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The Path to an Orally Administered Protein Therapeutic for the Treatment of Diabetes Mellitus

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The Path to an Orally Administered Protein Therapeutic for the Treatment of Diabetes Mellitus Syracuse University SURFACE Chemistry - Dissertations College of Arts and Sciences 12-2012 The Path to an Orally Administered Protein Therapeutic for the Treatment of Diabetes Mellitus Susan Clardy James Syracuse University Follow this and additional works at: https://surface.syr.edu/che_etd Part of the Chemistry Commons Recommended Citation James, Susan Clardy, "The Path to an Orally Administered Protein Therapeutic for the Treatment of Diabetes Mellitus" (2012). Chemistry - Dissertations. 196. https://surface.syr.edu/che_etd/196 This Dissertation is brought to you for free and open access by the College of Arts and Sciences at SURFACE. It has been accepted for inclusion in Chemistry - Dissertations by an authorized administrator of SURFACE. For more information, please contact [email protected]. Abstract Protein therapeutics like insulin and glucagon-like peptide-1 analogues are currently used as injectable medications for the treatment of diabetes mellitus. An orally administered protein therapeutic is predicted to increase patient adherence to medication and bring a patient closer to metabolic norms through direct effects on hepatic glucose production. The major problem facing oral delivery of protein therapeutics is gastrointestinal tract hydrolysis/proteolysis and the inability to passage the enterocyte. Herein we report the potential use of vitamin B12 for the oral delivery of protein therapeutics. We first investigated the ability of insulin to accommodate the attachment of B12 at the B1 vs. B29 amino acid position. The insulinotropic profile of both conjugates was evaluated in streptozotocin induced diabetic rats. Oral administration of the conjugates produced significant drops in blood glucose levels, compared to an orally administered insulin control, but no significant difference was observed between conjugates. We also report, for the first time, a dose dependent response of a B12-insulin conjugate. We then explored the implications of B12 conjugation on the biological activity of the potent peptide glucagon-like peptide-1 (7-36) amide, with a K34R amino acid substitution. Various in vitro bioassays utilizing human embryonic kidney cells and human pancreatic islets were conducted and indicated B12 has a minimally negative effect on GLP-1 biological activity. Finally we modified the structure of B12 for future conjugation work. The modification of the 5’hydroxyl group of the ribose unit of B12 to a carboxylic acid is predicted to benefit the field of B12 conjugation significantly with the ability to produce higher yielding and more stable B12 conjugates. The Path to an Orally Administered Protein Therapeutic for the Treatment of Diabetes Mellitus By Susan Clardy-James B.S. Chemistry, Millikin University 2008 DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate School of Syracuse University December 2012 Copyright 2012 © Susan Clardy-James All rights reserved Acknowledgements It would not have been possible to write this thesis without the guidance and support of many kind people around me, to only some of whom it is possible to give particular mention here. I am truly indebted and thankful to my advisor, Professor Robert P. Doyle, whose encouragement, wisdom and commitment to the highest standards inspired and motivated me to become the scientist I am today. I will forever be thankful for his patience, guidance and support throughout my time at Syracuse University. I could not have imagined having a better advisor and mentor for my Ph. D studies and am incredibly grateful to have had the opportunity to work with him. I would like to thank all of the individuals in the Doyle lab. Each one brought a different perspective that helped me grow and learn in unexpected ways. I especially want to thank Professor Nadia Marino for the wonderful conversation about research, life and the future. I also want to thank Professor G. G. Holz, Dr. Colin Leech and Dr. Oleg Chepurny for allowing me the opportunity learn a new skill, in a new lab. I would also like to thank my friends and family for their love and support. My dearest friend, Jessica Seely, for being there any time even with thousands of miles between us. My parents, Theresa and Richard Clardy, for always loving me and encouraging me to never stop learning. My brother, Richard Clardy Jr., for putting things into perspective and reminding that there is a world outside the lab. Last but far from least, my husband, Stephen James, for his tireless devotion and complete understanding through the good and bad times. I am without a doubt blessed to have such an amazing support system and am truly thankful to all of them. iv Table of Contents Page Chapter 1 General Introduction 1 1.1 Diabetes Mellitus 1 1.2 Injectable Peptides and Proteins for the Treatment of Diabetes Mellitus 2 1.2.1 Insulin 3 1.2.1.1 History and Discovery 3 1.2.1.2 Biosynthesis and Secretion of Insulin 4 1.2.1.3 Structure and Sequence of Bovine Insulin 8 1.2.1.4 The Insulin Receptor 10 1.2.2 Glucagon-Like Peptide-1 12 1.2.2.1 Discovery of the Incretin Effect 12 1.2.2.2 Biosynthesis and Secretion 13 1.2.2.3 Structure and Sequence of Human GLP-1 15 1.2.2.4 The Glucagon-Like Peptide Receptor 16 1.3 A Pursuit of a Noninvasive Peptide/Protein Treatment for Diabetes Mellitus 17 1.3.1 Strategies for Oral Delivery of Peptides and Proteins 19 1.4 Vitamin B12 20 1.4.1 History 20 1.4.2 Structure and Chemistry of Vitamin B12 21 1.4.3 Physiological Roles of B12 23 1.4.4 The Dietary Uptake Pathway Overview 25 1.4.5 Haptocorrin 27 v 1.4.6 Intrinsic Factor 27 1.4.7 Cubilin and Amnionless 29 1.4.8 Transcobalamin 31 1.4.9 Megalin and TCblR/CD320 34 1.5 Using the B12 Pathway for Drug Delivery 35 1.5.1 Targeted Delivery 35 1.5.1.1 The Use of B12 for Imaging 37 1.5.1.2 Vitamin B12 Therapeutics 38 1.5.2 Oral Delivery of Peptides and Proteins 40 1.6 Points of Modification on B12 40 1.7 Summary 43 1.8 References 43 Chapter 2 Investigating the B12 Uptake Pathway for the Oral Delivery of Insulin 54 2.1 Introduction 54 2.2 Results and Discussion 58 1 29 2.2.1 Synthesis of B12-B Insulin (1) and B12-B Insulin (2) 57 2.2.2 MALDI-ToF Mass Spectrometry 62 2.2.3 Electronic Absorption Spectroscopy 65 2.2.4 In vivo experimentation 66 2.3 Conclusion 72 2.4 References 74 Chapter 3 Exploring the effect of B12 on Glucagon-Like Peptide-1 Biological Activity 77 3.1 Introduction 77 vi 3.2 Results and Discussion 80 3.2.1 Synthesis and Purification of B12-K34R-GLP-1 (7-36) amide (3) 80 3.2.2 MALDI-ToF Mass Spectrometry 82 3.2.3 In vitro Experimentation 83 3.3 Conclusion 90 3.4 References 90 Chapter 4 Synthesis of a B12 Derivative Capable of Amide Bond Formation 93 4.1 Introduction 93 4.2 Results and Discussion 95 4.2.1 Synthesis of B12 5’-carboxylic acid (4) 95 4.2.2 1D and 2D NMR analysis of 4 100 4.2.3 Synthesis of B12 5’-carboxylic acid-benzylamine (5) 104 4.3 Conclusion 110 4.4 References 110 Chapter 5 Experimental Procedure 113 5.1 Materials and Methods 113 5.2 Experimental Procedure 114 1 5.2.1 Synthesis of B12-B Insulin (1) 114 29 5.2.2 Synthesis of B12-B Insulin (2) 115 5.2.3 Synthesis of B12-K34R-GLP-1(7-36) amide (3) 116 5.2.4 Synthesis of B12-5’-carboxylic acid (4) 116 5.2.5 Synthesis of B12 5’-carboxylic acid-benzylamine (5) 117 5.3 In vitro Experimentation of 3 118 vii 5.3.1 Cell Cultures 118 5.3.2 Luciferase Assay 118 5.3.3 Fura-2 Assay 118 5.3.4 Insulin Secretion in Human Pancreatic Islets 119 5.4 In vivo Experimentation of 1 and 2 120 5.5 References 120 Chapter 6 Future Work 122 6.1 Vitamin B12 122 6.2 Glucagon-Like Peptide-1 127 6.3 C-peptide 128 6.4 References 129 Appendix A Publications arising from this work in chronological order viii List of Figures Page Figure 1. The primary treatment goal for DM is to maintain glycemic control 2 (indicated as normal glucose level). Glycemic control in a patient with diabetes can be challenging since the proper amount of insulin must be administered after meals. Not injecting enough or injecting too much insulin will result in hyperglycemia or hypoglycemia, respectively. While persistent hyperglycemia causes microvascular complications, hypoglycemia can cause diabetic coma or death. Figure 2. The subcellular organization of the insulin secretory pathway. 5 Figure 3. The post-translational modifications of preproinsulin, which results 6 in active monomeric insulin and C-peptide. Figure 4. Glucose induced insulin secretion (GSIS) is regulated by three 8 pathways: triggering pathway, metabolic amplify pathway and neurohormonal amplify pathway. Gs: Gs α subunit, AC: adenyl cyclase. Figure 5. A representation of the primary structure of bovine insulin including 9 the disulfide bonds formed between the A- and B-chains and the A- chain intrachain disulfide bond. ix Figure 6. A carton representation of the domain organization within the 11 homodimeric insulin receptor. L1, L2: first and second leucine-rich- repeat domains, CR: cysteine-rich region, Fn0-1-2: fibronectin domains, Ins: Insulin insert domain, TM/JM: trans- and juxta-membrane regions, TK: tyrosine kinase domain and CT: C-terminal domain.
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