Binding of novel peptide inhibitors of type IV collagenases to phospholipid membranes and use in liposome targeting to tumor cells in vitro.

We have recently described a novel cyclic peptide inhibitor CTTHWGFTLC (CTT) for matrix metalloproteinases (MMP)-2 and MMP-9, also called type IV collagenases or gelatinases (E. Koivunen et al., NAT: BIOTECHNOL:, 17: 768-774, 1999). As indicated by its amino acid composition, CTT is hydrophobic, and its partitioning into phospholipid films could be verified by the monolayer technique. Augmented fluorescence emission anisotropy (from 0.064 to 0.349) and reduced collisional quenching by I(-) of the Trp residue in CTT was evident in the presence of unilamellar phosphatidylcholine/phosphatidylethanolamine liposomes, revealing the association of CTT with the lipid bilayers. Gelatinases are potential targets of therapeutic intervention in cancer, and inhibitors of these enzymes can prevent tumor progression in animal models. CTT enhanced 3- to 4-fold the cellular uptake of liposome-encapsulated water-soluble fluorescent marker, rhodamine B by gelatinase-expressing cells. Gelatinase targeting seems to be essential, as modified peptides that were less potent gelatinase inhibitors were also less efficient in promoting the cellular uptake of liposomes. Augmented killing ( approximately 4-fold) of U937 leukemia and HT1080 sarcoma cells was obtained by the CTT-enhanced delivery of Adriamycin-containing liposomes, compared with control liposomes administered without the peptide. These results suggest a novel type of utility for small gelatinase inhibitors in targeted cancer therapy.

Sphingomyelinase activity associated with human plasma low density lipoprotein.

Isolated human plasma low density lipoprotein (LDL) was observed to possess sphingomyelinase activity. Accordingly, the formation of ceramide was catalyzed by LDL at 37 degrees C using tertiary liposomes composed of sphingomyelin (mole fraction (x) = 0.2), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (x = 0.7), 1, 2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (x = 0.1), and either the fluorescent sphingomyelin analog Bodipy-sphingomyelin or [(14)C]sphingomyelin as substrates. However, this activity was not present in either very low density lipoprotein or the high density lipoprotein subfractions HDL(2) and HDL(3). Oxidation of LDL abrogated its sphingomyelinase activity. Aggregation of the liposomes upon incubation with LDL was evident from the light scattering measurements. Microinjection of LDL to the surface of giant liposomes composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), N-palmitoyl-d-sphingomyelin (C16:0-sphingomyelin), and Bodipy-sphingomyelin as a fluorescent tracer (0.75:- 0.20:0.05, respectively) revealed the induction of vectorial budding of vesicles, resembling endocytosis.

Tumor targeting with a selective gelatinase inhibitor.

Several lines of evidence suggest that tumor growth, angiogenesis, and metastasis are dependent on matrix metalloproteinase (MMP) activity. However, the lack of inhibitors specific for the type IV collagenase/gelatinase family of MMPs has thus far prevented the selective targeting of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) for therapeutic intervention in cancer. Here, we describe the isolation of specific gelatinase inhibitors from phage display peptide libraries. We show that cyclic peptides containing the sequence HWGF are potent and selective inhibitors of MMP-2 and MMP-9 but not of several other MMP family members. Our prototype synthetic peptide, CTTHWGFTLC, inhibits the migration of human endothelial cells and tumor cells. Moreover, it prevents tumor growth and invasion in animal models and improves survival of mice bearing human tumors. Finally, we show that CTTHWGFTLC-displaying phage specifically target angiogenic blood vessels in vivo. Selective gelatinase inhibitors may prove useful in tumor targeting and anticancer therapies.