Optimization of Transcardiac Perfusion for More Accurately Evaluating Biodistribution of Large Molecules

Abstract

The accurate assessment of drug concentrations in biodistribution studies is crucial for evaluating the efficacy and toxicity of compounds in drug development. As the concentration of biologics in plasma can be higher than in tissue due to their potentially low volume of distribution, transcardiac perfusion is commonly employed to reduce the influence of excess drugs in residual blood. However, there is a lack of consistency in the literature on the conditions and methods of perfusion. To enhance blood removal during transcardiac perfusion, sodium nitrite (NaNO2), a vasodilator, has been widely used with concentrations up to 5% in publications. However, we found that such high NaNO2 could disrupt the BBB during perfusion, which should be avoided in experiments. In this study, we examined the impact of various vasodilators on blood–brain barrier integrity and vascular permeability using the ratio of FITC-Dextran to Texas Red-Dextran (FITC/Texas Red). Additionally, we optimized perfusion conditions—including euthanasia method and perfusion flow rate—based on hemoglobin levels and the FITC/Texas Red ratio in tissues. Despite the superiority of NaNO2 in terms of solubility and cost over other vasodilators, we found that 2% NaNO2 disrupted blood–brain barrier integrity, significantly altering the FITC/Texas Red ratio. In contrast, 100 mM NaNO2 did not significantly affect this ratio. Moreover, under Ketamine/Xylazine (Ket/Xyl) anesthesia, which reduced blood clot formation compared to CO2 euthanasia, 100 mM NaNO2 achieved the lowest hemoglobin levels in the brain. Compared to other vasodilators and the PBS control group, 100 mM NaNO2 decreased the tissue/plasma ratio (Kp,t) but not brain/plasma ratio (Kp,b) of hIgG1 and human transferrin. We have developed a method to efficiently evaluate blood–brain barrier integrity during transcardiac perfusion. The combination of Ket/Xyl anesthesia and 100 mM NaNO2 effectively removes residual blood from tissues without significantly affecting blood vessel permeability.