lipo-MP-lPS

Bacterial lipopolysaccharides (LPS) are potent innate immunostimulants targeting the Toll-like receptor 4 (TLR4), an attractive and validated target for immunostimulation in cancer therapy. Although LPS possess anti-tumor activity, toxicity issues prevent their systemic administration at effective doses in humans. We first demonstrated that LPS formulated in liposomes preserved a potent antitumor activity per se upon systemic administration in syngeneic models, and significantly enhance the antitumor activity of the anti-CD20 antibody rituximab in mice xenografted with the human RL lymphoma model. Liposomal encapsulation also allowed a 2-fold reduction in the induction of pro-inflammatory cytokines by LPS. Mice receiving an intravenous administration demonstrated a significant increase of neutrophils, monocytes and macrophages at the tumor site as well as an increase of macrophages in spleen. Further, we chemically detoxified LPS to obtain MP-LPS that was associated with a 200-fold decrease in the induction of proinflammatory cytokines. When encapsulated in a clinically approved liposomal formulation, toxicity, notably pyrogenicity (10-fold), was limited while the antitumor activity and immunoadjuvant effect were maintained. This improved tolerance profile of liposomal MP-LPS was associated with the preferential activation of the TLR4-TRIF pathway. Finally, in vitro studies demonstrated that stimulation with encapsulated MP-LPS reversed the polarization of M2 macrophages towards an M1 phenotype, and a phase 1 trial in healthy dogs validated its tolerance upon systemic administration up to very high doses (10µg/kg). Altogether, our results demonstrate the strong therapeutic potential of MPLPS formulated in liposomes as a systemically active anticancer agent, supporting its evaluation in patients with cancer.

The preparation of cross-linked conjugate microparticles of N-succinyl-chitosan (Suc) or 6-O-carboxymethylchitin (CM) with mitomycin C (MMC), which showed an adequate si-e for liver targeting (0.2-3 microm), was attempted by a combination of water-soluble carbodiimide (EDC) coupling and emulsification technique. As for Suc, microparticles with a diameter less than a few micrometers could be obtained easily, while the preparation of CM microparticles (CM-MPs) of the same diameter was not necessarily easy. First, preparation conditions were compared for CM-MPs, and some conditions gave CM-MPs with a diameter less than a few micrometers. As to CM-MMC conjugate microparticles, the method by addition of EDC after emulsification using CM with low molecular weight (CML) gave more appropriate microparticles with a mean diameter of 0.97microm (CM1-MP-MMC). Suc-MMC conjugate microparticles adequate for liver targeting could be produced by the addition of EDC both before and after emulsification, especially, the conjugate microparticles with a mean diameter of 0.45 microm (Suc-MP-MMC) were derived by the addition of EDC before emulsification. Suc-MP-MMC exhibited a higher drug content than CML-MP-MMC. CML-MP-MMC and Suc-MP-MMC exhibited 50% drug release times of 2.87h and 42.1 h, respectively.