implies the involvement of His residues in proton-dependent peptide transport. Interestingly, there are two His residues in the putative transmembrane domains of the PepT1 sequence (His 57,121 ) . It is our strategy to mutate these residue(s) and so 2 examine their role in the proton dependence of D-PHE-L-GLU transport. Acknowledgements We wish to thank M. A. Hediger for the PepT1 clone, R. A. Davey for the pRD67 /pRD67 TPF vectors and the Wellcome Trust for financial support. References 1. C.S. Temple, J.R. Bronk, P.D. Bailey and C.A.R. Boyd. Substrate charge dependence of stoichiometry shows membrane potential is the driving force for proton-peptide cotransport in rat renal cortex, Pflugers Arch. (1995) in press. 2. Y. Miyamoto, V. Ganapathy and F.H. Leibach. Identification of histidyl and thiol groups at the active site of rabbit renal dipeptide transporter. J. Biol. Chem. 261 (1986) 16133–16140. 3. Y.J. Fei, Y. Kanai, S. Nussberger, V. Ganapathy, F.H. Leibach, F.F. Romero, S.K. Singh, W.F. Boron and M.A. Hediger. Expression cloning of a mammalian proton-coupled oligopeptide transporter, Nature 368 (1994) 563–566.
improved tumour targeting. Further studies should elucidate if different impact of PEGylation on tissue and protein specific mechanisms operating in their biodistribution provide the basis for the change in tissue selectivity. Acknowledgements This work was supported by the Cancer Research Campaign. A.H. was an academic visitor from Bioquimica y Biologia Molecular, Univ. Alcala de Henares ( Madrid), Spain. References 1. G.E. Francis, C. Delgado and D. Fisher. PEG-modified proteins. Stability of protein pharmaceuticals: in-vivo pathways of degradation and strategies for protein stabilisation. In: Pharmaceutical Biotechnology, Vol. 3, R.T. Borchardt, T.J. Ahern and M. Manning (Editors). Plenum Press, New York, 1991, pp. 235–263. 2. C. Delgado, G.E. Francis and D. Fisher. Uses and properties of PEG linked proteins. Crit. Rev. Ther. Drug Carrier Syst. (1992) 249–304. PROTECTION OF LUTENISING HORMONE-RELEASING HORMONE (LH-RH) FROM DEGRADATION BY THE ENZYMES OF THE SMALL INTESTINE
PEGYLATION OF PROTEINS BY AN IMPROVED COUPLING METHOD FOR SELECTIVE TISSUE TARGETING: APPLICATION FOR ENHANCED TUMOUR SPECIFICITY
S.J. Kenworthy, J.F. Woodley and J. Pato´ Drug Delivery Group, Department of Biological Sciences, Keele University, Staffordshire, UK
C. Delgado, A. Herraez, F. Malik, D. Briones, A.K. Agrawal, D. Fisher and G.E. Francis Molecular Cell Pathology, Royal Free Hospital School of Medicine, University of London, UK
The oral delivery of peptide drugs would be enormously valuable if these molecules could be protected from the hostile environment of the small intestine. This research is investigating the use of polymers for protection of the peptide LH-RH from degradation by gastrointestinal peptidases. LH-RH, a ten amino acid peptide (Pro-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly) is currently used in the treatment of prostate cancer.The basic degradation assay consisted of 100 m l LH-RH, 100 m l 0.2 M Tris buffer (pH 7.2) and 100 m l enzyme preparation. Two enzyme preparations were used: the luminal contents enzyme preparation containing mainly pancreatic enzymes and the brush border membrane enzyme preparation. The reaction mixture was incubated at 378C for 0 and 5 min. At each time point 2350 m l samples were taken from the reaction mixture and added to 10 m l of 1 M HCl. To precipitate the polymer before analysis, 10 m l of 5 M CaCl 2 was added and the samples spun at 10 0003g for 15 min. The resulting supernatant was analysed for LH-RH and its degradation products by reverse phase HPLC and the kinetics of degradation evaluated.Four polymers, Carbopol, Chitosan and an anionic and a cationic derivative of Poly(N-vinylpyrrolidone-co-maleic acid) (NVPMA) were added to the assay to assess their effect on enzyme degradation. It was shown at 0.1, 0.5 and 1% both Carbopol and NVPMA 2 afforded up to 60% protection to the LH-RH, where as the positively charged polymers gave no protection. Kinetic analysis with Carbopol showed that the degrading enzymes were inhibited in a non-competitive fashion.This work showed that as a peptide LH-RH is subject to severe degradation in the small intestine. However, some protection is given to the LH-RH molecule by association with negatively
PEGylation of proteins has proven an effective means to increase their plasma half-life, reduce their immunogenicity / antigenicity and to increase their resistance to proteolytic degradation. Since some of the biological activity is retained (depending on the coupling method used) PEGylated proteins have advantages over their unmodified counterparts as therapeutic agents [1,2].We have used an improved coupling method to PEGylate two proteins, transferrin and a chimeric Fab fragment (F9) of A5B7 (an antiCEA antibody) and studied the influence of PEGylation in their biodistribution in nude mice bearing the human colon carcinoma LS174T by comparison of the area under the concentration-time curve (AUC 0-` ) in each tissue. PEGylation increased the AUC 0-` for F9 in all tissues but there were significant differences (variance ratio test, F533.62, P,0.001) between the proportional increments in the AUC 0-` with the tumour showing the greatest increase. There was therefore, increased tumour specificity of F9 by PEGylation. In contrast, PEGylation of transferrin led to increased AUC 0-` (blood, liver, lung, spleen, kidney, tumour) and reduced / unchanged AUC 0-` (muscle, skin, colon). The proportional changes in AUC 0-` promoted by PEGylation of transferrin also showed significant differences (F5112, P,0.001) but the tumour did not show the greatest increase.PEGylation can therefore, be exploited to change the selectivity of a protein for the different tissues and in the case of F9 to produce a conjugate for