A recently developed inhibitor of the retrograde transport, namely Retro-2.1, proved to be a potent and broad-spectrum lead in vitro against intracellular pathogens such as toxins, parasites, intracellular bacteria, and viruses. In order to circumvent its low aqueous solubility, a formulation in poly(ethylene glycol)-bloc-poly(D,L)lactide micelles was developed. This formulation allowed studying Retro-2.1 pharmacokinetic parameters in mice following intravenous or intraperitoneal injection, revealing a low blood circulation time. To explain these poor pharmacokinetic parameters, Retro-2.1 metabolic stability was studied in vitro and in vivo revealing a fast cytochrome P-450-mediated metabolism into a less potent hydroxylated analog. Subcutaneous injection of Retro-2.1 formulated in a biocompatible and bioresorbable polymer-based thermosensitive hydrogel allowed for a sustained-release of the drug and a better control of its metabolism. This study gives a guideline on how to administer this promising lead in vivo in order to study its efficacy.