Complement activation as a bioequivalence issue relevant to the development of generic liposomes and other nanoparticulate drugs

• Outlining of difficulties in generic development of liposomal drugs.
• New regulatory requirements to evaluate CARPA in preclinical studies.
• Review of complement activation by liposomes and its adverse consequences (CARPA).
• Assays of C activation in vitro and CARPA in vivo, with the porcine test in focus.
• Decision tree how to handle the risk of CARPA assessed by a battery of tests.

Liposomes are known to activate the complement (C) system, which can lead in vivo to a hypersensitivity syndrome called C activation-related pseudoallergy (CARPA). CARPA has been getting increasing attention as a safety risk of i.v. therapy with liposomes, whose testing is now recommended in bioequivalence evaluations of generic liposomal drug candidates. This review highlights the adverse consequences of C activation, the unique symptoms of CARPA triggered by essentially all i.v. administered liposomal drugs, and the various features of vesicles influencing this adverse immune effect. For the case of Doxil, we also address the mechanism of C activation and the opsonization vs. long circulation (stealth) paradox. In reviewing the methods of assessing C activation and CARPA, we delineate the most sensitive porcine model and an algorithm for stepwise evaluation of the CARPA risk of i.v. liposomes, which are proposed for standardization for preclinical toxicology evaluation of liposomal and other nanoparticulate drug candidates.