title: The role of citric acid in oral peptide and protein formulations: relationship between calcium chelation and proteolysis inhibition.
abstract: The excipient citric acid (CA) has been reported to improve oral absorption of peptides by different mechanisms. The balance between its related properties of calcium chelation and permeation enhancement compared to a proteolysis inhibition was examined. A predictive model of CA's calcium chelation activity was developed and verified experimentally using an ion-selective electrode. The effects of CA, its salt (citrate, Cit) and the established permeation enhancer, lauroyl carnitine chloride (LCC) were compared by measuring transepithelial electrical resistance (TEER) and permeability of insulin and FD4 across Caco-2 monolayers and rat small intestinal mucosae mounted in Ussing chambers. Proteolytic degradation of insulin was determined in rat luminal extracts across a range of pH values in the presence of CA. CA's capacity to chelate calcium decreased ~10-fold for each pH unit moving from pH 6 to pH 3. CA was an inferior weak permeation enhancer compared to LCC in both in vitro models using physiological buffers. At pH 4.5 however, degradation of insulin in rat luminal extracts was significantly inhibited in the presence of 10mM CA. The capacity of CA to chelate luminal calcium does not occur significantly at the acidic pH values where it effectively inhibits proteolysis, which is its dominant action in oral peptide formulations. On account of insulin's low basal permeability, inclusion of alternative permeation enhancers is likely to be necessary to achieve sufficient oral bioavailability since this is a weak property of CA.
authors.affiliation: Diabetes Research Unit, Novo Nordisk A/S, Måløv, Denmark; Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Søren H
authors.initials: SH
authors.lastname: Welling
authors.affiliation: Diabetes Research Unit, Novo Nordisk A/S, Måløv, Denmark.
authors.forename: František
authors.initials: F
authors.lastname: Hubálek
authors.affiliation: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Jette
authors.initials: J
authors.lastname: Jacobsen
authors.affiliation: UCD School of Veterinary Medicine and UCD Conway Institute, University College Dublin, Dublin, Ireland.
authors.forename: David J
authors.initials: DJ
authors.lastname: Brayden
authors.affiliation: Diabetes Research Unit, Novo Nordisk A/S, Måløv, Denmark.
authors.forename: Ulrik L
authors.initials: UL
authors.lastname: Rahbek
authors.affiliation: Diabetes Research Unit, Novo Nordisk A/S, Måløv, Denmark. Electronic address: spby@novonordisk.com.
authors.forename: Stephen T
authors.initials: ST
authors.lastname: Buckley
mesh_terms: D000284:Administration, Oral; D000818:Animals; D018938:Caco-2 Cells; D002118:Calcium / Q000378:metabolism*; D065096:Calcium Chelating Agents / Q000008:administration & dosage / Q000378:metabolism*; D002626:Chemistry, Pharmaceutical; D019343:Citric Acid / Q000008:administration & dosage / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000008:administration & dosage / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism; D008297:Male; D010455:Peptides / Q000008:administration & dosage / Q000378:metabolism; D059748:Proteolysis / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D012710:Serum Albumin, Bovine / Q000008:administration & dosage / Q000378:metabolism*
chemical_list: D065096:Calcium Chelating Agents; D007328:Insulin; D010455:Peptides; D012710:Serum Albumin, Bovine; D019343:Citric Acid; D002118:Calcium
keywords: Chelation; Citric acid; Insulin; Intestinal drug permeability; Oral peptide delivery; Proteolysis inhibition