Gastrointestinal (Gl) absorption is a desirable route for administration of drugs including therapeutic peptides and proteins, because it can reduce the inconvenience, cost, and complications associated with injection, and increase patient compliance especially in chronic administration. However, no acceptable oral formulation is currently available for peptide and protein drugs. In this application, recombinant fusion proteins with a transferrin (Tf) domain will be designed and developed as a new class of protein drugs that can be orally administered. This novel protein drug design is based on our recent findings on the recombinant fusion protein between human Tf and granulocyte colony stimulating factor (G-CSF), which demonstrated an effective myelopoietic activity when orally administered to BDF1 mice. In this application, G-CSF-Tf will be studied, with a multidisciplinary approach including molecular biology, computer modeling, cell biology, and protein chemistry, to optimize the biological activity, as well as the applicability, of the recombinant products. In addition, the pharmacokinetics and biodistribution of the fusion protein will be investigated in order to elucidate the underlying mechanism of TfR-mediated protein absorption from the Gl tract. Thus, the specific aims during the next 4 years are first to prepare G-CSF-Tf constructs with different spacers and to determine their in vitro biological activities, i.e., Tf receptor binding and NFS-60 cell proliferation. These peptide spacers will be designed either to maintain a distance between the two domains to minimize the mutual interference, or to allow for separation of the two domains in the blood to regenerate the intact and fully bioactive protein drug. Subsequently, the selected fusion proteins with optimal linkers will be compared for in vivo myelopoietic activity with both subcutaneous and oral administration. Finally, pharmacokinetics and biodistribution of the selected fusion proteins will be investigated in BDF1 mice using a specially designed ELISA method. The potential Gl alteration and toxicity from repeated oral adminsitration in mice will be studied. The previously observed prolonged in vivo effect of orally administered G-CSF-Tf fusion protein will also be examined in correlation with dietary iron levels. These data will provide information regarding the deposition, bioavailability, and transport mechanism. Our long term goal is to develop Tf-fusion proteins into a new generation of protein drugs that can be administered orally to treat 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 linking the protein drug with transferrin to promote oral absorption. Such a new generation of protein drugs for effective oral delivery will overcome patient incompliance, cost, and side effects currently associated with injectable dosage forms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063647-08
Application #
7793358
Study Section
Gene and Drug Delivery Systems Study Section (GDD)
Program Officer
Okita, Richard T
Project Start
2002-04-01
Project End
2011-07-14
Budget Start
2010-04-01
Budget End
2011-07-14
Support Year
8
Fiscal Year
2010
Total Cost
$258,192
Indirect Cost
Name
University of Southern California
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Shao, Juntang; Zaro, Jennica L; Shen, Wei-Chiang (2016) Proinsulin-Transferrin Fusion Protein Exhibits a Prolonged and Selective Effect on the Control of Hepatic Glucose Production in an Experimental Model of Type 1 Diabetes. Mol Pharm 13:2641-6
Shao, Juntang; Zaro, Jennica L; Shen, Wei-Chiang (2016) Tissue barriers and novel approaches to achieve hepatoselectivity of subcutaneously-injected insulin therapeutics. Tissue Barriers 4:e1156804
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
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
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
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

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