Nojima Yasuhiro, Suzuki Yosuke, Iguchi Kazuma, Shiga Tuneo, Iwata Aya, Fujimoto Tomohito, Yoshida Kazuhiro, Shimizu Hirohiko, Takeuchi Takashi, Sato Atsushi
School of Bioscience and Biotechnology, Tokyo University of Technology, Hachioji, Tokyo, Japan.
Bioconjug Chem. 2008 Nov 19;19(11):2253-9. doi: 10.1021/bc800258v.
Lactoferrin (LF) is an iron-binding glycoprotein that possesses multifunctional biological activities. Recent reports from clinical trials suggest that LF is potentially effective as a therapeutic protein against cancer and gangrene. However, pharmaceutical proteins such as LF are unstable in vivo. Therefore, to improve stability, we developed mono-PEGylated bovine LF (20k-PEG-bLf) with branched 20 kDa (2 x 10 kDa) poly(ethylene glycol) (PEG). We examined in vitro activities such as iron binding, IL-6 cell based assay, and resistance to a proteolytic enzyme in artificial gastric fluid. The 20k-PEG-bLf protein was fully active in iron binding and exhibited 69.6 +/- 2.9% (mean +/- S.E., n = 6) of the original anti-inflammatory activity. The proteolytic half-life increased 2-fold over that of unmodified LF. In vivo pharmacokinetic analyses were performed to examine absorption from the intestinal epithelium and serum clearance. Direct administration of 20k-PEG-bLf (30 mg/kg) into rat stomachs demonstrated that the amount of absorption from the intestinal tract increased approximately 10-fold relative to unmodified LF. Intravenous injection of the protein (1 mg/kg) revealed that 20k-PEG-bLf prolongs serum half-life by approximately 5.4-fold, and that the area under the curve (AUC) was increased approximately 9.2-fold compared to that of unmodified LF. PEGylation improved the physical and pharmacokinetic properties of bovine LF. This is the first report on the use of bioconjugation of LF for the development of a promising oral pharmaceutical agent.
乳铁蛋白（LF）是一种具有多功能生物活性的铁结合糖蛋白。近期临床试验报告表明，LF作为一种抗癌和抗坏疽的治疗性蛋白质具有潜在疗效。然而，像LF这样的药用蛋白质在体内不稳定。因此，为提高稳定性，我们研发了带有分支20 kDa（2×10 kDa）聚乙二醇（PEG）的单聚乙二醇化牛乳铁蛋白（20k-PEG-bLf）。我们检测了其体外活性，如铁结合能力、基于IL-6细胞的检测以及在人工胃液中对蛋白水解酶的抗性。20k-PEG-bLf蛋白在铁结合方面完全具有活性，并且表现出原始抗炎活性的69. ± 2.9%（平均值 ± 标准误，n = 6）。其蛋白水解半衰期比未修饰的LF增加了2倍。进行了体内药代动力学分析以检测肠道上皮的吸收和血清清除情况。将20k-PEG-bLf（30 mg/kg）直接注入大鼠胃中表明，相对于未修饰的LF，肠道吸收量增加了约10倍。静脉注射该蛋白（1 mg/kg）显示，20k-PEG-bLf使血清半衰期延长了约5.4倍，并且与未修饰的LF相比，曲线下面积（AUC）增加了约9.2倍。聚乙二醇化改善了牛乳铁蛋白的物理和药代动力学性质。这是关于利用LF的生物共轭开发一种有前景的口服药剂的首次报道。
