02506nas a2200241   4500000000100000008004100001260001500042100002200057700002100079700001600100700002400116700002000140700002000160700002200180700002000202700001700222245008500239856003900324300001400363490000700377520186600384022001402250       2025                            d  c2025-04-071 aFrancesca Fanizza1 aSimone Perottoni1 aLucia Boeri1 aFrancesca Donnaloja1 aFrancesca Negro1 aFrancesca Pugli1 aGianluigi Forloni1 aCarmen Giordano1 aDiego Albani00aA gut–brain axis on-a-chip platform for drug testing challenged with donepezil  uhttps://doi.org/10.1039/d4lc00273c  a1854-18740 v253 aCurrent drug development pipelines are time-consuming and prone to a significant percentage of failure, partially due to the limited availability of advanced human preclinical models able to better replicate the in vivo complexity of our body. To contribute to an advancement in this field, we developed an in vitro multi-organ-on-a-chip system, that we named PEGASO platform, which enables the dynamic culturing of human cell-based models relevant for drug testing. The PEGASO platform is composed of five independent connected units, which are based on a previously developed millifluidic organ-on-a-chip device (MINERVA 2.0), hosting human primary cells and iPSC-derived cells recapitulating key biological features of the gut, immune system, liver, blood–brain-barrier and brain that were fluidically connected and challenged to model the physiological passage of donepezil, a drug prescribed for Alzheimer's disease. The nutrient medium flow rate of the connected units was tuned to obtain suitable oxygenation and shear stress values for the cells cultured in dynamic condition. A computational model was at first developed to simulate donepezil transport within the platform and to assess the drug amount reaching the last organ-on-a-chip. Then, we demonstrated that after 24 hours of donepezil administration, the drug was actually transported though the cell-based models of the platform which in turn were found viable and functional. Donepezil efficacy was confirmed by the decreased acetylcholinesterase activity at the brain model and by the increased expression of a donepezil-relevant multi-drug transporter (P-gp). Overall, the PEGASO platform is an innovative in vitro tool for drug screening and personalized medicine applications which holds the potential to be translated to preclinical research and improve new drug development pipelines.  a1473-0197