Due to the advancement of molecular biology and biotechnology, proteins and peptides are an increasingly important category of therapeutic drugs. Currently, there are at least 120 protein and peptide drugs on the market and an additional 150 in clinical trials. Although gastrointestinal absorption is a desirable route for administration, protein and peptide drugs are administered almost exclusively by injection since no acceptable oral formulations are currently available. This proposal utilizes a fusion protein drug design for oral delivery based on transferrin (Tf) receptor (TfR)-mediated transcytosis in intestinal epithelial cells, and focuses on 3 Tf- fused protein drugs, i.e., granulocyte colony-stimulating factor (G-CSF), human growth hormone (hGH) and proinsulin (ProINS). Various peptide spacers will be designed and inserted between the two domains in the fusion proteins to improve pharmacokinetics, bioavailability and bioactivity. Although the feasibility of TfR- mediated intestinal absorption of protein drugs has been demonstrated in our and others'laboratories, the in vivo modification of either protein domain during trans-epithelial transport is still largely unknown. Thus, part of our 1st specific aim is to explore and identify the modifications, both on carbohydrates or peptide bonds, of Tf during transepithelial transport in the intestine in order to improve bioavailability and efficacy of fusion proteins. Also, plasma half-lives of Tf and of each fusion protein in oral and i.v. administration will be determined, and compared and correlated with the modifications in intestinal epithelial cells that we identify. In our 2nd specific aim, we will investigate the activation of ProINS-Tf into insulin-Tf fusion protein via a TfR-mediated recycling pathway in the intestine and the liver. This activation by selective proteolysis will provide a novel approach to deliver ProINS as an oral hypoglycemic prodrug with slow activation and sustained release from intestinal epithelial cells and/or hepatocytes. Finally, in our 3rd specific aim, we will exploit findings that multivalent TfR binding increases intracellular accumulation and, consequently, transcytosis of Tf. Disulfide-linked oligomers of Tf-fusion protein will be prepared using a novel recombinant, rather than chemical, crosslinking method. Both bioactivity and plasma half-life of oligomeric fusion protein will be compared to its monomeric counterpart. Furthermore, the site of accumulation of oligomeric fusion protein in the oral absorption pathway, i.e., either the intestine or liver, will be identified. At the end of the 5-year project, our overall goal is to identify one or more Tf- fusion protein candidates for further development into effective oral therapeutics for treatment of human diseases. Relevance: Proteins and peptides are an important class of drugs for treating various human diseases;however, they are presently administered only by injection. The objective of this application is to develop recombinant proteins by fusing protein drugs with an iron-carrying protein, transferrin, to promote oral absorption. Such a new generation of protein drugs for convenient effective oral delivery will overcome patient incompliance, high cost, and adverse side effects currently associated with injectable dosage forms.
Proteins and peptides are an important class of drugs for treating various human diseases;however, they are presently administered only by injection. The objective of this application is to develop recombinant proteins by fusing protein drugs with an iron-carrying protein, transferrin, to promote oral absorption. Such a new generation of protein drugs for convenient effective oral delivery will overcome patient incompliance, high cost, and adverse side effects currently associated with injectable dosage forms.
|Wang, Yan; Shao, Juntang; Zaro, Jennica L et al. (2014) Proinsulin-transferrin fusion protein as a novel long-acting insulin analog for the inhibition of hepatic glucose production. Diabetes 63:1779-88|
|Chen, Xiaoying; Zaro, Jennica L; Shen, Wei-Chiang (2013) Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev 65:1357-69|
|Zhang, Deshui; Lee, Hsin-Fang; Pettit, Steven C et al. (2012) Characterization of transferrin receptor-mediated endocytosis and cellular iron delivery of recombinant human serum transferrin from rice (Oryza sativa L.). BMC Biotechnol 12:92|
|Chen, Xiaoying; Zaro, Jennica L; Shen, Wei-Chiang (2012) Pharmacokinetics of recombinant bifunctional fusion proteins. Expert Opin Drug Metab Toxicol 8:581-95|
|Bobst, Cedric E; Wang, Shunhai; Shen, Wei-Chiang et al. (2012) Mass spectrometry study of a transferrin-based protein drug reveals the key role of protein aggregation for successful oral delivery. Proc Natl Acad Sci U S A 109:13544-8|
|Chen, Xiaoying; Lee, Hsin-Fang; Zaro, Jennica L et al. (2011) Effects of receptor binding on plasma half-life of bifunctional transferrin fusion proteins. Mol Pharm 8:457-65|
|Wang, Yan; Chen, Yu-Sheng; Zaro, Jennica L et al. (2011) Receptor-mediated activation of a proinsulin-transferrin fusion protein in hepatoma cells. J Control Release 155:386-92|
|Amet, Nurmamet; Wang, Wei; Shen, Wei-Chiang (2010) Human growth hormone-transferrin fusion protein for oral delivery in hypophysectomized rats. J Control Release 141:177-82|
|Chen, Xiaoying; Bai, Yun; Zaro, Jennica L et al. (2010) Design of an in vivo cleavable disulfide linker in recombinant fusion proteins. Biotechniques 49:513-8|
|Norouziyan, Fariba; Shen, Wei-Chiang; Hamm-Alvarez, Sarah F (2008) Tyrphostin A8 stimulates a novel trafficking pathway of apically endocytosed transferrin through Rab11-enriched compartments in Caco-2 cells. Am J Physiol Cell Physiol 294:C7-21|
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