Abstract
Keratinocyte growth factor 1 (KGF-1) has proven useful in the treatment of pathologies associated with dermal adnexae, liver, lung, and the gastrointestinal tract diseases. However, poor stability and short plasma half-life of the protein have restricted its therapeutic applications. While it is possible to improve the stability and extend the circulating half-life of recombinant human KGF-1 (rhKGF-1) using solution-phase PEGylation, such preparations have heterogeneous structures and often low specific activities due to multiple and/or uncontrolled PEGylation. In the present study, a novel solid-phase PEGylation strategy was employed to produce homogenous mono-PEGylated rhKGF-1. RhKGF-1 protein was immobilized on a Heparin-Sepharose column and then a site-selective PEGylation reaction was carried out by a reductive alkylation at the N-terminal amino acid of the protein. The mono-PEGylated rhKGF-1, which accounted for over 40% of the total rhKGF-1 used in the PEGylation reaction, was purified to homogeneity by SP Sepharose ion-exchange chromatography. Our biophysical and biochemical studies demonstrated that the solid-phase PEGylation significantly enhanced the in vitro and in vivo biostability without affecting the over all structure of the protein. Furthermore, pharmacokinetic analysis showed that modified rhKGF-1 had considerably longer plasma half-life than its intact counterpart. Our cell-based analysis showed that, similar to rhKGF-1, PEGylated rhKGF-1 induced proliferation in NIH 3T3 cells through the activation of MAPK/Erk pathway. Notably, PEGylated rhKGF-1 exhibited a greater hepatoprotection against CCl4-induced injury in rats compared to rhKGF-1.
Highlights
Keratinocyte growth factor 1 (KGF-1), known as fibroblast growth factor 7 (FGF-7), is a paracrine-acting mitogen produced by cells of mesenchymal origin in response to pro-inflammatory cytokines and steroid hormones [1,2,3,4]
Preparation of Recombinant human KGF-1 (rhKGF-1) The cDNA of rhKGF-1 was cloned into the NdeI and BamHI
A wealth of pharmacological studies has been previously shown that KGF-1, a member of the FGF family, has a high therapeutic potential in preventing oral mucositis in patients with haematological malignancies [8,26]
Summary
Keratinocyte growth factor 1 (KGF-1), known as fibroblast growth factor 7 (FGF-7), is a paracrine-acting mitogen produced by cells of mesenchymal origin in response to pro-inflammatory cytokines and steroid hormones [1,2,3,4]. KGF-1 is a heparin binding growth factor that acts exclusively through a splicing variant of FGF receptor 2, FGFR2-IIIb, which is expressed predominantly by epithelial cells in the tongue, oral mucosa and gastrointestinal tract as well as liver, lung and pancreas [5]. Since KGF-1 impacts proliferation and differentiation in parenchymal epithelial cells of differentiated tissues, it has been proposed for treatment of pathologies associated with dermal adnexae, liver, lung, and the gastrointestinal tract diseases, wound healing in various tissues and organs [8,9]. RhKGF-1, seems a major breakthrough in the management of patients receiving intensive treatment for solid tumours and haematological malignancies This has led the Food and Drug Administration (FDA) to approve rhKGF-1, known as Palifermin, for the prevention of oral mucositis of patients with haematological malignancies and boosted the development of other mucosa-protective growth factors, such as repifermin (KGF-2) and velafermin (FGF-20)
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