title: Conjugation of cell-penetrating peptides to parathyroid hormone affects its structure, potency, and transepithelial permeation.
abstract: Delivery of therapeutic peptides and proteins by the use of cell-penetrating peptides (CPPs) as carriers has been suggested as a feasible strategy. The aim of the present study was to investigate the effect of conjugating a series of well-known CPPs to the biologically active part of parathyroid hormone, i.e., PTH(1-34), and to evaluate the effect with regard to secondary structure, potency in Saos-2 cells, immunogenicity, safety, as well as the transepithelial permeation across monolayers by using the Caco-2 cell culture model. Further, co-administration of CPP and PTH(1-34) as an alternative to covalent conjugation was compared with regard to the transepithelial permeation. CPP-conjugated PTH(1-34) fusion peptides were successfully expressed in Escherichia coli and purified from inclusion bodies. No clear correlation between the degree of secondary structure of the CPP-conjugated PTH(1-34) fusion peptides and their potency was found, albeit a general decrease in permeation was observed for both N- and C-terminally CPP-conjugated PTH(1-34) as compared to native PTH(1-34). However, attachment of CPP to the N-terminus significantly increased permeation across Caco-2 cell monolayers as compared to the corresponding C-terminally CPP-conjugated PTH(1-34). In addition, the nonaarginine sequence proved to be the only CPP capable of increasing permeation when conjugated to PTH(1-34) as compared to co-administration of CPP and PTH(1-34). This enhancement effect was, however, associated with an unacceptably low level of cell viability. In conclusion, covalent conjugation of CPPs to PTH(1-34) influenced the secondary structure, potency, and transepithelial permeation efficiency of the resulting conjugate, and hence this approach appears not to be favorable as compared to co-administration when optimizing CPP-mediated permeation of PTH(1-34) across an intestinal epithelium.
authors.affiliation: †Section for Biologics, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
authors.forename: Mie
authors.initials: M
authors.lastname: Kristensen
authors.affiliation: §Division of Immunology, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University. Yalelaan 1, 3584 CL Utrecht, The Netherlands.
authors.forename: Anne Marit
authors.initials: AM
authors.lastname: de Groot
authors.affiliation: ‡Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen, Denmark.
authors.forename: Jens
authors.initials: J
authors.lastname: Berthelsen
authors.affiliation: ∥Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Franzyk
authors.affiliation: §Division of Immunology, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University. Yalelaan 1, 3584 CL Utrecht, The Netherlands.
authors.forename: Alice
authors.initials: A
authors.lastname: Sijts
authors.affiliation: †Section for Biologics, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
authors.forename: Hanne Mørck
authors.initials: HM
authors.lastname: Nielsen
mesh_terms: D000818:Animals; D018938:Caco-2 Cells; D002463:Cell Membrane Permeability / Q000187:drug effects* / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D057846:Cell-Penetrating Peptides / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D002478:Cells, Cultured; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D010281:Parathyroid Hormone / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology
chemical_list: D057846:Cell-Penetrating Peptides; D010281:Parathyroid Hormone