August 2010 PROTECTION of AUTHOR ' S C O P Y R I G H T This

THE UNIVERSITY LIBRARY PROTECTION OF AUTHOR ’S COPYRIGHT This copy has been supplied by the Library of the University of Otago on the understanding that the following conditions will be observed: 1. To comply with s56 of the Copyright Act 1994 [NZ], this thesis copy must only be used for the purposes of research or private study. 2. The author's permission must be obtained before any material in the thesis is reproduced, unless such reproduction falls within the fair dealing guidelines of the Copyright Act 1994. Due acknowledgement must be made to the author in any citation. 3. No further copies may be made without the permission of the Librarian of the University of Otago. August 2010 Oral Delivery of Peptides to Salmon Greg F. Walker A thesis submitted for the degree of Doctor of Philosophy of the University of Otago, Dunedin, New Zealand August 1999 11 Abstract Purpose. Oral delivery of peptide and protein drugs to fish has potential advantages to the aquaculture industry. However, the bioavailability of proteins and peptides from the intestinal tract is very low. This can be attributed inpart to the extensive lumenal and mucosa! proteolytic activities of the intestine. In this study the metabolic activities of the Quinnat salmon (Oncorhynchus tshawytscha) intestinal tract towards bovine serum albumin (BSA), human (hLHRH) and salmon (sLHRH) luteinizing-hormone releasing hormones were investigated. To overcome this metabolic barrier a number of proteolytic inhibitors were investigated to stabilise the protein and peptides drugs in the intestine, improving their opportunity for absorption across the salmon intestine. Methods. The extent of BSA degradation was determined by the formation of acid soluble peptides from BSA, measured by the method of Lowry. The metabolic stability hLHRH and sLHRH were determined using a capillary electrophoresis (CE) assay. Binding of proteins and proteolytic substrates to the polyacrylic acid, specifically Carbopol ® 934P (CP934P), was studied by centrifugal filtration. Gel filtration and reverse phase HPLC was used to determine the stability of trypsin in the presence of CP934P. Results. The lower intestinal tract of the salmon is divided into three anatomically distinct sections: anterior, middle and posterior. The lumenal proteolytic activities of the posterior intestinal section towards BSA were approximately half that of the anterior and middle sections. The half-lives of the LHRH analogues in the posterior intestinal sections were 2-fold longer than for the anterior and middle sections. Intestinal lumenal proteolytic activities towards BSA and the LHRH analogues correlated with higher activities towards the synthetic substrates of chymotrypsin, trypsin and elastase. LHRH analogues were shown to be rapidly hydrolysed by bovine a-chymotrypsin, whereas bovine trypsin and porcine elastase hydrolysed the LHRH analogues slowly by companson. 111 Proteolytic activity of the posterior mucosal homogenates towards BSA was 4-fold higher than for the middle mucosal homogenates. LHRH analogues were hydrolysed by the posterior mucosal homogenate, whereas in the middle mucosal homogenate they were stable. The higher metabolic instability of LHRH analogues in .the poste1ior mucosal homogenate correlated with higher chymotryptic-lik:e activity. Soybean trypsin inhibitor (SBTI) was the most effective inhibitor, stabilising the LHRH analogues against the lumenal proteolytic activity of the intestine and increasing their half-lives in the anterior, middle and posterior section between 7.5..: and 15-fold. CP934P (0.35% w/v, pH 8.0 at 15°C) increased the half-lives of the LHRH analogues in the anterior, middle and posterior sections between 1. 7- and 2. 7-fold. The rates of degradation in the presence of CP934P followed pseudo-zero order kinetics, indicating that the mechanism of inhibition was not autolysis. CP934P (0.35% w/v, pH 7 at 37°C) inhibited the rate of hydrolysis of N-a-benzyol-L arginine ethyl ester (BAEE) and hLHRH by trypsin to 34% and 28% of the control activity, respectively. CP934P completely stabilised hLHRH in the presence of bovine a chymotrypsin. Binding studies showed 68% of the trypsin and 22% of chymotrypsin; 10% ofBAEE was bound to CP934P, but no LHRH was bound. Lower specific activities of the unbound CP934P treated proteases indicate that inactivation of the proteases is also occurring. No low molecular weight autolysis products of trypsin could be identified by gel filtration. Reverse phase HPLC analysis of the unbound CP934P treated trypsin suggests a number of conformational forms of trypsin. The equilibrium binding capacity was 30 mg of trypsin to lg of CP934P. IV Conclusions. Chymotryptic-like activity of the salmon intestinal tract is a major metabolic barrier to the oral absorption of the LHRH analogues. The chymotryptic-like activities of the intestinal lumen and the intestinal mucosa are most likely due to the pancreatic secreted chymotrypsin and the intestinal brush-border membrane protease endopeptidase 24.18, respectively. The co-administration of SBTI with LHRH analogues in oral formulations could improve the oral absorption ofLHRH analogues in salmon. The inhibition of lumenal proteolytic activity by CP934P is the result of enzyme-polymer interaction, thereby reducing the free concentration of protease and in part denaturing the protease. The posterior intestine of salmon is the most metabolically favourable site for the oral delivery of peptides and proteins; however the LHRH analogues will have to overcome the higher endopeptidase 24.18 activity present in the posterior mucosa. V Acknowledgments My sincere thanks to my supervisors, Prof. Ian Tucker and Dr Robin Ledger for their guidence, help and advice throughout the course of study leading to this thesis. I am especially grateful to my friend and colleague Paul O Donnell (instrumentation tech.) who frequently fixed the capillary electrophoresis machine while others ran. I would also like to thank Dr Nigel Davies for his advice, support and friendship during my time at the School of Pharmacy. I acknowledge all the past and present postgraduate students of the School of Pharmacy, in particular fellow salmon researcher Leo Schep for his support and cooperation. I am grateful for the financial assistance given by the Otago University Targeted Research Scholarship. I also take the opportunity to thank my friends and flatmates, who over the many years have helped me through with many good times and memories. Without them I would have been cast against a padded cell. Finally, I would like to acknowledge the support from my family, in particular my parents who have always encouraged and supported me in what ever I have done. Vl Publications arising from this thesis Refereed journals Walker, G.F., Ledger, R. & Tucker, LG. (1999). Carbomer inhibits tryptic proteolysis of luteinizing hormone-releasing hormone and benzoyl-L-arginine ethyl ester by binding the enzyme. Pharmaceutical Research 16, 1074-1080. Refereed conference abstracts Walker, G.F., Ledger, R. & Tucker, LG. (1996) Proteolytic activity in the lower gastrointestinal tract of salmon. The Australian Journal of Hospital Pharmacy 26, P50. Walker, G.F., Ledger, R. & Tucker, LG. (1997). Distribution of proteolytic enzymes in the lumen of the lower intestinal tract of salmon. Pharmaceutical Research 14S, 1445. Walker, G.F., Ledger, R. & Tucker, LG. (1998). Susceptibility of human and salmon luteinizing hormone-realeasing hormone (LHRH) to metabolism in the salmon intestinal lumen and mucosal homogenates and their protection by various protease inhibitors. Proceedings ofthe Australian Society of Clinical Experimental Pharmacologists and Toxicologists 5, 1-57. Vll Table of contents Title page 1 Abstract 11 Acknowledgements V Publications arising from this thesis Vl Table of contents Vll List of tables Xl List of figures XIV List of abbreviations XXll Chapter 1 : General Introduction 1 ?' 1.1 Preface 2 1.2 Intestinal barriers 3 1.2.1 Biochemical barriers 3 1.2.2 Transport barrier 5 1.2.3 P-glycoprotein efflux pump 8 1.3 Strategies to improve peptide and protein delivery 9 1.3.1 Protease inhibitors 9 1.3 .1.1 Serine protease inhibitors 9 1.3 .1.2 Low molecular weight peptidase inhibitors 10 1.3.1.3 Bile salts 11 1.3.1.4 Chelating agents 12 1.3.1.5 Mucoadhesive polymers 13 1.3.2 Permeation enhancers 15 1.3.3 Chemical modification 18 1.3.4 Formulation 20 1.4 Oral delivery of peptides and proteins to teleosts 23 1.4. l Digestive tract of gastric teleosts 23 1.4.2 Biochemical barriers of the intestinal tract of teleosts 27 1 A.2.1 Lumenal proteases 27 1.4.2.2 Intestinal membrane bound proteases 31 1.4.2.3 Intracellular digestion of proteins and peptides 31 1.4.2.4 Biochemical barriers to the immunological system 32 1.4.3 Absorption of proteins and peptides 34 1.4.3 .1 Peptide carrier mediated transport 34 1.4.3.2 Paracellular route 34 1.4.3 .3 Transcytosis 34 1.4.3.4 P-glycoprotein efflux pump in teleosts 36 1.4.4 Oral peptide and protein delivery to teleosts 36 1.5 Aims of the study 40 Chapter 2: Capillary Electrophoretic Assay ofLuteinizing Hormone-Releasing Hormones 41 2.1 Introduction 42 2.2 Materials 48 vm 2.3 Methods 48 2.3 .1 Instrumentation and standard conditions 48 2.3.2 Capillaries 49 2.3 .3 Buffers 49 2.3.4 LHRH digests 50 2.3.5 Silanisation · 50 2.3.6 hLHRH standards-in the presence of a-chymotrypsin 50 2.3. 7 hLHRH standards- variability studies 51 2.4 Results 52 2.4.1 Precision 52 2.4.2 Optimisation of sample stacking 56 ~ 2.4.3 Optimisation of run buffer pH 63 2.4.4 Effect of chymotrypsin on the reproducibility 66 2.4.5 Storage stability 67 2.4.6 CE analysis ofLHRH 67 2.5 Discussion 68 Chapter 3: The Metabolic Barriers 73 3 .1 Introduction 74 3 .2 Materials 75 3.3 Methods 76 3 .3 .1 General 76 3.3.1.1 Fish 76 3.3.1.2 Collection and preparation of lumenal homogenates 76 3.3.1.3 In situ pH measurements 77 3.3.1.4 Preparation ofmucosal homogenates from frozen intestine 77 3.3.1.5 Preparation ofmucosal homogenates from fresh intestine 77 3.3.1.6 Protein estimations 78 3 .3 .1.

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