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Previous studies show that chronic ethanol treatment induces prominent changes in brain neuropeptide Y (NPY). The purpose of the present study was to explore ethanol effects at a deeper NPY-system level, measuring expression of NPY and its receptors (Y1, Y2, Y5) as well as NPY receptor binding and NPY-stimulated [(35)S]GTPgammaS functional binding. Rats received intragastric ethanol repeatedly for 4 days, and the NPY system was studied in the hippocampal dentate gyrus (DG), CA3, CA1, and piriform cortex (PirCx) and neocortex (NeoCx) during intoxication, peak withdrawal (16 hr), late withdrawal (3 days), and 1 week after last ethanol administration. NPY mRNA levels decreased during intoxication and at 16 hr in hippocampal regions but increased in the PirCx and NeoCx at 16 hr. NPY mRNA levels were increased at 3 days and returned to control levels in most regions at 1 week. Substantial changes also occurred at the receptor level. Thus Y1, Y2, and Y5 mRNA labelling decreased at 16 hr in most regions, returning to control levels at 3 days, except for PirCx Y2 mRNA, which increased at 3 days and 1 week. Conversely, increases in NPY receptor binding occurred in hippocampal regions during intoxication and in functional binding in the DG and NeoCx during intoxication and at 16 hr and in PirCx during intoxication and at 1 week. Thus this study shows that ethanol intoxication and withdrawal induce complex plastic changes in the NPY system, with decreased/increased gene expression or binding occurring in a time- and region-specific manner. These changes may play an important role in mediating ethanol-induced changes in neuronal excitability.

BackgroundRecombinant interleukin-2 (IL-2, aldesleukin) is an approved cancer immunotherapy but causes severe toxicities including cytokine storm and vascular leak syndrome (VLS). IL-2 promotes antitumor function of IL-2Rβ/γ+ natural killer (NK) cells and CD8+, CD4+ and gamma delta (γδ) T cells. However, IL-2 also potently activates immunosuppressive IL-2Rα+ regulatory T cells (Tregs) and IL-2Rα+ eosinophils and endothelial cells, which may promote VLS. Aldesleukin is rapidly cleared requiring frequent dosing, resulting in high Cmax likely potentiating toxicity. Thus, IL-2 cancer immunotherapy has two critical drawbacks: potent activation of undesired IL-2Rα+ cells and suboptimal pharmacokinetics with high Cmax and short half-life.MethodsTransCon IL-2 β/γ was designed to optimally address these drawbacks. To abolish IL-2Rα binding yet retain strong IL-2Rβ/γ activity, IL-2 β/γ was created by permanently attaching a small methoxy polyethylene glycol (mPEG) moiety in the IL-2Rα binding site. To improve pharmacokinetics, IL-2 β/γ was transiently attached to a 40 kDa mPEG carrier via a TransCon (transient conjugation) linker creating a prodrug, TransCon IL-2 β/γ, with sustained release of IL-2 β/γ. IL-2 β/γ was characterized in binding and primary cell assays while TransCon IL-2 β/γ was studied in tumor-bearing mice and cynomolgus monkeys.ResultsIL-2 β/γ demonstrated selective and potent human IL-2Rβ/γ binding and activation without IL-2Rα interactions. TransCon IL-2 β/γ showed slow-release pharmacokinetics with a low Cmax and a long (>30 hours) effective half-life for IL-2 β/γ in monkeys. In mouse tumor models, TransCon IL-2 β/γ promoted CD8+ T cell and NK cell activation and antitumor activity. In monkeys, TransCon IL-2 β/γ induced robust activation and expansion of CD8+ T cells, NK cells and γδ T cells, relative to CD4+ T cells, Tregs and eosinophils, with no evidence of cytokine storm or VLS. Similarly, IL-2 β/γ enhanced proliferation and cytotoxicity of primary human CD8+ T cells, NK cells and γδ T cells.SummaryTransCon IL-2 β/γ is a novel long-acting prodrug with sustained release of an IL-2Rβ/γ-selective IL-2. It has remarkable and durable pharmacodynamic effects in monkeys and potential for improved clinical efficacy and tolerability compared with aldesleukin. TransCon IL-2 β/γ is currently being evaluated in a Phase 1/2 clinical trial (NCT05081609).