Novel Liposaccharide Conjugates for Drug and Peptide Delivery BRUNO DROUILLAT, ² ANYA M. HILLERY, ‡ GYULA DEKANY, ² ROBERT FALCONER, ² KAREN WRIGHT, ² AND ISTVAN TOTH* ,² Contribution from the the Department of Pharmaceutical and Biological Chemistry, The School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, U.K., and the Department of Pharmacy, University of Brighton, Moulsecoomb, Brighton BN2 4GJ, U.K. Received May 29, 1997. Accepted for publication September 26, 1997 X . Abstract 0 Sugar-lipid conjugates with general structure 1-4 were prepared by coupling amino sugars with N-Boc-protected lipoamino acids and oligomers. Conjugates with general structure 5 were also prepared from glucuronic acid and methyl 2-aminohexadecanoate. The physicochemical properties of the conjugates were modified by varying the nature and number of sugars or the number of lipoamino acids or their alkyl chain length. The ability of the liposaccharides to aggregate was examined. These preliminary experiments have demonstrated the ability of the liposaccharides to form particulate systems per se and also their ability to be incorporated into conventional liposomal systems. The structure of the respective liposaccharides and the molar ratio of liposaccharide to dimyristoyl lecithin and cholesterol were found to have a profound effect on the type of colloidal systems produced. Introduction The efficient oral delivery of poorly absorbed biomol- ecules such as peptides and proteins represents a major challenge. Such compounds have considerable potential both as therapeutic drugs and as vaccines, but various factors associated with the GI (gastrointestional) tract, including the presence of degradative enzymes, extremes of pH, and hydrophobic membranes, combine to make their oral delivery extremely difficult. The basis of our strategy to improve the oral bioavail- ability of such compounds has been to conjugate them with lipoamino acids (LAAs), i.e., R-amino acids having long alkyl side chains. 1 Conjugation with an LAA increases the lipophilicity of a synthetic peptide, thereby increasing the membrane permeability of the drug. The degree of lipidic character of the LAA system can be modified to allow selection of a drug-LAA conjugate possessing optimal lipophilicity for oral absorption. In addition to increasing the lipophilicity, the long alkyl chains of the LAAs are also capable of conferring protection from enzymatic attack on a labile drug moiety. The LAA system has been used to increase the oral absorption of various drug molecules, including peptides, 2 -lactam antibiotics 3 , and benzoquino- lizine alkaloids. 4 Covalent coupling of drugs with LAAs may result in highly hydrophobic conjugates which are insufficiently soluble in water for use in drug delivery. To overcome the problems of low water solublility, LAAs have been conju- gated with mono- and oligosaccharides, to form liposac- charides 3,5 which represent novel carbohydrate-based sys- tems, whose amphipathic structure raises the possibility that they may form particulate systems, with applications as colloidal carriers for the oral route. By exploiting the potential of the liposaccharides to form colloidal carriers, peptide antigens or drugs may be pro- tected from the harsh conditions of the GI tract. Many modifications to the liposaccharides are possible (molecular weight, charge, lipophilicity, number of sugars, etc.) allow- ing tailoring of the system to allow the preparation of particulate systems with optimal properties for each po- tential drug or antigen candidate. This approach may be of particular interest for the oral delivery of synthetic peptide vaccines, as the demonstrated accumulation of particles in the lymphoid sections of the GI tract, particularly the Peyer’s patches of the GALT, 6,7 may facilitate the passive targeting of peptide antigens to these immunocompetent sites. Due to the common mu- cosal immune system (CMIS), specific antigen-activated lymphocytes from the Peyer’s patches can subsequently migrate to various other mucosal and glandular tissues, producing a disseminated mucosal immune response. 8,9 Sugar-lipid conjugates can act as immunostimulators, 10 and colloidal systems such as liposomes and microspheres can have adjuvant activity, 11-13 which may further potenti- ate the desired effects. The presence of the sugar moieties may facilitate targeting of the antigens to specific immu- nocompetent regions of the GI tract, further optimizing antigenic delivery and presentation. We describe here the synthesis of 1-N-substituted de- rivatives of 1-deoxy--D-glycopyranosylamine and lipidic amino acids (Chart 1), investigations into their particle- forming behavior with particular reference to the influence of the sugar and the 1-N-substituent on the type of supramolecular assemblies produced, and also their inter- action with phospholipid systems. Experimental Methods Chemical Synthesiss 1 H NMR spectra were obtained on Bru ¨ ker AM250 and Bru ¨ ker AM500 instruments operat- ing at fields of 250 and 500 MHz, respectively; chemical shifts are reported in ppm downfield from internal TMS. Mass spectra were run on a VG Analytical ZAB-SE instrument, using fast atom bombardment (FAB) tech- niques and VG Analytical TofSpec, using matrix-assisted laser desorption (MALD) ionization at a wavelength of 337 nm generated by a N 2 laser. The Reaction progress was monitored by thin-layer chromatography (TLC) on Kiesel- gel PF 254 . Purification was achieved by column chroma- tography, through Kieselgel G 60 35-75 µm. Solvents were evaporated under reduced pressure with a rotary evaporator. The purity of the compounds was determined by thin-layer chromatography and microanalysis. Method A: N-(2′,3′,4′,6′-Tetra-O-acetyl--D-glucopy- ranosyl)-2-[(tert-butoxycarbonyl)amino]-D,L-octade- * Corresponding author. Tel: (44) 171 753 5873. Fax: (44) 171 278 1939. E-mail: itoth@cua.ulsop.ac.uk. † University of London. ‡ University of Brighton. X Abstract published in Advance ACS Abstracts, November 15, 1997. © 1998, American Chemical Society and S0022-3549(97)00212-8 CCC: $15.00 Journal of Pharmaceutical Sciences / 25 American Pharmaceutical Association Vol. 87, No. 1, January 1998 Published on Web 01/02/1998