gcerezo@idibell.cat (G.C.); obestard@vhebron.net (O.B.); emelilli@bellvitgehospital.cat (E.M.); n.montero@bellvitgehospital.cat (N.M.); acoloma@bellvitgehospital.cat (A.C.); amanonelles@bellvitgehospital.cat (A.M.); jtorras@bellvitgehospital.cat (J.T.); jmcruzado@bellvitgehospital.cat (J.M.C.) 2 Nephrology Laboratory, Department of Clinical Sciences, Campus Bellvitge, University of Barcelona, 08907 Barcelona, Spain; jgrinyo@ub.edu 3 Biochemistry Department, IDIBELL, Hospital Universitari de Bellvitge, 08907 Barcelona, Spain; raulr@bellvitgehospital.cat 4 Hospital Hartziekenhuis, 2500 Lier, Belgium; stefaan.van.oevelen@heilighartlier.be 5 Biopharmaceutics and Pharmacokinetics Unit, Department of Pharmacy and Pharmaceutical Technology and Physical Chemistry, School of Pharmacy, University of Barcelona, 08028 Barcelona, Spain * Correspondence: helena.colom@ub.edu (H.C.); nlloberas@ub.edu (N.L.) † These authors contributed equally to this work. Abstract: Despite intensive monitoring of whole blood tacrolimus concentrations, acute rejection after kidney transplantation occurs during tacrolimus therapy. Intracellular tacrolimus concentrations could better reflect exposure at the site of action and its pharmacodynamics (PD). Intracellular phar- macokinetic (PK) profile following different tacrolimus formulations (immediate-release (TAC-IR) and extended-release (TAC-LCP)) remains unclear. Therefore, the aim was to study intracellu- lar tacrolimus PK of TAC-IR and TAC-LCP and its correlation with whole blood (WhB) PK and PD. A post-hoc analysis of a prospective, open-label, crossover investigator-driven clinical trial (NCT02961608) was performed. Intracellular and WhB tacrolimus 24 h time-concentration curves were measured in 23 stable kidney transplant recipients. PD analysis was evaluated measuring calcineurin activity (CNA) and simultaneous intracellular PK/PD modelling analysis was conducted. Higher dose-adjusted pre-dose intracellular concentrations (C 0 and C 24 ) and total exposure (AUC 0–24 ) values were found for TAC-LCP than TAC-IR. Lower intracellular peak concentration (C max ) was found after TAC-LCP. Correlations between C 0 ,C 24 and AUC 0–24 were observed within both formu- lations. Intracellular kinetics seems to be limited by WhB disposition, in turn, limited by tacrolimus release/absorption processes from both formulations. The faster intracellular elimination after TAC- IR was translated into a more rapid recovery of CNA. An Emax model relating % inhibition and intracellular concentrations, including both formulations, showed an IC 50 , a concentration to achieve 50% CNA inhibition, of 43.9 pg/million cells. Keywords: tacrolimus; leukocytes; mononuclear; kidney transplantation; pharmacokinetics; pharmacodynamics 1. Introduction Tacrolimus (TAC) is a calcineurin inhibitor and key immunosuppressant prescribed after kidney transplantation to prevent graft rejection. TAC has a narrow therapeutic Pharmaceutics 2023, 15, 1481. https://doi.org/10.3390/pharmaceutics15051481 https://www.mdpi.com/journal/pharmaceutics