RECOMBINANT HUMAN TUMOR-NECROSIS-FACTOR-ALPHA COVALENTLY CONJUGATED TO LONG-CIRCULATING LIPOSOMES

Recombinant tumor necrosis factor-alpha (rHuTNF) was covalently conjug ated to a phospholipid, N-glutaryl phosphatidylethanolamine (NGPE). Th e resultant rHuTNF-NGPE conjugates were incorporated into liposomes co mposed of phosphatidylcholine (PC) and cholesterol (Chol) with or with out polyethyleneglycol conjugated to phosphatidylethanolamine (PEG3000 -PE). Efficient incorporation (35-50%) of rHuTNF-NGPE conjugates into liposomes was obtained for both PC/Chol and PC/Chol/PEG3000-PE liposom es. An in vitro cytotoxicity assay showed that rHuTNF-NGPE conjugates incorporated into liposomes exhibit a reduced biological activity as c ompared to the free rHuTNF. Biodistribution studies using I-125-labele d rHuTNF showed a significant increase in the circulation time of rHuT NF by incorporation into PC/Chol/PEG3000-PE liposomes, but not convent ional PC/Chol liposomes. However, studies using a radioactive lipid as a liposome marker showed that incorporation of rHuTNF-NGPE conjugates resulted in increased clearance from tile blood and accumulation in t he spleen and liver of both liposomal formulations. The liposome clear ance from the blood depends on the protein/lipid ratio of liposomes. T he higher the protein/lipid ratio, the higher the liposome clearance f rom the blood and accumulation in the spleen and liver, suggesting tha t accumulation of rHuTNF-bound liposomes in the spleen and liver invol ves interactions with TNF-receptors in these organs.