Lactoferrin (LF) is an 80-kDa member of the transferrin family of iron-binding glycoproteins and is found in various biological fluids, especially milk.2) The multifunctional LF protein exhibits a wide variety of activities including immunomodulation, 3) iron binding, 4) anti-microbial, 5) anti-inflammatory, 6,7) cell proliferation 8) and anti-oxidation. 9) This multitude of biological activities has created great interest in the use of LF in drug discovery.10) Recently, we have developed PEGylated LF with high biological activities and enhanced pharmacokinetics properties.11) PEGylation refers to the modification of biological molecules by the linking of one or more polyethylene glycol (PEG) groups. This approach is utilized in many pharmaceutical and biotechnical applications. 12,13) N-Hydroxysuccinimide (NHS) active esters of PEG (PEG-NHS) are frequently used for the amino group modification of target proteins. NHS activated esters produce stable amide linkages between PEG and primary amines such as N-terminal a-amine and lysine e-amine residues. PEG-NHS typically couples to the free amino group of the targeted protein at physiological pH, ranging from pH 7 to 9. NHS active esters are known to possess highly hydrolytic activities under basic conditions. Although bioconjugation by NHS active esters is generally considered to be the mechanism of PEGylation, detailed reports of actual PEGylated reactions by NHS active esters are rare. Thus, the aim of this study was to investigate the kinetics of the conjugation reaction between branched PEG-NHS and bovine lactoferrin (bLF), focusing on pH dependency. The present investigation demonstrates the importance of the pH values in the PEGylated reactions. MATERIALS AND METHODSpH-Dependent PEGylation of Bovine Lactoferrin (bLF) PEGylated reaction mixtures contained bLF (MG nutritionals, Melbourne, Australia) and branched 40-kDa PEG-NHS reagent (SUNBRIGHT GL2-400GS2, NOF Corporation, Tokyo, Japan) at a 1 : 10 molar ratio. Reaction mixtures were dissolved in the following buffers at the indicated pH: 50 mM acetate buffer (pH 4.0, 5.0); 50 mM phosphate buffer (pH 6.0, 7.0, 8.0); or 50 mM borate buffer (pH 9.0). The final bLF concentration was 0.5 mg/ml. Reactions were carried out for 1 h at 25°C and stopped by the addition of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. The reaction mixtures were then subjected to 7.5% SDS-PAGE under non-reducing conditions and visualized by Coomassie Brilliant Blue (CBB) staining. Conjugation Reaction Kinetics in Buffers of Different pH ValuesTwo kinds of branched PEG-NHS with average molecular weights of 20 kDa (SUNBRIGHT GL2-200GS2, NOF Corporation, Tokyo, Japan) or 40 kDa (see above) were used. Conjugation reactions were carried out as described above except that the incubation time was 24 h, and the buffers used were phosphate-buffered saline (PBS, pH 7.4), 50 mM borate (pH 9.0) and Good's buffers (BICINE, CHES and TAPS, pH 9.0). Reaction samples (corresponding to 2 mg bLF) were taken at indicat...

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.