A Rapid in Vitro Screening for Delivery of Peptide-Derived Peptidase Inhibitors as Potential Drug Candidates via Epithelial Peptide Transporters Martin Foltz, Antje Meyer, Stephan Theis, Hans-Ulrich Demuth, and Hannelore Daniel Molecular Nutrition Unit, Institute of Nutritional Sciences, Technical University of Munich, Freising-Weihenstephan, Germany (M.F., S.T., H.D.); and Probiodrug AG, Halle, Germany (A.M., H.-U.D.) Received February 3, 2004; accepted March 29, 2004 ABSTRACT Targeting drugs or prodrugs to a specific enzyme by simulta- neously targeting cell membrane carriers for efficient transport should provide the highest bioavailability along with specificity at the site of action. The peptide transporters PEPT1 and PEPT2 are expressed in a variety of tissues, including the brush-border membranes of epithelial cells of the small intes- tine and kidney. The transporters accept a wide range of sub- strates and are therefore good targets for a transporter-medi- ated drug delivery. Here, we report a screening procedure for peptidomimetic drug candidates combining two independent expression systems: 1) a competition assay in transgenic Pichia pastoris yeast cells expressing either mammalian PEPT1 or PEPT2 for identifying substrate interaction with the transporter binding site; and 2) a Xenopus laevis-based oocyte expression of the peptide transporter for assessing electrogenic transport of drug candidates. Based on the known oral availability and in vivo efficacy of the dipeptidyl peptidase IV (DPIV) inhibitor isoleucine-thiazolidide and its peptide-like structure, we first tested whether this compound is a substrate of epithelial pep- tide transporters. Additionally, a series of structurally related inhibitors were analyzed for transport. We identified various compounds that serve as substrates of the intestinal peptide transporter PEPT1. In contrast, none of these DPIV inhibitors showed electrogenic transport by PEPT2, although a variety of the compounds displayed good affinities for competition in peptide uptake in PEPT2-expressing cells, suggesting that they may serve as efficient inhibitors. In conclusion, we have applied an in vitro screening system that predicts efficient intestinal absorption of peptide-derived peptidase inhibitors via PEPT1 in vivo. The mammalian proton/peptide symporters PEPT1 and PEPT2 mediate the proton-driven and membrane potential (V m )-dependent cellular uptake of dipeptides and tripeptides into a variety of epithelial tissues. PEPT1 and PEPT2 have been characterized with respect to expression, tissue local- ization, and function (Amidon and Lee, 1994; Leibach and Ganapathy, 1996; Adibi, 1997; Daniel and Herget, 1997; Nussberger et al., 1997; Covitz et al., 1998; Fei et al., 1998). It is known that in addition to di- and tripeptides, both mammalian peptide transporters have the capability to ac- cept a large number of peptidomimetic drugs such as -lac- tam antibiotics, angiotensin-converting enzyme inhibitors, selected peptidase inhibitors, and prodrugs (Rubio-Aliaga and Daniel, 2002). PEPT1 possesses a high transport capac- ity, is expressed along the entire small intestine epithelium, and is central for the known high oral availability of peptido- mimetic drug substrates. The function of the high-affinity type transporter PEPT2 in drug transport is mainly reab- sorption of the compounds after glomerular filtration in the kidney. PEPT2 expression also has been shown in other tissues such as epithelium of the choroid plexus, lung, mam- mary gland, and glia cells of the central nervous system. Recent studies have identified major differences in the struc- tural requirements for binding and transport in substrates of PEPT1 and PEPT2. Whereas -amino fatty acids and amino acid-arylamides are recognized and transported by PEPT1 as high-affinity substrates, PEPT2 does not accept -amino fatty acids and displays a wide range of affinities and differ- ent transport characteristics when studied with amino acid- arylamides (Borner et al., 1998; Doring et al., 1998a). How- ever, modifications, such as the introduction of a carbonyl group into the backbone of omega-amino fatty acids, can transform a substrate with very low affinity into a high- affinity compound that is transported by PEPT2 electrogeni- cally (Theis et al., 2002), suggesting that PEPT2 has more This work was supported by the Federal Department of Science and Tech- nology (Bundesministerium fu ¨ r Bildung und Forschung Grant 0312302). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.104.066480. ABBREVIATIONS: PEPT, peptide transporter; V m , membrane potential; DPIV, dipeptidyl peptidase IV; PEP, prolyl endopeptidase; Thia, thiazo- lidide; Ac, acetate; Pyrr, pyrrolidide; Boc, tert-butyloxycarbonyl; Bz, benzoyl; Z, benzyloxycarbonyl; PPB, potassium phosphate buffer. 0022-3565/04/3102-695–702$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 310, No. 2 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 66480/1157386 JPET 310:695–702, 2004 Printed in U.S.A. 695 at ASPET Journals on July 19, 2018 jpet.aspetjournals.org Downloaded from