Among developing drugs, the most promising new drug candidates may be targeting protein-protein interactions (PPIs) in cytosol space. Cyclic peptides are leading categorized compounds as inhibitors of PPIs for their structural rigidity and chemical stability. In order to inhibit efficiently, the cyclic peptides should be larger than conventional small molecule drugs to cover larger surface area of PPI. However, the larger candidate peptide to be, the less it permeable passively to get through cell membrane.¹ We have been developing novel cell penetrating peptides (CPPs)² and investigating mechanisms.³ One of our aims is to facilitate translocation of such large molecular weight PPI inhibitors that have intrinsically poor cell-permeability, when it compares with small molecules.

As a proof of concept, we try to improve permeability of cyclosporine A (CsA), which is an exceptional example of a bioavailable cyclic peptide that has been the focus of drug development. However, the aqueous solubility and membrane permeability of this peptide are low in comparison to those of small molecule drugs, which together narrow its therapeutic index limit its use as a drug.  In this study, we observed that the cell penetrating peptide (CPP), LK-3, facilitates the delivery of nanomolar concentrations of CsA into cells. Observations suggest that LK-3 induces active transport of CsA through macropinocytosis that differs from the conventional passive diffusion followed by CsA. As a result, LK-3 causes a ca. 10-fold increase in cell penetration of CsA at nanomolar concentrations, which leads to an improvement of the efficacy of this cyclic peptide for reduction of inflammatory stimuli such as IL-6 and IL-8. Moreover, the results of in vivo dry eye mouse model demonstrated that a 100-fold lower dose of the CsA/LK-3 complex than that of Restasis® is sufficient to cause the same therapeutic effect.