title: Conjugation with cationic cell-penetrating peptide increases pulmonary absorption of insulin.
abstract: In this study, we determined if cell-penetrating peptides (CPPs) can be used to enhance the absorption rate of insulin (INS) across the alveolar epithelial barrier. Using a heterobifunctional cross-linker, INS was conjugated to a series of cationic CPPs, including Tat peptide, oligoarginine (r9) or oligolysine (k9), via disulfide bridge to a D-isoform cysteine (c) present at the N-terminal of the peptide sequence, yielding INS-cTat, INS-cr9, and INS-ck9, respectively. SDS-PAGE and MALDI-TOF mass spectroscopy confirmed homogeneous conjugates with a 1:1 ratio of INS and various CPPs. Transport of INS and INS-CPPs across primary cultured rat alveolar epithelial cell monolayers was in the order INS-cr9 > INS-cTat > INS-ck9 > INS, with 27-, 19- and 4-fold increase compared to native INS, respectively. Transport of INS-cr9 was temperature- and time-dependent. Covalent conjugation between r9 and INS, as opposed to adding unconjugated INS and r9 together into donor fluid, was necessary to enhance transport of INS. Absorption of INS-cr9 across the alveolar epithelial barrier appeared to be in part transcellular, since INS-cr9 transport in the presence of heparin and protamine was decreased by approximately 20%. Adsorptive transcytosis appeared to be in part responsible for INS-cr9 absorption, as INS-cr9 did not compete with free INS in binding assays for INS receptors. Finally, intratracheal instillation of INS-cr9 in diabetic rats resulted in a steady decrease in blood glucose level that was more sustained over time when compared with INS. These results suggest that oligoarginine can be used to increase the alveolar absorption rate of insulin (and potentially other macromolecules as well).
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, Will Rogers Institute Pulmonary Research Center, Keck School of Medicine, University of Southern California, Los Angeles, California 90089-9121, USA.
authors.forename: Leena N
authors.initials: LN
authors.lastname: Patel
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Wang
authors.forename: Kwang-Jin
authors.initials: KJ
authors.lastname: Kim
authors.forename: Zea
authors.initials: Z
authors.lastname: Borok
authors.forename: Edward D
authors.initials: ED
authors.lastname: Crandall
authors.forename: Wei-Chiang
authors.initials: WC
authors.lastname: Shen
mesh_terms: D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000378:metabolism; D002478:Cells, Cultured; D003921:Diabetes Mellitus, Experimental / Q000188:drug therapy / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects; D006801:Humans; D007004:Hypoglycemic Agents / Q000737:chemistry* / Q000378:metabolism*; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism*; D008113:Liver Neoplasms / Q000188:drug therapy / Q000378:metabolism; D011650:Pulmonary Alveoli / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011972:Receptor, Insulin / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D007004:Hypoglycemic Agents; D007328:Insulin; D011994:Recombinant Proteins; D011972:Receptor, Insulin