Steatotic liver defatting by ex vivo perfusion with a novel ready-to-use rapamycin formulation: Preclinical results of the RAPAPERF study

Background: Steatotic liver grafts are increasingly prevalent due to obesity and metabolic syndrome and are associated with inferior post-transplant outcomes, leading to frequent organ discard and worsening donor shortage. Normothermic machine perfusion (NMP) enables ex situ pharmacological “defatting”, but translation is hampered by complex multi-agent cocktails and perfusion-circuit compatibility issues. Rapamycin, a clinically approved mTOR inhibitor, can restore lipid homeostasis by reducing de novo lipogenesis, enhancing autophagy, and promoting fatty acid β-oxidation through multiple distinct metabolic pathways. The study’s objective is to develop and assess a dedicated, ready-to-use rapamycin formulation compatible with the ex‑situ liver perfusion.

Methods: A pharmaceutical-grade rapamycin formulation optimized for perfusion use was developed and validated for solubility and stability. Defatting efficacy and cytotoxicity were assessed in steatotic primary human hepatocytes (PHHs) and precision-cut liver slices (PCLS). Ex vivo perfusions were performed using a controlled oxygenated rewarming protocol followed by normothermic perfusion (NMP) in porcine livers and discarded human livers. We evaluated adsorption and pharmacokinetics, tissue triglycerides (TG) content, mTOR pathway inhibition (p70S6K1 phosphorylation), histology, and mechanism-of-action gene expression (RT-qPCR).

Results: The selected formulation (200nM, 85% DMSO/15% water) remained stable for up to 24 months when stored at −20°C. In vitro, rapamycin reduced intracellular TG content by 20% in steatotic PHHs and by 28% in PCLS, without affecting viability. In a blood-containing circuit without an organ, target rapamycin concentrations were reached within minutes and remained stable, with no detectable adsorption to circuit components. In discarded steatotic human livers, rapamycin decreased tissue TG by 22% versus no significant change in vehicle controls, without evidence of treatment-related histological injury. On-target activity was confirmed by decreased phosphorylation of p70S6K1 and transcriptional changes consistent with increased autophagy (LC3, SIRT1) and reduced de novo lipogenesis (FAS, SREBP1), alongside activation of lipid oxidation/export markers.

Conclusion: A perfusion-compatible, ready-to-use rapamycin formulation enables a single-agent defatting approach of potential translational interest, achieving a measurable TG reduction and mTOR pathway inhibition in preclinical models and discarded human livers. As an exploratory proof-of-concept, this strategy warrants further functional and transplantation studies before clinical evaluation.