Reversing protonation of weakly basic drugs greatly enhances intracellular diffusion and decreases lysosomal sequestration

Abstract

Drug efficacy depends not only on delivery but also on intracellular diffusion, distribution and availability. Yet, more than a half of pharmaceutical drugs are weakly basic and thus, protonated and sequestered at intracellular pH. Protonation reduces their diffusion rates due to macromolecular crowding and nonspecific interactions, and subcellular sequestration prevents them from reaching their target due to ion trapping. Here, we explored the intracellular diffusion rates of a series of small molecular fluorescent drugs by fluorescence recovery after the photobleaching (FRAP). We found a strong correlation between drug pKaand intracellular diffusion and distribution. Weakly basic, small-molecule drugs displayed slower fraction recovery after photobleaching and 10-to-20-fold lower diffusion rates in cells than in aqueous solutions, suggesting sequestration. But while our imaging studies showed that acidic organelles, particularly the lysosome, captured these molecules, blocking lysosomal import did not improve diffusion. Instead, blocking protonation enhanced their diffusion, as shown by theirN-acetylated analogues. Moreover, co-administration with basic quinacrine, which is accumulated in lysozymes, prevented the sequestration of the anti-cancer drug mitoxantrone, thereby improving its biodistribution. Based on these results, combiningN-acetylation with co-administration may improve the intracellular diffusion and distribution of weakly basic, small molecule drugs.