To design a releasable PEGylated TNF-α(rPEG-TNF-α ), a cathepsin B-sensitive dipeptide (Val-Cit moiety) was inserted into conventional PEG-modified TNF- (PEG-TNF- ), facilitating its clinical use for anti-tumor therapy. Comparative pharmaco- kinetic and pharmacodynamic studies showed that the half-lives of both PEGylated forms of TNF-α were ~60-fold greater than that of unmodified TNF-α . In addition, the in vitro bioactivity of rPEG-TNF-α was greater than that of PEG-TNF-α with the same degree of PEG modification. Release of TNF-α from rPEG-TNF-α in vitro was dependent on the presence of cathepsin B and was inhibited by a cathepsin B inhibitor. Despite the potent cytotoxicity of unmodified TNF-α against normal cells, its PEGylated forms at higher TNF-α concentrations showed low cytotoxic activity against these cells. In contrast, both forms of PEGylated TNF-α showed potent cytotoxic activity against the B16 and L929 cell lines, with rPEG-TNF-α being 5- and 9- fold more potent, respectively, than PEG-TNF-α . Moreover, rPEG-TNF-α was a more potent in vivo antitumor agent than PEG-TNF-α .
To improve the pharmacological profile of tumor necrosis factor alpha(TNF-α),we have synthesized a new PEGylated prodrug,PEG-vcTNF-α,using a cathepsin B-sensitive dipeptide(valine-citrulline,vc) to link branched PEG and TNF-α.PEG-modified TNF-α without the dipeptide linker(PEG-TNF-α) and unconjugated TNF-α were also tested as controls.It was found for the first time that TNF-α released from PEG-vcTNF-α was specifically dependent on the presence of cathepsin B.PEG-vcTNF-α induced higher cytotoxicity and greater apoptosis against L929 murine fibrosarcoma cells than PEG-TNF-α.Reversal of these effects by a cathepsin-B inhibitor confirmed that these effects were mediated by cathepsin B-specific release of TNF-α.In vivo pharmacokinetics studies demonstrated that the plasma stability of PEG-vcTNF-α was significantly increased compared to TNF-α.Finally,the improved anticancer efficacy of PEG-vcTNF-α and the distinct activities among the three formulations confirmed the positive contribution of both PEGylation and the dipeptide linkage to the improved drug-like properties of PEG-vcTNF-α.The results here indicate that linking proteins and PEG via the cathepsin B-sensitive dipeptide may be a promising strategy for developing protein therapeutics.
DAI ChuanYunFU YaLI BiaoWANG YiGuangZHANG XuanWANG JianChengZHANG Qiang
