The Development of Gut Hormones As Drugs for the Treatment of Obesity

The Development of Gut Hormones as Drugs for the Treatment of Obesity

A thesis submitted for the Degree of Doctor of Philosophy from Imperial College London

Joyceline Cuenco

2014

Division of Diabetes, Endocrinology and Metabolism Section of Investigative Medicine Faculty of Medicine

ABSTRACT

Obesity is an ever-increasing problem with limited effective treatments available that are safe and tolerable. Gut hormones are naturally occurring endogenous satiety factors that are released in response to food consumption. As well as playing an important role in the regulation of appetite, food intake and body weight, they also have roles in glucose disposal, gastric motility, fuel-type utilisation and energy expenditure. These characteristics make gut hormones attractive drugable targets for the treatment of obesity. However, the vulnerability of gut hormones to degradative enzymes results in rapid clearance rates and limited bioactivity which limits their viability as anti-obesity therapies.

This work aims to address two important properties to consider when developing gut hormones as drugs: cause of degradation and immunogenicity. Using pancreatic polypeptide (PP), I developed in vitro and in vivo testing systems to identify the physiologically important mechanisms involved in PP degradation. DPPIV and NEP were identified as important enzymes and enzyme-resistant analogues of PP were designed which demonstrated greater efficacy and slower clearance rates.

Subcutaneous (SC) administration of foreign exogenous peptides is known to potentially cause immunogenic reactions against the foreign peptide. The dangers of this reaction can range from mild to lethal. Currently peptide therapeutics require SC administration as oral bioavailability is low. Therefore, an aim of my studies was to explore the impact of modifications to PYY with regard to immunogenicity. To do this I assessed the impact of changes to different regions and specific amino acids of PYY. I successfully identified a region of PYY which, when modified, caused immunogenicity and what substitutions resulted in the most changes to immunogenic properties.

In summary my work demonstrated that by understanding the causes of peptide clearance and immunogenicity, safer and more efficacious analogues of gut hormones can be designed to take forward as potential treatments for obesity.

The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work

DECLARATION OF CONTRIBUTORS

All of the work in this thesis was performed by the author. All collaborations are listed below.

Chapter 3:

Development of the PP analogue was in collaboration with Professor Stephen Bloom. Testing of the analogues in animal studies was carried out by the Drug Discovery team, Imperial College (Drs. James Minnion, Tricia Tan, Jordan Baxter, Mike Tilby, Sejal Patel and Miss Claire Gibbard). Dr. James Gardiner assisted in setting up the over-expressing cell lines. Drs. Mélisande Addison and Jordan Baxter assisted in preparations of RBB. MALDI-MS was outsourced to the Proteomic facility (ABC, Imperial College, London, UK).

Chapter 4:

Development of the PYY analogue was in collaboration with Professor Stephen Bloom. Testing of the analogues in animal studies was carried out by the Drug Discovery team, Imperial College (Drs. James Minnion, Tricia Tan, Mélisande Addison, Mohammed Hankir, Samar Ghourab, Natacha Germain-Zito, and Keisuke Suzuki). Dr. James Gardiner assisted in setting up the over-expressing cell lines.

All radioimmunoassays were performed under the guidance and supervision of Professor Mohammad Ghatei who established and maintained all of the assays.

Radiolabelled peptides for all competitive assays were also provided by Professor Mohammad Ghatei.

ACKNOWLEDGEMENTS

I would like to thank Professors Steve Bloom and Mohammad Ghatei for giving me the opportunity to carry out this work and for their supervision and encouragement.

Much appreciation to Dr James Minnion for his supervision, continuing guidance and advice.

Big thanks to the Drug Discovery Team in all their evolving shapes and guises for all their hard work and assistance.

I am particularly grateful to members of the lab for their support and huge amounts of encouragement to the bitter end.

A special thank you, of course, to my family and those who have listened regardless.

“Just because it binds, doesn’t mean it’s doing anything. Shit sticks to the bottom of your shoe; doesn’t mean it has receptors on it.” - Anon

Abbreviations

5-HT 5-hydroxytryptamine or Serotonin

5-HT2C Serotonin receptor type 2C AcN Acetonitrile ACTH Adrenocorticotrophic hormone ADA Anti-drug antibody AgRP Agouti related protein AIB aminoisobutyric acid AMP Adenosine monophosphate AMPA/KA Kainate receptor ANOVA Analysis of variance AP Area postrema ARC Arcuate nucleus ATP Adenosine triphosphate AUC Area under curve BAT Brown adipose tissue BBB Blod brain barrier BDNF Brain-derived BMI Body mass index BPD Biliopancreatic diversion BSA Bovine serum albumin BW Body weight cAMP Cyclic adenosine monophosphate CART Cocaine- and amphetamine-regulated transcript CB1 Canabinnoid receptor 1 CCK Cholecystokinin CHO Chinese hamster ovary CNS Central nervous system

CO2 Carbon dioxide CRF Corticotrophin-releasing factor CsCl Caesium chloride CYP Cytochome DMEM Dulbecco’s modified Eagle medium DMF Dimethylformamide DMN Dorsomedial nucleus DMSO Dimethyl sulphoxide DNA Deoxyribonucleic acid DPPIV Dipeptidyl peptidase IV

DVC Dorso-vagal comples EDTA Ethylene diamine tetra-acetic acid EE Energy expenditure ELISA Enzyme-linked immunosorbant assay EMA European Medicines Agency EtBr Ethidium bromide Ex-4 Exendin 4 FBS Foetal bovine serum FDA US Food and Drug Administration FGF Fibroblast growth factor FI Food intake Fmoc Fluorenyl Methoxy-Carbonyl FW Food weight GABA Gamma aminobutyric acid GCG Glucagon GDW Glass distilled water GEE Generalised estimating equations GHSR Growth hormone secretagogue receptor GI Gastrointestinal GIP Glucose-dependent insulinotropic peptide GLP-1 Glucagon-like peptide-1 GLP-1r Glucagon-like peptide-1 receptor GLP-2 Glucagon-like peptide-2 GPCR G-protein coupled receptor GRPP Glicentin-related pancreatic polypeptide GTE Glucose tris EDTA HEK Human embryonic kidney HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPLC High performance liquid chromatography IBMX 3-Isobutyl-1-methylxanthine ICV Intracerebroventricular Insl5 Insulin-like peptide 5 IP Intraperitoneal IV Intravenous LB Lysogeny broth LHA Lateral hypothalamic area MALDI-ToF Matrix-assisted laser desorption/ionisation Time-of-flight MCH Melanin-concentrating hormone MCR Melanocortin receptor MC3r Melanocortin-3 receptor MC4r Melanocortin-4 receptor

ME Median eminence MEMRI Manganese-enhanced magnetic resonance imaging MPGF Major proglucagon fragment MPOA Medial preoptic area mRNA Message ribonucleic acid m/z Mass to charge ratio MSH Melanocyte-stimulating hormone NB Naltrexone buproprion NDMA Neurotransmitter glutamate NEP Neprilysin or Neutral endopeptidase 24.11 NHS National health service NICE National institute for health and clinical excellence NPY Neuropeptide Y NTS Nucleus of the solitary tract Ob-Rb Leptin receptor OXM Oxyntomodulin PBS Phosphate buffered saline PC1/2/3 Prohormone convertase PEG Polyethylene glycol PEI Polyethylenimine PMSF Phenylmethylsulphonylfluoride POMC Proopiomelanocortin PP Pancreatic polypeptide PVN Paraventricular nucleus PYY Peptide tyrosine tyrosine RBA Receptor binding assay RBB Renal brush border RIA Radioimmunoassay RIPA Radioimmunoprecipitation assay RLM Rat liver microsomes RNA Ribonucleic acid RT Retention time RYGB Roux–en-Y gastric bypass SC Subcutaneous SDS Sodium dodecyl sulphate SEM Standard error of the mean SP Spacer peptide SPPS Solid phase peptide synthesis T2DM Type 2 diabetes mellitus TAE Tris, acetic acid, EDTA TFA Trifluoroacetic acid

UCP-1 Uncoupling protein 1 VIP Vasoactive intestinal peptide VMN Ventromedial nucleus VSG Vertical sleeve gastrectomy WHO World health oranisation Y1/2/4/5r NPY1/2/4/5 receptor

TABLE OF CONTENTS

ABSTRACT ...... 2 DECLARATION OF CONTRIBUTORS ...... 4 ACKNOWLEDGEMENTS ...... 5 ABBREVIATIONS ...... 7 TABLE OF CONTENTS ...... 11 INDEX OF FIGURES ...... 18 INDEX OF TABLES...... 25 CHAPTER 1 ...... 27

GENERAL INTRODUCTION ...... 27 1.1 OBESITY ...... 28

1.1.2 CURRENT THERAPIES ...... 28 1.2 REGULATION OF FOOD INTAKE ...... 29

1.2.1 CENTRAL STRUCTURES INVOLVED IN THE ENERGY BALANCE ...... 30 1.2.1.1 The Hypothalamus ...... 30 1.2.1.1.1 Arcuate Nucleus (ARC) ...... 30 1.2.1.1.2 Paraventricular Nucleus (PVN) ...... 32 1.2.1.1.3 Dorsomedial Nucleus (DMN) ...... 33 1.2.1.1.4 Ventromedial Nucleus (VMN) ...... 33 1.2.1.1.5 Lateral Hypothalamic (LH) Area ...... 34 1.2.1.2 Role of Brainstem and vagus nerve in Energy Homeostasis ...... 34

1.2.2 PERIPHERAL CONTROL ...... 35 1.2.2.1 Adipose-derived signals ...... 35 1.2.2.1.1 Leptin ...... 35 1.2.2.1.2 Adiponectin ...... 37 1.2.2.2 Pancreatic signals ...... 37 1.2.2.3 Gastrointestinal-derived signals ...... 38 1.2.2.3.1 Ghrelin ...... 38 1.2.2.3.2 Cholecystokinin (CCK) ...... 39 1.2.2.4 Pancreatic Polypeptide (PP)-fold peptides ...... 41 1.2.2.4.1 Neuropeptide Y (NPY) ...... 42 1.2.2.4.2 Pancreatic Polypeptide (PP) ...... 43 1.2.2.4.3 Peptide YY (PYY) ...... 46 1.2.2.5 Proglucagon-derived products ...... 48

1.2.2.5.1 Glucagon ...... 49 1.2.2.5.2 Glucagon-like peptide 1 (GLP-1) ...... 51 1.2.2.5.3 Oxyntomodulin (OXM) ...... 53 1.3 CURRENT STRATEGIES IN ANTI-OBESITY THERAPEUTICS ...... 55

1.3.1 LIFESTYLE MODIFICATIONS ...... 55

1.3.2 SURGICAL ...... 55

1.3.3 DRUG TREATMENT ...... 58

1.3.4 CURRENT AND PREVIOUSLY LICENSED PHARMACOLOGICAL TREATMENTS FOR OBESITY ...... 58

1.3.5 DRUG IN DEVELOPMENT FOR OBESITY ...... 60 1.3.5.1 Small Molecules ...... 60 1.3.5.2 Biologics ...... 61 1.3.5.2.1 GLP-1 Agonists ...... 61 1.3.5.2.2 MC4r Agonists ...... 62 1.3.5.2.3 Leptin ...... 63 1.3.5.2.4 FGF-21 ...... 63 1.4 DEVELOPMENT OF GUT HORMONES AS THERAPEUTIC AGENTS ...... 64

1.4.1 CONFERRING ENZYME RESISTANCE IN GUT PEPTIDES ...... 64

1.4.2 CONTROLLED RELEASE AND CONJUGATION OF DRUGS...... 66

1.4.3 TACHYPHYLAXIS ...... 67 1.5 AIMS AND HYPOTHESES ...... 68 CHAPTER 2 ...... 69

MATERIALS AND METHODS ...... 69 2.1 PEPTIDES ...... 70

2.1.2 CUSTOM SYNTHESIS OF PEPTIDES ...... 70 2.2 ANIMAL STUDIES ...... 71

2.2.1 ANIMALS ...... 71 2.2.1.1 C57BL/6 mice ...... 71 2.2.1.2 Diet-induced obese (DIO) C57BL/6 mice ...... 72 2.2.1.3 Wistar rats ...... 72 2.3 ACUTE FEEDING STUDIES ...... 73 2.4 CHRONIC FEEDING STUDIES ...... 73

2.4.1 CHRONIC ADMINISTRATION TO DIO MICE ...... 73

2.4.2 CHRONIC ADMINISTRATION TO RATS ...... 74 2.5 RAT PHARMACOKINETICS ...... 74

2.6 .PRODUCTION OF HUMAN Y2 AND HUMAN Y4 RECEPTOR OVEREXPRESSING CELL LINES ...... 75

2.6.1 PRODUCTION OF COMPETENT BACTERIA ...... 75

2.6.2 TRANSFORMATION OF COMPETENT BACTERIA ...... 76

2.6.3 SMALL SCALE PREPARATION OF PLASMID ...... 76

2.6.4 RESTRICTION ENDONUCLEASE DIGESTION OF PLASMID DNA ...... 78

2.6.5 ELECTROPHORESIS OF DNA FRAGMENTS ...... 78

2.6.6 LARGE SCALE PLASMID PURIFICATION ...... 79

2.6.7 CAESIUM CHLORIDE GRADIENT PURIFICATION ...... 80

2.6.8 QUANTIFICATION OF DNA BY SPECTROPHOTOMETER...... 82

2.6.9 POLYETHYLENIMINE (PEI) MEDIATED IN VITRO GENE TRANSFER ...... 82

2.6.10 MAINTENANCE OF CELLS ...... 82

2.6.11 TRANSFECTION OF CELLS ...... 83 2.7 RECEPTOR BINDING ASSAYS...... 84

2.7.1 PREPARATION OF MEMBRANES FROM CELLS ...... 85 2.7.1.1 Maintenance of cells...... 85 2.7.1.2 Receptor purification ...... 85

2.7.2 IODINATION OF PEPTIDES ...... 86

2.7.3 Y1, Y2 AND Y4 RECEPTOR BINDING STUDIES ...... 87 2.8 PROTEOLYTIC DEGRADATION OF PEPTIDES ...... 87

2.8.1 LIVER MICROSOMES PREPARATION ...... 88 2.8.1.1 Biuret assay ...... 89

2.8.2 LIVER MICROSOME HYDROLYSES ...... 89

2.8.3 RENAL BRUSH BORDER (RBB) MEMBRANE PREPARATION ...... 90

2.8.4 RENAL BRUSH BORDER MEMBRANE HYDROLYSES ...... 91

2.8.5 DIPEPTIDYL-PEPTIDASE IV (DPPIV) HYDROLYSES ...... 91 2.8.5.1 Matrix-assisted laser desorption/ionization-Time of flight (MALDI-TOF) analysis of DPPIV proteolytic breakdown products ...... 92

2.8.6 HUMAN TRYPSIN HYDROLYSES ...... 93

2.8.7 HUMAN RECOMBINANT NEPRILYSIN HYDROLYSES ...... 93 2.9 RADIOIMMUNOASSAY (RIA) ...... 94

2.9.1 PRODUCTION OF PP-X AND PYY2-36(ΑLTX-4H) POLYCLONAL ANTIBODY ...... 94

2.9.2 PYY AND PYY ANALOGUES RIA ...... 95

2.9.3 PP AND PP ANALOGUES RIA ...... 97

2.10 DETECTION OF IMMUNOGENICITY OF PYY ANALOGUES ...... 98

2.10.1 ADA COMPETITION ASSAY WITH PYY ANALOGUES ...... 99 2.11 STATISTICAL ANALYSIS ...... 100 CHAPTER 3 ...... 101

DEVELOPING ANALOGUES OF PANCREATIC POLYPEPTIDE AS A PHARMACOTHERAPY FOR OBESITY . 101 3.1 INTRODUCTION ...... 102

3.1.1 HEPATIC METABOLISM ...... 104

3.1.2 RENAL METABOLISM ...... 106

3.1.3 DPPIV DEGRADATION ...... 106

3.1.4 NEPRILYSIN DEGRADATION ...... 107

3.1.5 TRYPSIN DEGRADATION ...... 108

3.1.6 DESIGN OF ENZYME-RESISTANT PEPTIDE ANALOGUES ...... 110

3.1.7 HYPOTHESIS AND AIMS ...... 111 3.1.7.1 Hypothesis ...... 111 3.1.7.2 Aims ...... 111 3.2 RESULTS ...... 112 3.2.1 CHARACTERISATION OF PP DEGRADATION BY TISSUE ENZYME PREPARATIONS...... 112 3.2.1.1 Investigation into RBB-mediated breakdown of PP (HPLC) ...... 112 3.2.1.2 Identification of RBB target sites on PP (MALDI) ...... 113 3.2.1.3 HPLC analysis of PP degradation by RLM ...... 115 3.2.1.5 Identification of RLM target sites on PP – mass spectrometry of HPLC fractions ...... 119 3.2.2 CHARACTERISATION OF PP DEGRADATION BY INDIVIDUAL RECOMBINANT ENZYMES .... 126 3.2.2.1 Investigations into the breakdown of PP by DPPIV ...... 126 3.2.2.1.1 Breakdown of PP by DPPIV ...... 126 3.2.2.1.2 Effect of PP and DPPIV fragment PP3-36 on acute food intake in mice ...... 128 3.2.2.1.3 Effect of N-terminal modifications on DPPIV-mediated degradation ...... 129 3.2.2.1.4 Effect of N-terminal modifications on Y4 receptor affinity ...... 130 3.2.2.1.5 Effect of N-terminal modifications on acute food intake in mice ...... 131 3.2.2.2 Investigations into the breakdown of PP by Trypsin ...... 133 3.2.2.2.1 Breakdown of PP by trypsin ...... 133 3.2.2.2.2 Effect of modifications at trypsin-target sites on Y4 receptor affinity ...... 136 3.2.2.2.3 Breakdown of PP analogues by trypsin ...... 137 3.2.2.2.4 Effect of modifications at trypsin-target sites on acute food intake in mice ...... 139 3.2.2.3 Investigations into the breakdown of PP by neprilysin (NEP) ...... 145 3.2.2.3.1 Breakdown of PP by NEP ...... 145 3.2.2.3.2 Effect of NEP inhibition on acute food intake in mice ...... 150

3.2.2.3.3 Pharmacokinetics of PP in mice ...... 151 3.2.2.3.4 Effect of phosphoramidon on plasma levels of PP in mice ...... 152 3.2.3 EFFECT OF PP MODIFICATIONS ON RBA, NEP DIGESTS, ACUTE FEEDING AND PHARMACOKINETICS ...... 154 3.2.4 INVESTIGATION OF PLASMA LEVELS OF PP ANALOGUES IN MICE ...... 163 3.2.4.1 Effect of phosphoramidon on plasma levels of a DPPIV-resistant PP analogue in mice 163 3.2.4.2 Effect of phosphoramidon on plasma levels of PP analogues with global modifications in mice ...... 164 3.2.4.3 Investigation of plasma levels of PP analogues with different susceptibilities to NEP and contrasting effects in vivo ...... 169 3.2.4.4 Investigation of plasma levels of PP analogues with similar in vivo bioefficacy and different affinities to the Y4 receptor ...... 173 3.3 DISCUSSION ...... 178 3.4 CONCLUSIONS ...... 190 CHAPTER 4 ...... 191

INVESTIGATION OF THE IMMUNOGENICITY OF A LONG-ACTING PYY3-36 ANALOGUE IN THE

DEVELOPMENT OF A POTENTIAL ANTI-OBESITY AGENT ...... 191 4.1 INTRODUCTION ...... 192

4.1.1 PYY AS AN ANTI-OBESITY AGENT ...... 192 4.1.2 IMMUNOGENICITY ...... 198

4.1.3 HYPOTHESES AND AIMS ...... 205 4.1.3.1 Hypotheses ...... 205 4.1.3.2 Aims ...... 205 4.2 RESULTS ...... 206

4.2.1 CHARACTERISATION OF PYY2-36(ΑLTX-4H) ...... 206 4.2.1.1 Y1r and Y2r Receptor affinity ...... 206

4.2.1.2 Comparison of PYY2-36(αLTX-4H) against PYY3-36 on food intake in mice ...... 208

4.2.1.3 Acute effect of a dose response of PYY2-36(αLTX-4H) on food intake in mice ...... 209

4.2.1.4 The importance of Zn in the pharmacokinetic of a single SC injection of PYY2-36(αLTX-4H) in rat ...... 212

4.2.1.5 Effect of chronic administration of PYY2-36(αLTX-4H) on food intake and body weight in DIO mice ...... 215

4.2.1.6 Effects of chronic administration of PYY2-36(αLTX-4H) on food intake and body weight in male Wistar rats ...... 217

4.2.2 IMMUNOGENICITY ...... 219

4.2.2.1 Immunogenicity of chronic administration of PYY2-36(αLTX-4H) ...... 219 4.2.2.2 Effect of Zn precipitation on immunogenicity ...... 220

4.2.2.3 Immunogenicity of PYY2-36(αLTX-4H) fragments ...... 221

4.2.2.4 Immunogenicity of PYY2-36(αLTX-4H) analogues ...... 225

4.2.3 ASSESSMENT OF ALTERNATIVES TO PYY2-36(ΑLTX-4H) ...... 230

4.2.3.1 PEGylation of PYY2-36(αLTX-4H) – PYY2-36(αLTX-4H)-PEG ...... 230 4.2.3.1.1 Y1r and Y2r RBA ...... 230

4.2.3.1.2 Effect of PYY2-36(αLTX-4H)-PEG on food intake in mice ...... 232

4.2.3.1.3 Pharmacokinetics of PYY2-36(αLTX-4H)-PEG with Zn in rat ...... 234

4.2.3.1.4 Immunogenicity of PYY2-36(αLTX-4H)-PEG ...... 235

4.2.3.2 Removal of αLTX – PYY2-36(4H) ...... 236 4.2.3.2.1 Y1r and Y2r RBA ...... 236

4.2.3.2.2 Comparison of PYY2-36(4H) against PYY3-36 on food intake in mice ...... 238

4.2.3.2.3 Effect of acute administration of PYY2-36(4H) on food intake in mice (dose response study without Zn formulation) ...... 239

4.2.3.2.4 Effect of Zinc on acute administration of PYY2-36(4H) on food intake in mice ...... 241

4.2.3.2.5 Pharmacokinetics of PYY2-36(4H) investigating dose response of Zn formulation ...... 243

4.2.4 FURTHER CHARACTERISATION OF PYY2-36(4H) ...... 245

4.2.4.1 Comparison of acute effect of subcutaneous administration of PYY2-36(4H) and PYY2-

36(αLTX-4H) on food intake in mice ...... 245

4.2.4.2 Pharmacokinetics of PYY2-36(4H) at high concentrations – Zn dose range finding ...... 247

4.2.4.3 Comparison of pharmacokinetics of PYY2-36(4H) and PYY2-36(αLTX-4H) at high concentrations with 1:1 Zn ...... 248

4.2.4.4 Effect of chronic administration of PYY2-36(4H) on food intake and body weight in DIO mice ...... 249

4.2.4.5 Effect of chronic administration of PYY2-36(4H) on food intake and body weight in rats 252

4.2.4.6 Investigation of immunogenicity of PYY2-36(4H) ...... 254 4.3 DISCUSSION ...... 255 4.4 CONCLUSIONS ...... 264

CHAPTER 5 ...... 265

GENERAL DISCUSSION ...... 265 5.1 INTRODUCTION ...... 266 5.2 DEVELOPMENT OF AN ENZYME-RESISTANT PP ANALOGUE ...... 268 FUTURE WORK ...... 271

5.3 IMMUNOGENICITY OF A LONG-ACTING ANALOGUE OF PYY3-36 ...... 272 FUTURE WORK ...... 276 REFERENCES ...... 278 APPENDIX A – AMINO ACID CODES ...... 309 APPENDIX B – ANALOGUE SEQUENCES ...... 310 APPENDIX C – BUFFERS AND SOLUTIONS ...... 311 APPENDIX D – CLONING MAP OF HY2 AND HY4 CDNA ...... 313 APPENDIX E – GENERAL PRINCIPLE OF A RADIOIMMUNOASSAY ...... 315 ORIGINAL ARTICLES ...... 317

INDEX OF FIGURES

FIGURE 1.1 SCHEMATIC TREE-DIAGRAM OF THE HYPOPTHALAMUS AND THE VARIOUS NUCLEI ...... 31

FIGURE 1.2 A SCHEMATIC DIAGRAM OF THE BASIC STRUCTURE OF PP ...... 41

FIGURE 1.3 A DIAGRAMMATIC REPRESENTATION OF THE TISSUE-SPECIFIC PROCESSING OF PREPROGLUCAGON IN THE

PANCREAS AND THE CNS/INTESTINE ...... 49

FIGURE 1.4 SIMPLIFIED DIAGRAMS OF VSG AND RYGB ...... 57

FIGURE 1.5 CHALLENGES OF FORMULATION: BOLUS VS SUSTAINED-DELIVERY FORMULATION ...... 66

FIGURE 2.1 NOMENCLATURE FOR ENZYME CLEAVAGE SITES WITHIN A SUBSTRATE ...... 88

FIGURE 2.2 GRAPHICAL REPRESENTATION OF THE ACN GRADIENT IN WATER ...... 90

FIGURE 2.3 EQUATION TO MEASURE RELATIVE DISPLACEMENT STRENGTH AGAINST RADIOLABELLED DRUG ...... 99

FIGURE 3.1 DIAGRAMMATIC REPRESENTATION OF THE 3 LAYERS WHICH COMPRISE THE SKIN ...... 108

FIGURE 3.2 HPLC CHROMATOGRAM OF A TIME COURSE OF PP DIGEST WITH RBB AT 37OC ...... 112

FIGURE 3.3 (A) MALDI MS ANALYSIS OF PP DIGEST WITH RBB AT 37°C FOR 10MINS ...... 114

FIGURE 3.3 (B) MALDI MS ANALYSIS OF PP DIGEST WITH RBB AT 37°C FOR 60MINS ...... 114

FIGURE 3.3 (D) SEQUENCES OF FRAGMENTS FROM PP DIGEST WITH RBB FOR 10 AND 60MINS...... 114

FIGURE 3.4 (A) HPLC CHROMATOGRAM OF PP DIGEST WITH RLM AT 37OC FOR 0MINS ...... 115

FIGURE 3.4 (B) HPLC CHROMATOGRAM OF PP DIGEST WITH RLM AT 37OC FOR15MINS ...... 115

FIGURE 3.4 (C) HPLC CHROMATOGRAM OF PP DIGEST WITH RLM AT 37OC FOR 30MINS ...... 115

FIGURE 3.4 (D) HPLC CHROMATOGRAM OF PP DIGEST WITH RLM AT 37OC FOR 60MINS ...... 115

FIGURE 3.5 (A) MALDI MS ANALYSIS OF PP DIGEST WITH RLM AT 37°CFOR 10MINS ...... 116

FIGURE 3.5 (C) SEQUENCES OF FRAGMENTS FROM PP DIGESTS WITH RLM FOR 10MINS ...... 116

FIGURE 3.6 (A) MALDI MS ANALYSIS OF PP DIGEST WITH RLM AT 37°C FOR 10MINS ...... 118

FIGURE 3.6 (C) SEQUENCES OF FRAGMENTS FROM PP DIGEST WITH RLM AT 60MINS ...... 118

FIGURE 3.7 HPLC CHROMATOGRAM OF PP DIGEST WITH RLM AT 37OC FOR 60MINS ...... 119

FIGURE 3.8 (A) MALDI MS ANALYSIS OF FRACTION RT=16-17M OF PP DIGESTS WITH RLM AT 37°C

FOR 60MINS ...... 120

FIGURE 3.8 (C) SEQUENCES OF FRAGMENTS FROM FRACTION RT=16-17M OF PP DIGESTS WITH RLM

FOR 60MINS ...... 120

FIGURE 3.9 DIAGRAM OF THE PP SEQUENCE SHOWING THE TERTIARY STRUCTURE WITH THE STABILISING BONDS .... 121

FIGURE 3.10 (A) MALDI MS ANALYSIS OF FRACTION RT=21-22M OF PP DIGESTS WITH RLM AT 37°C

FOR 60MINS ...... 123

FIGURE 3.10 (C) SEQUENCES OF FRAGMENTS FROM FRACTION RT=21-22M OF PP DIGESTS WITH RLM

FOR 60MINS ...... 123

FIGURE 3.11 (A) MALDI MS ANALYSIS OF FRACTION RT=24-25M OF PP DIGESTS WITH RLM AT 37°C

FOR 60MINS ...... 125

FIGURE 3.11 (C) SEQUENCES OF FRAGMENTS FROM FRACTION RT=24-25M OF PP DIGESTS WITH RLM

FOR60MINS ...... 125

FIGURE 3.12 HPLC ANALYSIS OF PP WITH 10, 20 AND 50MU DPPIV FOR 2H AT 37°C ...... 126

FIGURE 3.13 (A) MALDI-TOF MS ANALYSIS OF PP ALONE FOR 2H AT 37°C ...... 127

FIGURE 3.13 (B) MALDI-TOF MS ANALYSIS OF PP + 10MU DPPIV FOR 2H AT 37°C ...... 127

FIGURE 3.14 THE EFFECT OF SC ADMINISTRATION OF PP AND PP3-36 (50NMOL/KG) ON FOOD INTAKE IN

C57BL/6 MICE FASTED OVERNIGHT ...... 128

FIGURE 3.15 (A) MALDI-TOF MS ANALYSIS OF PP-ALA0 + 10MU DPPIV FOR 2H AT 37°C ...... 129

FIGURE 3.15 (B) MALDI-TOF MS ANALYSIS OF PP2-36 + 10MU DPPIV FOR 2H AT 37°C ...... 129

FIGURE 3.16 BINDING AFFINITIES OF PP AND ANALOGUES TO HUMAN Y4R ...... 130

FIGURE 3.17 THE EFFECT OF SC ADMINISTRATION OF PP AND PP2-36 (150NMOL/KG) ON FOOD INTAKE IN

C57BL/6 MICE FASTED OVERNIGHT ...... 131

FIGURE 3.18 THE EFFECT OF SC ADMINISTRATION OF PP AND PP-ALA0 (300NMOL/KG) ON FOOD INTAKE IN

C57BL/6 MICE FASTED OVERNIGHT ...... 132

FIGURE 3.19 HPLC ANALYSIS OF PP WITH 0.1MU TRYPSIN FOR 15MINS AT 37°C ...... 133

FIGURE 3.20 MALDI MS ANALYSIS OF FRACTION PPF1 OF PP IN THE PRESENCE OF TRYPSIN FOR 15MINS

AT 37°C ...... 134

FIGURE 3.21 MALDI MS ANALYSIS OF FRACTION PPF2 OF PP IN THE PRESENCE OF TRYPSIN FOR 15MINS

AT 37°C ...... 135

FIGURE 3.22 (A) HPLC CHROMATOGRAM OF PP DIGEST WITH TRYPSIN AT 37OC FOR 15MINS ...... 138

FIGURE 3.22 (B) HPLC CHROMATOGRAM OF PP-LYS25 DIGEST WITH TRYPSIN AT 37OC FOR 15MINS ...... 138

FIGURE 3.22 (C) HPLC CHROMATOGRAM OF PP-LYS26 DIGEST WITH TRYPSIN AT 37OC FOR 15MINS ...... 138

FIGURE 3.22 (D) HPLC CHROMATOGRAM OF PP-GLN25,26 DIGEST WITH TRYPSIN AT 37OC FOR 15MINS ...... 138

FIGURE 3.23 THE EFFECT OF SC ADMINISTRATION OF PP, PP-LYS25 AND PP-LYS26 (150NMOL/KG)

ON FOOD INTAKE IN C57BL/6 MICE FASTED OVERNIGHT ...... 139

FIGURE 3.24 THE EFFECT OF SC ADMINISTRATION OF PP (150NMOL/KG), PP-ALA25 AND

PP-ALA26 (300NMOL/KG) ON FOOD INTAKE IN C57BL/6 MICE FASTED OVERNIGHT ...... 140

FIGURE 3.25 THE EFFECT OF SC ADMINISTRATION OF PP (150NMOL/KG) AND PP-HIS26 (300NMOL/KG)

ON FOOD INTAKE IN C57BL/6 MICE FASTED OVERNIGHT ...... 141

FIGURE 3.26 THE EFFECT OF SC ADMINISTRATION OF PP, PP-HIS35, AND PP-HIS25,26 (300NMOL/KG)

ON FOOD INTAKE IN C57BL/6 MICE FASTED OVERNIGHT ...... 142

FIGURE 3.27 THE EFFECT OF SC ADMINISTRATION OF PP AND PP-GLN25,26 (150NMOL/KG), AND PP-ALA35

(600NMOL/KG) ON FOOD INTAKE IN C57BL/6 MICE FASTED OVERNIGHT ...... 143

FIGURE 3.28 THE EFFECT OF SC ADMINISTRATION OF PP (150NMOL/KG), PP-ALA33, PP-LYS35, AND PP-LYS33

(300NMOL/KG) ON FOOD INTAKE IN C57BL/6 MICE FASTED OVERNIGHT ...... 144

FIGURE 3.29 HPLC ANALYSIS OF PP WITH 200NG OF NEP FOR 15, 45, 60, AND 120MINS AT 37°C ...... 146

FIGURE 3.30 (A) MALDI MS ANALYSIS OF PP DIGEST WITH NEP AT 37°CFOR 30 AND 120MINS ...... 147

FIGURE 3.30 (C) SEQUENCES OF FRAGMENTS FROM PP DIGEST WITH NEP AT 37°C FOR 30 AND 120MINS ...... 147

FIGURE 3.31 (A) MALDI MS ANALYSIS OF FRACTION RT=17-19M OF PP DIGESTS WITH NEP AT 37°C

FOR 120MINS ...... 149

FIGURE 3.31 (C) MALDI MS ANALYSIS OF FRACTION RT=17-19M OF PP DIGESTS WITH NEP AT 37°C

FOR 120MINS ...... 149

FIGURE 3.32 THE EFFECT OF SC ADMINISTRATION OF PP (150NMOL/KG), PHOSPHORAMIDON (5MG/KG), AND

PP + PHOSPHORADMIDON ON FOOD INTAKE IN C57BL/6 MICE FASTED OVERNIGHT ...... 150

FIGURE 3.33 PHARMACOKINETIC PROFILE OF A SINGLE SUBCUTANEOUS INJECTION OF PP AT 1000NMOL/KG

TO MALE C57BL/6 MICE...... 151

FIGURE 3.34 (A) PLASMA LEVELS OF PP AT 45MINS FOLLOWING AN SC INJECTION OF PP AT 150NMOL/KG TO MALE

C57BL/6 MICE IN COMBINATION WITH A SINGLE IP INJECTION OF PHOSPHORAMIDON

AT 7MG/KG, 20MG/KG AND 60MG/KG ...... 153

FIGURE 3.34 (B) PLASMA LEVELS OF PP AT 90MINS FOLLOWING AN SC INJECTION OF PP AT 150NMOL/KG TO MALE

C57BL/6 MICE IN COMBINATION WITH A SINGLE IP INJECTION OF PHOSPHORAMIDON

AT 7MG/KG, 20MG/KG AND 60MG/KG ...... 153

FIGURE 3.34 (C) COMPARISON OF PLASMA LEVELS OF PP AT 45 AND 90MINS FOLLOWING AN SC INJECTION OF PP AT

150NMOL/KG TO MALE C57BL/6 MICE IN COMBINATION WITH A SINGLE IP INJECTION OF PHOSPHORAMIDON

AT 7MG/KG, 20MG/KG AND 60MG/KG ...... 153

FIGURE 3.35 THE EFFECT OF SC ADMINISTRATION OF PP (300NMOL/KG), PP-PRO0 (150NMOL/KG) AND PP-CYS0

DIMER (100NMOL/KG) ON FOOD INTAKE IN C57BL/6 MICE FASTED OVERNIGHT...... 156

FIGURE 3.36 PLASMA LEVELS OF PP-ALA0 AT 45 AND 90MINS FOLLOWING AN SC INJECTION OF PP-ALA0 AT

150NMOL/KG TO MALE C57BL/6 MICE AND IN COMBINATION WITH A SINGLE IP INJECTION OF

PHOSPHORAMIDON AT 20MG/KG ...... 164

FIGURE 3.37 (A) PLASMA LEVELS OF PP AT 45 AND 90MINS FOLLOWING A SINGLE SUBCUTANEOUS INJECTION OF PP

AT 1000NMOL/KG TO MALE C57BL/6 MICE IN COMBINATION WITH A SINGLE IP INJECTION OF

PHOSPHORAMIDON AT 20MG/KG ...... 166

FIGURE 3.37 (B) PLASMA LEVELS OF PP AT 45 AND 90MINS FOLLOWING A SINGLE SUBCUTANEOUS INJECTION OF PP

AT 25NMOL/KG TO MALE C57BL/6 MICE IN COMBINATION WITH A SINGLE IP INJECTION OF

PHOSPHORAMIDON AT 20MG/KG ...... 166

FIGURE 3.38 (A) PLASMA LEVELS OF PP-LYS30 AT 45 AND 90MINS FOLLOWING A SINGLE SUBCUTANEOUS INJECTION

OF PP-LYS30 AT 1000NMOL/KG TO MALE C57BL/6 MICE IN COMBINATION WITH A SINGLE IP INJECTION OF

PHOSPHORAMIDON AT 20MG/KG ...... 168

FIGURE 3.38 (B) PLASMA LEVELS OF PP-P0,A23,K30 AT 45 AND 90MINS FOLLOWING A SINGLE SUBCUTANEOUS

INJECTION OF PP-P0,A23,K30 AT 25NMOL/KG TO MALE C57BL/6 MICE IN COMBINATION

WITH A SINGLE IP INJECTION OF PHOSPHORAMIDON AT 20MG/KG ...... 168

FIGURE 3.38 (C) PLASMA LEVELS OF PP-P0,K19,K30 AT 45 AND 90MINS FOLLOWING A SINGLE SUBCUTANEOUS

INJECTION OF PP-P0,K19,K30 AT 1000NMOL/KG TO MALE C57BL/6 MICE IN COMBINATION WITH A SINGLE

IP INJECTION OF PHOSPHORAMIDON AT 20MG/KG ...... 168

FIGURE 3.38 (D) PLASMA LEVELS OF PP-K6,NPY19-23 AT 45 AND 90MINS FOLLOWING A SINGLE SUBCUTANEOUS

INJECTION PP-K6,NPY19-23 AT 1000NMOL/KG TO MALE C57BL/6 MICE IN COMBINATION WITH A SINGLE

IP INJECTION OF PHOSPHORAMIDON AT 20MG/KG ...... 168

FIGURE 3.38 (E) PLASMA LEVELS OF PP-D6,NPY19-23 AT 45 AND 90MINS FOLLOWING A SINGLE SUBCUTANEOUS

INJECTION PP-D6,NPY19-23 AT 25NMOL/KG TO MALE C57BL/6 MICE IN COMBINATION

WITH A SINGLE IP INJECTION OF PHOSPHORAMIDON AT 20MG/KG ...... 168

FIGURE 3.39 BINDING AFFINITIES OF ANALOGUES PP-25 AND PP-26 TO MOUSE Y4R ...... 168

FIGURE 3.40 (A) THE EFFECT OF SC ADMINISTRATION OF 200NMOL/KG PP AND 25NMOL/KG PP-26 ON FOOD INTAKE

IN C57BL/6 MICE FASTED OVERNIGHT ...... 169

FIGURE 3.40 (B) THE EFFECT OF SC ADMINISTRATION OF 25NMOL/KG PP AND 25NMOL/KG PP-25 ON FOOD INTAKE

IN C57BL/6 MICE FASTED OVERNIGHT ...... 169

FIGURE 3.41 (A) PLASMA PEPTIDE LEVELS AT 30MINS AND 2HRS FOLLOWING A SC INJECTION OF

1000NMOL/KG PP ...... 171

FIGURE 3.41 (B) PLASMA PEPTIDE LEVELS AT 30MINS AND 2HRS FOLLOWING A SC INJECTION OF

1000NMOL/KG PP-25 ...... 171

FIGURE 3.41 (C) PLASMA PEPTIDE LEVELS AT 30MINS AND 2HRS FOLLOWING A SC INJECTION OF

1000NMOL/KG PP-26 ...... 171

FIGURE 3.42 (A) COMPARISON OF PLASMA PEPTIDE LEVELS OF PP, PP-25, AND PP-26 AT 30MINS POST-INJECTION ...... 172

FIGURE 3.42 (B) COMPARISON OF PLASMA PEPTIDE LEVELS OF PP, PP-25, AND PP-26 AT 120MINS POST-INJECTION ...... 172

FIGURE 3.43 BINDING AFFINITIES OF ANALOGUES PP-27 AND PP-28 TO MOUSE Y4R ...... 173

FIGURE 3.44 THE EFFECT OF SC ADMINISTRATION OF PP (50NMOL/KG) AND PP-27 (10NMOL/KG) ON FOOD INTAKE

IN C57BL/6 MICE FASTED OVERNIGHT ...... 174

FIGURE 3.45 THE EFFECT OF SC ADMINISTRATION OF PP (50NMOL/KG) AND PP-28 (10NMOL/KG) ON FOOD INTAKE

IN C57BL/6 MICE FASTED OVERNIGHT ...... 175

FIGURE 3.46 (A) PLASMA PEPTIDE LEVELS AT 15, 45 AND 120MINS FOLLOWING A SC INJECTION OF

1000NMOL/KG PP ...... 176

FIGURE 3.46 (B) PLASMA PEPTIDE LEVELS AT 15, 45 AND 120MINS FOLLOWING A SC INJECTION OF

1000NMOL/KG PP-27 ...... 176

FIGURE 3.46 (C) PLASMA PEPTIDE LEVELS AT 15, 45 AND 120MINS FOLLOWING A SC INJECTION OF

1000NMOL/KG PP-28 ...... 176

FIGURE 3.47 (A) COMPARISON OF PLASMA PEPTIDE LEVELS OF PP, PP-27, AND PP-28 AT 15MINS POST-INJECTION ...... 177

FIGURE 3.47 (B) COMPARISON OF PLASMA PEPTIDE LEVELS OF PP, PP-27, AND PP-28 AT 45MINS POST-INJECTION ...... 177

FIGURE 3.47 (C) COMPARISON OF PLASMA PEPTIDE LEVELS OF PP, PP-27, AND PP-28 AT 120MINS POST-INJECTION ...... 177

FIGURE 4.1 PRIMARY AMINO ACID SEQUENCE OF PYY AND SCHEMATIC REPRESENTATION OF STABILISING BONDS .... 193

FIGURE 4.2 DESIGN OF PYY ANALOGUE ...... 197

FIGURE 4.3 PRODUCTION OF ANTIBODIES BY B CELLS ...... 200

FIGURE 4.4 (A) BINDING AFFINITIES OF PYY AND ANALOGUES TO HUMAN Y2R ...... 207

FIGURE 4.4 (B) BINDING AFFINITIES OF PYY AND ANALOGUES TO HUMAN Y1R ...... 207

FIGURE 4.4 (C) BINDING AFFINITIES OF PYY AND ANALOGUES TO MOUSE Y2R ...... 207

FIGURE 4.4 (D) BINDING AFFINITIES OF PYY AND ANALOGUES TO HUMAN Y1R ...... 207

FIGURE 4.5 EFFECT OF SUBCUTANEOUS ADMINISTRATION OF 100NMOL/KG PYY3-36 AND PYY2-36(ΑLTX-4H)

ON FOOD INTAKE IN FASTED C57BL/6 MICE ...... 209

FIGURE 4.6 EFFECT OF SUBCUTANEOUS DOSE RESPONSE OF 100, 300, AND 500NMOL/KG PYY2-36(ΑLTX-4H)

ON FOOD INTAKE IN FASTED C57BL/6 MICE ...... 210

FIGURE 4.7 CUMULATIVE 24HOUR FOOD INTAKE AFTER ADMINISTRATION OF 100, 300, AND 500NMOL/KG

PYY2-36(ΑLTX-4H) ...... 211

FIGURE 4.8 (A) PHARMACOKINETICS AFTER A SINGLE S.C. INJECTION OF 50MG/ML PYY3-36 ...... 212

FIGURE 4.8 (B) PHARMACOKINETICS AFTER A SINGLE S.C. INJECTION OF 50MG/ML PYY2-36(ΑLTX-4H) ...... 212

FIGURE 4.9 PHARMACOKINETICS AFTER ADMINISTRATION OF 20MG/ML PYY2-36(ΑLTX-4H)

WITH AND WITHOUT 1:1 ZN ...... 213

FIGURE 4.10 PHARMACOKINETIC PROFILE OF A SINGLE SUBCUTANEOUS INJECTION PYY2-36(ΑLTX-4H)

AT 50MG/ML 1:1 ZN ...... 214

FIGURE 4.11 (A) EFFECT OF SUBCUTANEOUS ADMINISTRATION OF PYY2-36(ΑLTX-4H) (3000NMOL/KG) ON

CUMULATIVE FOOD INTAKE IN C57BL/6 DIO MICE ...... 215

FIGURE 4.11 (B) EFFECT OF SUBCUTANEOUS ADMINISTRATION OF PYY2-36(ΑLTX-4H) (3000NMOL/KG) ON BODY

WEIGHT IN C57BL/6 DIO MICE ...... 216

FIGURE 4.12 (A) EFFECT OF SUBCUTANEOUS ADMINISTRATION OF PYY2-36(ΑLTX-4H) (500 AND 3000NMOL/KG) ON

CUMULATIVE FOOD INTAKE IN MALE WISTAR RATS ...... 217

FIGURE 4.12 (B) EFFECT OF SUBCUTANEOUS ADMINISTRATION OF PYY2-36(ΑLTX-4H) (500 AND 3000NMOL/KG) ON

BODY WEIGHT IN MALE WISTAR RATS ...... 218

FIGURE 4.13 FRAGMENTS OF PYY2-36(ΑLTX-4H) TESTED AGAINST ADAS ...... 222

FIGURE 4.14 ANALOGUES OF PYY2-36(ΑLTX-4H) TESTED AGAINST ADAS...... 225

FIGURE 4.15 (A) BINDING AFFINITIES OF PYY AND ANALOGUES TO HUMAN Y2R ...... 231

FIGURE 4.15 (B) BINDING AFFINITIES OF PYY AND ANALOGUES TO HUMAN Y1R ...... 231

FIGURE 4.15 (C) BINDING AFFINITIES OF PYY AND ANALOGUES TO MOUSE Y2R ...... 231

FIGURE 4.15 (D) BINDING AFFINITIES OF PYY AND ANALOGUES TO HUMAN Y1R ...... 231

FIGURE 4.16 EFFECT OF SUBCUTANEOUS ADMINISTRATION OF 200, 1000 AND 4000NMOL/KG

PYY2-36(ΑLTX-4H)-PEG ON FOOD INTAKE IN FASTED MICE C57BL/6 MICE ...... 232

FIGURE 4.17 CUMULATIVE 24HOUR FOOD INTAKE AFTER ADMINISTRATION OF 200, 1000, AND 4000NMOL/KG

PYY2-36(ΑLTX-4H)-PEG ...... 233

FIGURE 4.18 PHARMACOKINETIC PROFILE OF A SINGLE SUBCUTANEOUS INJECTION OF 50MG/ML

PYY2-36(ΑLTX-4H)-PEG TO MALE WISTAR RATS ...... 234

FIGURE 4.19 (A) BINDING AFFINITIES OF PYY AND ANALOGUES TO HUMAN Y2R ...... 237

FIGURE 4.19 (B) BINDING AFFINITIES OF PYY AND ANALOGUES TO HUMAN Y1R ...... 237

FIGURE 4.19 (C) BINDING AFFINITIES OF PYY AND ANALOGUES TO MOUSE Y2R ...... 237

FIGURE 4.19 (D) BINDING AFFINITIES OF PYY AND ANALOGUES TO HUMAN Y1R ...... 237

FIGURE 4.20 EFFECT OF SUBCUTANEOUS ADMINISTRATION OF 100NMOL/KG PYY3-36 AND PYY2-36(4H) ON FOOD

INTAKE IN FASTED C57BL/6 MICE ...... 238

FIGURE 4.21 EFFECT OF SUBCUTANEOUS DOSE RESPONSE OF 100, 300, AND 500NMOL/KG PYY2-36(4H) ON FOOD

INTAKE IN FASTED MICE C57BL/6 MICE ...... 239

FIGURE 4.22 CUMULATIVE 24HOUR FOOD INTAKE AFTER ADMINISTRATION OF 100, 300, AND 500NMOL/KG

PYY2-36(4H) ...... 240

FIGURE 4.23 EFFECT OF ZN ON SUBCUTANEOUS ADMINISTRATION OF 500NMOL/KG PYY2-36(4H) ON FOOD INTAKE IN

FASTED C57BL/6 MICE ...... 241

FIGURE 4.24 CUMULATIVE 24HOUR FOOD INTAKE AFTER ADMINISTRATION OF 500NMOL/KG PYY2-36(4H)

WITH AND WITHOUT 1:1 ZN ...... 242

FIGURE 4.25 PHARMACOKINETIC PROFILE IN RAT OF PYY2-36(4H) WHEN ADMINISTERED SC AT 50MG/ML DOSE WITH

VARIOUS AMOUNTS OF ZN IN THE FORMULATION ...... 243

FIGURE 4.26 PHARMACOKINETIC PROFILE OF A SINGLE SUBCUTANEOUS INJECTION 50MG/ML PYY2-36(4H) ...... 244

FIGURE 4.27 EFFECT OF SUBCUTANEOUS ADMINISTRATION OF 100NMOL/KG PYY2-36(4H) AND

PYY2-36(ΑLTX-4H) ON FOOD INTAKE IN FASTED C57BL/6 MICE ...... 246

FIGURE 4.28 CUMULATIVE 24HOUR FOOD INTAKE AFTER ADMINISTRATION OF 100NMOL/KG PYY2-36(4H) AND

PYY2-36(ΑLTX-4H) ...... 246

FIGURE 4.29 PHARMACOKINETICS AFTER ADMINISTRATION OF PYY2-36(4H) AT 200MG/ML DOSE CONTAINING

VARIOUS AMOUNTS OF ZN IN THE FORMULATION ...... 248

FIGURE 4.30 PHARMACOKINETIC PROFILE AFTER ADMINISTRATION OF PYY2-36(4H) AND PYY2-36(ΑLTX-4H)

BOTH AT 200MG/ML DOSE CONTAINING 1:1 ZN ...... 249

FIGURE 4.31 (A) EFFECT OF SUBCUTANEOUS ADMINISTRATION OF PYY2-36(4H) ON CUMULATIVE FOOD INTAKE IN

C57BL/6 DIO MICE...... 250

FIGURE 4.31 (B) EFFECT OF SUBCUTANEOUS ADMINISTRATION OF PYY2-36(4H) ON BODY WEIGHT IN C57BL/6

DIO MICE ...... 251

FIGURE 4.32 (A) EFFECT OF SUBCUTANEOUS ADMINISTRATION OF PYY2-36(4H) ON CUMULATIVE FOOD INTAKE

IN MALE WISTAR RATS ...... 252

FIGURE 4.32 (B) EFFECT OF SUBCUTANEOUS ADMINISTRATION OF PYY2-36(4H) ON BODY WEIGHT IN MALE WISTAR

RATS ...... 253

FIGURE B.1 SUMMARY OF SEQUENCES OF PYY ANALOGUES ...... 310

FIGURE B.2 SEQUENCE OF PP-X USED TO RAISE THE ANTIBODY FOR THE PP ANALOGUES RIA ...... 310

FIGURE D.1 MAP OF PCDNA3.1+ VECTOR ...... 313

FIGURE D.2 INSERT MAP OF THE HY2 AND HY4 CDNA CLONES ...... 313

FIGURE D.3 (A) RESTRICTION ENDONUCLEAS DIGESTIONS OF PLASMID DNA EXTRACTED FROM E.COLI TRANSFORMED

WITH PLASMID CONTAINING Y2 RECEPTOR CDNA ...... 314

FIGURE D.3 (B) RESTRICTION ENDONUCLEAS DIGESTIONS OF PLASMID DNA EXTRACTED FROM E.COLI TRANSFORMED

WITH PLASMID CONTAINING Y4 RECEPTOR CDNA ...... 314

INDEX OF TABLES

TABLE 1.1 SUMMARY OF LIGAND PREFERENCES AT THE Y RECEPTORS ...... 42

TABLE 1.2 AMINO ACID SEQUENCE OF Y RECEPTOR LIGANDS ...... 44

TABLE 2.1 COMPONENTS OF THE PYY AND PYY ANALOGUES RADIOIMMUNOASSAY USED ...... 95

TABLE 2.2 COMPONENTS OF THE PP AND PP ANALOGUES RADIOIMMUNOASSAY USED ...... 97

TABLE 3.1 IDENTIFIED PEAKS FROM MALDI MS OF PP IN THE PRESENCE OF RBB FOR 10 AMD 60MINS AT 37°C ... 114

TABLE 3.2 IDENTIFIED PEAKS FROM MALDI MS OF PP DIGEST WITH RLM FOR 10MINS AT 37°C ...... 116

TABLE 3.3 IDENTIFIED PEAKS FROM MALDI MS OF PP DIGEST WITH RLM FOR 60MINS AT 37°C ...... 118

TABLE 3.4 IDENTIFIED PEAKS FROM MALDI MS OF FRACTION RT=16-17M OF PP DIGEST WITH RLM

FOR 60MINS AT 37°C ...... 120

TABLE 3.5 IDENTIFIED PEAKS FROM MALDI MS OF FRACTION RT=21-22M OF PP DIGEST WITH RLM

FOR 60MINS AT 37°C ...... 123

TABLE 3.6 IDENTIFIED PEAKS FROM MALDI MS OF FRACTION RT=24-25M OF PP DIGEST WITH RLM

FOR 60MINS AT 37°C ...... 125

TABLE 3.7 IDENTIFIED PEAKS FROM MALDI MS OF FRACTION PPF1 OF PP DIGEST WITH 0.1MU TRYPSIN

FOR 15MINS AT 37°C ...... 134

TABLE 3.8 IDENTIFIED PEAKS FROM MALDI MS OF FRACTION PPF2 OF PP DIGEST WITH 0.1MU TRYPSIN

FOR 15MINS AT 37°C ...... 135

TABLE 3.9 BINDING AFFINITIES OF PP AND ANALOGUES WITH SUBSTITUTIONS FOR TRYPSIN RESISTANCE TO HUMAN Y4R ...... 137

TABLE 3.10 IDENTIFIED PEAKS FROM MALDI MS OF PP DIGEST WITH NEP

FOR 30 AND 120MINS AT 37°C ...... 147

TABLE 3.11 IDENTIFIED PEAKS FROM MALDI MS OF FRACTION RT=17-19M OF PP DIGEST WITH NEP

FOR 120MINS AT 37°C ...... 149

TABLE 3.12 POSITION 0 (N-TERMINAL) MODIFICATIONS OF PP AND THE EFFECTS ON Y4R AFFINITY, SUSCEPTIBILITY TO

NEP DEGRADATION, IN VIVO BIOEFFICACY...... 155

TABLE 3.13 POSITION 30 (C-TERMINAL) MODIFICATIONS OF PP AND THE EFFECTS ON Y4R AFFINITY, SUSCEPTIBILITY TO

NEP DEGRADATION, IN VIVO BIOEFFICACY...... 157

TABLE 3.14 COMBINED MODIFICATIONS AT POSITIONS 0 AND 30 OF PP AND THE EFFECTS ON Y4R AFFINITY,

SUSCEPTIBILITY TO NEP DEGRADATION, IN VIVO BIOEFFICACY ...... 158

TABLE 3.15 SINGLE MID-SECTION MODIFICATIONS OF PP AND THE EFFECTS ON Y4R AFFINITY, SUSCEPTIBILITY TO NEP

DEGRADATION, IN VIVO BIOEFFICACY ...... 159

TABLE 3.16 THE EFFECT OF COMBINED SUBSTITUTIONS AT POSITIONS 6, 19, 23 AND 30 ON Y4R AFFINITY,

SUSCEPTIBILITY TO NEP DEGRADATION, IN VIVO BIOEFFICACY ...... 160

TABLE 3.17 THE EFFECT OF COMBINED SUBSTITUTIONS AT POSITIONS 0, 6, 19, 23 AND 30 ON Y4R AFFINITY,

SUSCEPTIBILITY TO NEP DEGRADATION, IN VIVO BIOEFFICACY ...... 161

TABLE 3.18 THE EFFECT OF COMBINED SUBSTITUTIONS AT POSITIONS 0, 6, 16, 17, 19, 23 AND 30 ON Y4R AFFINITY,

SUSCEPTIBILITY TO NEP DEGRADATION, IN VIVO BIOEFFICACY ...... 162

TABLE 3.19 THE EFFECT OF GLOBAL SUBSTITUTIONS ON Y4R AFFINITY, SUSCEPTIBILITY TO NEP DEGRADATION, IN VIVO

BIOEFFICACY ...... 165

TABLE 3.20 PP ANALOGUES WITH SIMILAR Y4R AFFINITY BUT DIFFERENT SUSCEPTIBILITIES TO DEGRADATION BY NEP,

AND DIFFERENCES IN IN VIVO BIOEFFICACY ...... 169

TABLE 3.21 PP ANALOGUES THAT ARE BOTH RELATIVELY NEP-RESISTANT BUT DIFFER AT THEIR AFFINITY TO THE Y4R

AND WITH MODEST DIFFERENCES IN IN VIVO BIOEFFICACY ...... 173

TABLE 4.1 PRIMARY AMINO ACID SEQUENCES OF THE HOMOLOGOUS Α-HELICAL REGIONS OF ΑLTX, EX4 AND

PYY-ΑLTX ...... 195

TABLE 4.2 IMMUNOGENICITY OF PLASMA FROM DOG PHARMACOKINETIC STUDY, 7 DAYS AFTER FINAL INJECTION ... 219

TABLE 4.3 IMMUNOGENICITY OF TERMINAL PLASMA SAMPLES FROM CHRONIC ADMINISTRATION OF PYY2-36(ΑLTX-4H)

TO MALE WISTAR RATS ...... 220

TABLE 4.4 EFFECT OF ZINC CHLORIDE ON IMMUNOGENICITY ...... 221

TABLE 4.5 SUMMARY OF THE DISPLACEMENT RATIO OF PYY2-36(ΑLTX-4H) FRAGMENTS WITH DOG, RAT AND SHEEP

ADAS ...... 223

TABLE 4.6 AVERAGE RATIOS OF PYY2-36(ΑLTX-4H) FRAGMENTS AGAINST DOG, RAT AND SHEEP ADAS ...... 224

TABLE 4.7 SUMMARY OF THE DISPLACEMENT RATIO OF PYY2-36(ΑLTX-4H) ANALOGUES WITH DOG, RAT AND SHEEP

ADAS ...... 227-228

TABLE 4.8 AVERAGE RATIOS OF PYY2-36(ΑLTX-4H) FRAGMENTS AGAINST DOG, RAT AND SHEEP ADAS ...... 229

TABLE 4.9 EFFECT OF PEGYLATION ON IMMUNOGENICITY ...... 235

TABLE 4.10 SUMMARY OF THE CHRONIC DATA STUDIES WITH PYY2-36(4H) AND IMMUNOGENICITY OF TERMINAL

PLASMA SAMPLES ...... 254

TABLE E.1 RADIOIMMUNOASSAY PLAN ...... 316

CHAPTER 1

GENERAL INTRODUCTION

1.1 OBESITY

Obesity is one of the world’s largest health concerns. Its prevelance is not only on the rise in developed nations but also in developing countries, particularly in urban settings. In 2008, the World Health Organisation stated that approximately 1.5 billion adults across the world were considered to be overweight and at least 500 million were considered to be obese. These figures are set to increase; projections estimate that by 2015, 2.3 billion adults will be overweight and 700 million will be obese (WHO, 2011).

Obesity can lead to serious health consequences such as cardiovascular disease (including heart disease and stroke), type 2 diabetes mellitus (T2DM), musculoskeletal disorders (especially osteoarthritis), obstructive sleep apnoea, and some cancers (including endometrial, breast and colon) (Flegal et al., 2007). These conditions put a tremendous financial strain on healthcare systems. In the UK, obesity and associated diseases are estimated to cost the NHS at least £4.2 billion each year (DoH, 2011). Recent strategies to combat obesity have either proved ineffective or harmful (Sacks et al., 2009, Sjostrom et al., 2007, Buchwald et al., 2004). New treatments with better efficacy which cause significant weight loss long-term would therefore be of great benefit to society.

The cause of obesity is an imbalance between energy input and energy output. Today, an increasingly large percentage of the population live in an obesogenic environment with high energy content food readily available plus a sedentary life style (Pearson and Biddle, 2011) (Prentice and Jebb, 2004). The biological systems that have evolved to regulate homeostasis were designed for a very different environment where food availability was limited and are therefore designed to favour body weight gain (Schwartz and Niswender, 2004).

1.1.2 CURRENT THERAPIES

Although overall life expectancy has continued to increase since the 1950s (Bjorbaek, 2009), obesity has been shown to decrease life expectancy by approximately seven years (Munzberg, 2010, Caro et al., 1996, Hindlet et al., 2009, Berg et al., 2002, Hu et al., 1996,

Walter et al., 2009). The social and economic cost of obesity is great and weight loss in overweight and obese patients in encouraged. The first strategy is to modify diet and lifestyle by reducing calories ingested and increasing calories expended. It has been shown, however, that there is limited compliance with very low calorie or low fat diets (Arita et al., 1999, Hotta et al., 2001), and there is often weight regain approximately 18 months after completion of the diet (Hotta et al., 2001). If diet and lifestyle modifications are ineffective, the next step is usually some form of medical treatment to aid weight loss. Medical treatments for the treatment of obesity are discussed in section 1.3.4. Currently, the most successful method of achieving long term weight loss is through surgery; typically gastric banding or gastric bypass (discussed further in section 1.3.2). National Institute for Health and Clinical Excellence (NICE) guidelines recommend that bariatric surgery should only be carried out if all appropriate non-surgical measures have failed to be effective over 6 months and if the patient has a BMI >40kg/m2, or a combination of a BMI between 40>35kg/m2 and other significant diseases (such as T2DM) that could be improved by weight loss (NICE, 2006). For individuals with BMI >50kg/m2, NICE recommends bariatric surgery as first line treatment. However, there are a number of groups that suggest bariatric surgery should be implemented at lower BMI and as first line therapy (Fruebis et al., 2001, Qi et al., 2004) as recommendations from NICE were based on limitations that are now outdated. Surgical techniques have greatly improved, are minimally invasive, and bariatric surgery in general has been found to have weight-independent benefits especially on T2DM which points to the insufficiency of using BMI as the qualifying criteria for surgery.

1.2 REGULATION OF FOOD INTAKE

Despite wide variations in daily food intake and energy expenditure, the majority of people maintain a stable weight for long periods. This balance is achieved through complex homeostatic mechanisms. Mechanoreceptors within the gut, and hormonal secretions by the intestine and pancreas, signal acute fluxes in energy intake and energy expenditure, while adipose tissue produces peptides such as leptin and adiponectin that reflect longer term energy stores. These signals are conveyed via nerve afferents to a variety of central

29 strucures. The control of food intake is regulated by two complimentary systems: the homeostatic and hedonic pathways. The homeostatic pathway increases the motivation to eat (hunger) in conditions of low energy stores, and induce satiety in a post-prandial setting. The hedonic control of food intake is rewards-based, and can supersede the homeostatic pathway when presented with highly palatable, energy-rich foods (Matsuda and Shimomura, 2013). These various regulatory strucutres will now be discussed.

1.2.1 CENTRAL STRUCTURES INVOLVED IN THE ENERGY BALANCE

1.2.1.1 The Hypothalamus

The hypothalamus regulates many homeostatic processes, including energy balance. It is situated above the pituitary gland and on either side of the third ventricle. It is composed of over forty morphologically distinct nuclei (Jilkova et al., 2013). Those nuclei with a role in energy homeostasis will be discussed in sections 1.2.1.1.1 to 1.2.1.1.5 (Fig 1.1).

1.2.1.1.1 Arcuate Nucleus (ARC)

The ARC is an area of the hypothalamus that has an important role in the regulation of appetite and body weight. It lies on either side of the ventral part of the third ventricle, towards the base of the hypothalamus and just above the median eminence (ME). The ME has an incomplete blood brain barrier (BBB) (Polonsky et al., 1988b, Polonsky et al., 1988a). This allows nutrients and hormones within the plasma to activate the ARC (Broadwell and Brightman, 1976).

The hypothalamus contains two distinct neuronal subtypes that regulate energy balance. These are the anorexigenic POMC/CART neurons (Porte et al., 2002) and the orexigenic NPY/AgRP neurons (Broberger et al., 1998, Baura et al., 1993).

Figure 1.1 Schematic tree-diagram of the hypopthalamus and the various nuclei. Blue arrows indicate anorexigenic signals and pink arrows indicate orexigenic signals and the different areas they signal to. Orexigenic NPY/AgRP neurons of the ARC are also in pink and anorexigenic POMC/CART neurons are blue. The ME in the hypothalamus and AP in the brainstem directly oppose areas of incomplete BBB where signals released in the periphery below, may pass. The hypothalamus and brain stem have neuronal connections, as does the brains stem with the periphery through the vagus nerve.

Anorexigenic POMC/CART neurons are located in the ventrolateral region of the ARC and co-express pro-opiomelanocortin (POMC) and cocaine and amphetamine-regulated transcript (CART). Alpha-melanocyte-stimulating hormone (α-MSH) is a post-translational product of the POMC transcript and is the neurotransmitter by which POMC/CART neurons exert their anorectic effect through activation of the melanocortin-4 receptor (MC4r) (Fekete et al., 2000).

Orexigenic NPY/AgRP neurons are located in the ventromedial part of the ARC and co- express neuropeptide Y (NPY) and Agouti-related protein (AgRP), both peptides which

31 potently stimulate feeding (Meister, 2007). AgRP is an inverse agonist at the MC4r, reversing the anorectic effects of α-MSH (Bagnol et al., 1999). NPY/AgRP neurones also relesase the inhibitory neurotransmitter gamma aminobutyric acid (GABA) which inhibits POMC neurons.

There is significant interaction between the neuronal populations. They project to similar areas within the brain, and indeed NPY/AgRP neurons synapse with POMC/CART neurons and can inibit them. There is also reciprocal regulation of the neurons; for example leptin both activates POMC and inhibits AgRP neurons (Cowley et al., 2001, van den Top et al., 2004).

1.2.1.1.2 Paraventricular Nucleus (PVN)

The PVN integrates signals from several CNS regions, with particular projections from the ARC, LHA and brainstem (Sawchenko and Swanson, 1983b, Sawchenko and Swanson, 1983a). In particular from the ARC, the PVN receives strong input from POMC/CART and NPY/AgRP neurons which release the MC4r agonist (α-MSH) and antagonist (AgRP) (Cowley et al., 1999).

In rodents, destruction of the PVN leads to hyperphagia and weight gain (Aravich and Sclafani, 1983, Leibowitz et al., 1981). Injection of orexigenic factors such as NPY, AgRP, ghrelin or orexin-A into the PVN stimulates food intake (Stanley and Leibowitz, 1985, Wirth and Giraudo, 2000, Melis et al., 2002, Olszewski et al., 2003, Dube et al., 1999), while anorectic peptides like leptin and GLP-1 reduce food intake when administered into the PVN (Elmquist et al., 1998, McMahon and Wellman, 1998).

MC4r is highly expressed in the PVN (Siljee et al., 2013) and MC4r-knockout mice exhibit an obese and hyperphagic phenotype (Huszar et al., 1997). Though the MC4r is widely expressed in the brain (including in the amygdala, cortex, thalamus, striatum, brainstem and spinal cord) (Mountjoy et al., 1994), the obese phenotype of MC4r-knockout mice can be reversed when the MC4r receptor is reactivated in the PVN alone (Balthasar et al., 2005).

1.2.1.1.3 Dorsomedial Nucleus (DMN)

The DMN mainly receives inputs from the lateral hypothalamic area (LHA described in more detail in section 1.2.1.1.5) and the ventromedial nucleus (VMN – described in section 1.2.1.1.4) and has strong projections to the PVN (Ter Horst and Luiten, 1987, Luiten et al., 1987). DMN lesions cause a short-term decrease in food intake which leads to a loss of body weight. This decrease in body mass is sustained even when caloric intake returns to normal (Dalton et al., 1981) suggesting that the DMN has regulatory functions concerned with body mass and growth rather than food intake. The short-term decrease in food intake is thought to be partly due to a decrease in response to mechanisms that would normally increase food intake e.g. hypoglycaemia (Bellinger and Bernardis, 2002). Therefore, in the long-term, rats with DMN lesions regulate their food intake around a new lower BW as they remain susceptible to diet-induced obesity if presented with more palatable food.

The DMN expresses α-MSH and its receptor, CART, as well as the orexigenic peptides melanin-concentrating hormone (MCH), orexin, and AgRP. When CART and AgRP are injected into the DMN, they increase food intake, whereas α-MSH agonists suppress food intake. The DMN also contains prolactin-releasing peptide (Roland et al., 1999) which suppresses food intake when injected into the DMN, possibly by increasing the release of α- MSH (Seal et al., 2001). The DMN may also be the site of action of the anorectic peptide GLP-2 (Tang-Christensen et al., 2001a).

1.2.1.1.4 Ventromedial Nucleus (VMN)

The VMN is part of the mid region of the hypothalamus adjacent to the ARC. Ablation of the VMN results in hyperphagia and obesity in rodents (Shimizu et al., 1987). POMC/CART and NPY/AgRP neurons project from the ARC to the VMN, as do neurons from the LHA, DMN and PVN (Ter Horst and Luiten, 1987). The VMN has receptors for α-MSH and leptin (King, 2006, Fu and van den Pol, 2008). MC4r activation stimulates expression of brain-derived- neurotrophic-factor (BDNF) (Xu et al., 2003); this then acts within the PVN to cause release of corticotropin-releasing factor (CRF), which subsequently suppresses food intake (Gotoh et al., 2013).

1.2.1.1.5 Lateral Hypothalamic (LH) Area

The LHA has an opposing role to the VMN. The LHA consists of neurons expressing orexigenic neuropeptides including orexin-A and -B, MCH, and NPY (Barson et al., 2013, Morris, 1989). Bilateral lesion to the LHA leads to an inhibition of food intake (Anand and Brobeck, 1951) and stimulation of the LHA, either electrically or with the excitatory neurotransmitter glutamate (via the NDMA receptor), causes an increase in food intake (Delgado and Anand, 1953, Stanley et al., 1993). The feeding effects of NPY (Lee and Stanley, 2005) and the orexins (Doane et al., 2007) are also thought to be partly mediated by NDMA receptors in the LHA.

The LHA also plays a role in glucose-sensing, with increased glucose levels causing increased neuronal activity in MCH neurons, and decreased activity in orexin and NPY neurons (Burdakov et al., 2005).

1.2.1.2 Role of Brainstem and vagus nerve in Energy Homeostasis

The brainstem is located in the posterior of the brain and is structurally continuous with the spinal cord. The brainstem relays information to and from the brain to the periphery via the cranial/spinal nerves. The brainstem regulates a number of essential functions, including breathing, consciousness, temperature regulation and acute nutritional status (Young, 2012). Extending from the brainstem, the vagus nerve lies alongside the internal jugular vein and into the chest and abdomen. Approximately 80-90% of vagus nerve fibres are afferent, conveying sensory information from the periphery to the brain. The vagus nerve plays an important role in the gut-brain axis (Laskiewicz et al., 2003), and vagotomy studies suggest it is essential for the actions of circulating factors such as ghrelin (Date, 2012), pancreatic polypeptide (Astrup et al., 2009), and peptide tyrosine-tyrosine (Lijnen et al., 2010). It has also been suggested that the brainstem can mediate the anorectic effects of gut hormones independent of the hypothalamus; CCK reduces food intake in decerebrated rats, which lack neuronal communication between the brainstem and the hypothalamus (Grill and Smith, 1988, Grill and Kaplan, 2002). Within the brainstem, the vagus nerve activates neurons in the dorsal vagal complex (DVC) which then relay signals to various parts of the brain.

The DVC includes the area postrema (AP), nucleus of solitary tract (NTS) and the dorsal motor nucleus. The AP is located posterior to the fourth ventricle, and has an incomplete blood brain barrier (Fig 1.1). It can therefore receive information from peripheral and central chemical mediators, as well as from the vagus nerve. Receptors for a number of peripheral satiety factors have been identified in the AP, including the PP Y4 receptor and the GLP-1 receptor (Goke et al., 1995, Menendez and Atrens, 1991, McGowan et al., 1992, Obici et al., 2002, Schwartz et al., 1992, Date, 2012, Wang et al., 2008). Lesion studies show the AP has a role in feeding and energy balance: ablation attenuates PYY3-36 induced inhibition of food intake in rats, and inhibits basal secretion of PP (Benoit et al., 2002). The NTS is situated next to the AP, and also lacks a blood brain barrier. Similarly, it integrates peripheral chemical signals and information from the vagus . Lesions here also affect feeding behaviour (Laskiewicz et al., 2003, Date, 2012, Yuan et al., 1999) and caused significant weight loss in rats (Grill and Smith, 1988).

1.2.2 PERIPHERAL CONTROL

Peripheral signals affecting caloric intake and energy homeostasis can be broadly divided into two categories: 1) those which are released in response to a meal and which act acutely to induce satiety (leading to meal termination) and/or satiation (prolonged satiety between meals); and 2) those which act over a longer time scale, in proportion to adiposity and overall nutritional status, to centrally regulate total body energy homeostasis. The interaction of satiety, energy expenditure and even glucose regulation, in turn, leads to the regulation of fat mass, body weight and nutrient availability.

1.2.2.1 Adipose-derived signals

1.2.2.1.1 Leptin

Leptin is a product of the ob gene which is expressed principally in the adipocytes in the white adipose tissue (Zhang et al., 1994). It is released into circulation in proportion to adipose tissue mass (Nicolaidis and Rowland, 1976), and as such is a marker of adiposity. It

35 therefore helps control a number of physiological proceses, including reproduction, the immune system, and energy homeostasis (Kojima et al., 1999). Leptin acts via the long form of the leptin receptor, Ob-Rb, which is widely expressed in the ARC, VMN, DMN and LHA of the hypothalamus and the medial preoptic area (MPOA) (Date et al., 2000, Fei et al., 1997) and in other areas of the CNS such as the brainstem (Mercer et al., 1998).

With regards to feeding, leptin reduces food intake. Chronic peripheral administration of leptin in rats decreases food intake, body weight and fat mass, and prevents the fall in energy expenditure associated with weight loss (Sakata et al., 2002). Circulating levels of leptin are suppressed by short term fasting and are restored by refeeding (Hosoda et al., 2000). Mutations or disruptions of the ob gene, and subsequent leptin deficiency, leads to hyperphagia and obesity in both rodents and humans (Jarkovska et al., 2004, Zhang et al., 1994, Wren et al., 2000), which can be reversed with exogenous leptin administration (Cummings et al., 2001, Sakata et al., 2002, Tsubone et al., 2005, Farooqi et al., 1999).

Leptin affects feeding via receptors in the hypothalamus and the brainstem. Within the ARC, leptin receptors are located on both orexigenic NPY/AgRP neurons and anorexigenic POMC/CART neurons. POMC/CART neurons are stimulated by leptin, whilst NPY/AgRP neurons are inhibited (Thompson et al., 2004, Otto et al., 2001). Knock-down of leptin receptors int the brainstem induces hyperphagia in rats (Hansen et al., 2002).

As leptin rises in proportion to adiposity, the majority of obese humans have high circulating leptin levels (Cummings et al., 2002, English et al., 2002). However, they remain overweight, and exogenous leptin is ineffective at reducing body weight in these subjects (Tamura et al., 2002, Chen et al., 2004). This is due to leptin resistance. This is caused by reduced active transport of leptin across the blood brain barrier, as well as reduced signalling within the hypothalamus (Date et al., 2002). Leptin resistance may also be due down-regulation of leptin receptor mRNA expression and leptin receptor protein following exposure to peristenstly high circulating levels of leptin (Traebert et al., 2002).

1.2.2.1.2 Adiponectin

Adiponectin is a fat-derived complement-like protein (Wang et al., 2002). In contrast to leptin (and insulin as discussed in 1.2.2.2), adiponectin levels are increased in response to weight loss and decreased in obesity (Pirnik et al., 2011, Williams et al., 2003, Hotta et al., 2001). However, perhaps counter-intuitively, peripheral treatment with adiponectin decreases weight gain in rodents. It increases EE by increasing fatty acid oxidation, but has no effect on food intake (Polak et al., 1975). The elevation of EE is mediated via adiponectin receptors in the PVN of the hypothalamus (Liddle et al., 1985). Adiponectin may also play a part in low-grade chronic inflammation that is associated with obesity. Resolution of inflammation in adipose tissue increases plasma levels of adiponectin (Jilkova et al., 2013). Additionally, adiponectin has anti-inflammatory properties (Gibbs et al., 1973) which can further help to resolve the inflammation however its physiological role remains unclear and requires further investigation.

1.2.2.2 Pancreatic signals

Insulin is a peptide hormone secreted by the β-cells of the pancreas in response to increased glucose levels in the circulation. Insulin regulates glucose homeostasis by stimulating glucose uptake and glycogen storage, and suppressing gluconeogenesis. Insulin levels increase acutely in response to meal ingestion (Kissileff et al., 1981). Circulating levels are proportional to total body fat and fat distribution, and it has been suggested that insulin acts as a signal of adiposity (West et al., 1984, West et al., 1987). However, circulating insulin levels are also inversely correlated with insulin sensitivity in the periphery: long term elevated circulating insulin levels are linked to insulin resistance and T2DM.

Insulin acts at the brain to have an anorectic effect and crosses the blood brain barrier via a saturable receptor-mediated process (Pardridge et al., 1985). Injection of insulin into the third ventricle decreases food intake and decreases overall body weight (Thompson et al., 1975). Further studies have suggested that the PVN and VMN are critical for this effect (Irwin et al., 2013a, McGowan et al., 1992).

Insulin binds to a tyrosine kinase receptor, a receptor which is distributed widely throughout the brain including sites in the PVN, VMN and the ARC. Stereotactic injection of antisense mRNA of the insulin receptor transcript into the ARC reverses the anorectic effect of insulin (Obici et al., 2002). Insulin receptors have been found on both POMC/CART and NPY/AgRP neurons of the ARC. Central insulin administration prevents the increase in NPY mRNA associated with fasting (Schwartz et al., 1992). This suggests that low body fat and, therefore, low insulin levels, contribute to fasting-induced increase in NPY and increased food intake. In contrast, central administration of insulin increases POMC mRNA expression in POMC/CART neurons (Benoit et al., 2002). The effects of central insulin are reversed by the administration of an MC4r antagonist, suggesting the anorectic central effects of insulin on appetite may be mediated via the the POMC product, α-MSH (Benoit et al., 2002).

The effect of peripherally administered insulin on the CNS is more difficult to investigate due to the peripheral effect of insulin on glucose homeostasis with consequent inhibition of food intake independent of any direct central effects. However, the use of a hyperinsulinaemic- euglycaemic clamp suggests that peripheral insulin inhibits food intake (Irwin et al., 2012).

1.2.2.3 Gastrointestinal-derived signals

1.2.2.3.1 Ghrelin

Until the recent discovery of Insl5 (Grosse et al., 2014), Ghrelin was the only known peripherally produced orexigenic hormone. It is secreted primarily from the stomach (Moran et al., 1998) and also at low levels from the duodenum, ileum, caecum and colon (Schwartz et al., 1999, McLaughlin et al., 1985). Circulating ghrelin is found in two forms: acylated ghrelin and the non-modified des-acyl ghrelin (Meereis-Schwanke et al., 1998). Acylated ghrelin is the active form and has the addition of the acyl side chain, n-octanoic acid, which is essential for receptor binding and subsequent effects on food intake (Kojima et al., 1999). Ghrelin is an endogenous agonist for the growth hormone secretagogue receptor (GHS-R) (Lo et al., 2010) and acts via this receptor to stimulate food intake and growth hormone secretion (Jordan et al., 2008). The GHS-R is highly expressed in the

38 anterior pituitary, but also in the ARC and the VMN of the hypothalamus (Tannenbaum et al., 1998).

Diurnal variation is seen in circulating ghrelin levels with the basal levels in humans at their highest in the morning and lowest at night (Shiiya et al., 2002). Plasma ghrelin levels also appear to increase on fasting and decrease post-prandially with sharp peaks before meals (Roses, 2009) suggesting a role for ghrelin in meal initiation. Central or peripheral administration of ghrelin dose dependently increases food intake in rats and humans (Crawley and Beinfeld, 1983, Matson et al., 2000, Irwin et al., 2013b, Plusczyk et al., 1997). As well as stimulating food intake, peripheral administration of acylated ghrelin also increases adipogenesis and decreases lipid oxidation and UCP1 expression (Pandol et al., 1999, Fuhlendorff et al., 1990).

Basal ghrelin may be a signal reflecting long-term energy stores with levels inversely correlating to adiposity. Anorexic individuals have high circulating levels (Gerald et al., 1996) while obese subjects have low plasma ghrelin levels (Blomqvist et al., 1992, Germain et al., 2013). However, obese subjects do not experience the normal rapid post-prandial fall in circulating ghrelin, which may contribute to their obesity (Erdmann et al., 2005).

Ghrelin acts on a number of central strucutres. Within the hypothalamus, it activates NPY/AgRP neurons within the ARC (Chen et al., 2004, Date et al., 2002, Traebert et al., 2002, Wang et al., 2002). Genetic ablation, or neonatal ablation with monosodium glutamate, abolishes the effects of peripherally administered ghrelin on feeding (Berglund et al., 2003, Chen et al., 2004). Peripheral ghrelin also activates neurons of the NTS and AP of the brainstem (Cabrele and Beck-Sickinger, 2000). GHS-R are found on peripheral vagal afferents and vagotomy in both humans and rodents abolishes the orexigenic effect of peripheral ghrelin (Date et al., 2002, Dumont et al., 1998).

1.2.2.3.2 Cholecystokinin (CCK)

Cholecystokinin (CCK) is produced by mucosal entero-endocrine I-cells of the upper small intestine (Ferrier et al., 2002) and was the first gut-derived hormone to be found to inhibit food intake (Gibbs et al., 1973). Circulating levels of CCK rise post-prandially, remain high for

39 a number of hours (McTigue et al., 1993). Release is stimulated by amino acids and free fatty acids (FFAs). Peripheral administration of CCK inhibits food intake in rodents (Gibbs et al., 1973), and in humans (Gellman and Woolfson, 2002).

The long-term effects of CCK on energy homeostasis and on body weight regulation are unclear. Peripheral CCK has a rapid but relatively short-lived effect on appetite (Shi et al., 2013, Witteman et al., 1994). While food intake is inhibited by chronic administration of CCK in rodent models, a compensatory increase in meal frequency is also observed (West et al., 1984, West et al., 1987). In rats, CCK-induced satiety occurs predominantly through the CCK-1 receptor (Moran et al., 1998, Feinle-Bisset et al., 2005). Rats lacking the CCK-1 receptor (CCK1r) are obese and hyperphagic, while CCK antibodies and CCK-1r antagonists cause rats to become overweight and obese, although no significant increase of food intake was observed (McLaughlin et al., 1985, Meereis-Schwanke et al., 1998). In contrast, CCK-1r is not involved in the physiological regulation of satiety in mice as CCK-1r knockout mice were not obese compared to wild-type littermates, and are actually resistant to high-fat diet induced obesity (Lo et al., 2010).

CCK-1r also appears to have minimal effects on energy balance in man. Obese humans given highly speciﬁc CCK-1r agonists demonstrated no increased weight loss compared to placebo controls, but had many undesirable gastrointestinal side effects (Jordan et al., 2008). However, it is difficult to draw conclusions from this study as the control group were placed on a hypocaloric diet, thus making it difficult to demonstrate a difference between the two groups (Roses, 2009).

The effect of CCK on food intake is transient. Rats given a continuous infusion of CCK-8 (a shorter yet active form of CCK) for seven or fourteen days failed to lose weight compared to controls, and after only four hours became resistant to the acute anorectic effects of a CCK bolus (Adrian et al., 1976). This suggested rapid development of tolerance to CCK and desensitisation to its anorectic effects which might explain the lack of efficacy seen in human trials of CCK agonists. However, more recent studies show that twice daily (but not once daily) administration of a degradation-resistant CCK analogue (with and without PEGylation) has been shown to reduce food intake and thus lowering body weight over 28 and 35 days (Irwin et al., 2012, Irwin et al., 2013a).

1.2.2.4 Pancreatic Polypeptide (PP)-fold peptides

The PP-fold family consists of three peptides each with roles in appetite regulation: NPY, PP and PYY. All three show high levels of sequence homology (Blomqvist et al., 1992) (Table 1.2). The common structures found in these peptides are a left-handed polyproline-like helix formed from amino acid residues 2–8 and an α-helix formed from amino acid residues 15- 32. The two helices are connected by a β-turn and together form the PP-fold (Germain et al., 2013) (Fig 1.2). The fold between these helical structures is strengthened by non-covalent interactions between highly conserved residues, in particular Pro2, Pro5, Pro8, Tyr20, Leu24 and Tyr27 (Nygaard et al., 2006).

Figure 1.2 A schematic diagram of the basic structure of PP. The backbone is represented by a ribbon, aromatic carbon in grey, oxygen atom in red, and proline atoms in purple. Amino acids 1-8 make up the polyproline helix, 9-14 for the β-turn and the amphipathic α-helix from position 15-32. Adapted from (Adrian, 1978).

In humans, PP-fold peptides mediate their effects on appetite regulation via 4 different G- protein coupled “Y-receptors”: Y1, Y2, Y4 and Y5 (Taylor et al., 1978). Y receptors are coupled to Gi or G0 proteins, which leads to the inhibition of adenylate cyclase and inhibition of the secondary messenger cyclic adenosine monophosphate (cAMP) accumulation (Berthoud and Powley, 1991).

Each receptor has a different affinity for each PP-fold ligand (Table 1.1), different function and tissue distribution, thereby presenting a rare example of a multi-ligand/multi-receptor system.

Y Receptor Ligand-binding heirarchy

Y1 NPY = PYY = [Leu31, Pro34]NPY1 > NPY2-36 > NPY3-36 ≥ PP > NPY13-36 Y2 PYY3-36 > NPY ≥ NPY2-36 = NPY3-36 = NPY13-36 >> [Leu31, Pro34]NPY1 Y4 PP > PYY ≥ NPY > NPY2-36 Y5 NPY = PYY = NPY2-36 > hPP > [D-Trp32]NPY2 > NPY13-36 > rPP

Table 1.1 Summary of ligand preferences at the Y receptors. PYY: full lenth PYY1-36. PYY3-36: fragment of full length PYY. 1specific Y1 agonist (Feurle et al., 1980). 2specific Y5 agonist (Kojima et al., 2007)

Y receptors are mainly expressed in the brain and digestive tract, though the Y1 receptor (Y1r) is also found in blood vessels and Y2r in the heart (Deng et al., 2001, Lin et al., 2009). Within the hypothalamus, Y1r are found predominately in the ARC, and Y2r in the PVN and the ARC (Parker and Herzog, 1999). Y2r are also expressed in the brainstem (Hernandez et al., 1994). Y4r is highly expressed in the AP and the dorsal vagal complex of the brainstem (Parker et al., 1999, Parker and Herzog, 1999) where there is limited expression of other Y receptors.

1.2.2.4.1 Neuropeptide Y (NPY)

NPY, a 36 amino acid polypeptide (Whitcomb et al., 1990), is most abundant in the CNS and acts as an orexigenic neuropeptide in the hypothalamus (Taylor et al., 1985). It acts through the Y family of receptors with particular high affinity to Y1r and Y5r. Acute intracerebroventricular (ICV) administration of NPY in rats increases appetite (Sainsbury et al., 2010) and repeated administration leads to weight gain (Lassmann et al., 1980). As mentioned in section 1.2.1.1.1, NPY neurons are found in the ARC with projections

42 throughout the hypothalamus (Morris, 1989). Levels of hypothalamic NPY inversely correlate with food intake: NPY levels increase with fasting and decrease post-prandially (Uhe et al., 1992). Hormones involved in the control of energy balance, such as insulin and leptin (section 1.2.2.1.1), have a negative feedback effect on hypothalamic NPY expression (Fujimoto et al., 1997, Stephens et al., 1995). In addition to its effects as an orexigenic neuropeptide, NPY also reduces energy expenditure when injected ICV in rats (Egawa et al., 1991), promotes storage of energy as fat (Kuo et al., 2007), reduces anxiety and stress, has affects on the circadian rhythm, alcohol intake, and blood pressure (Tatemoto, 2004), and has a role in epilepsy (Colmers and El Bahh, 2003).

1.2.2.4.2 Pancreatic Polypeptide (PP)

PP is an amidated 36 amino acid PP-fold peptide released from F cells in the islets of Langerhans of the pancreas in response to meal ingestion. Protein, and particularly fat, are potent stimulators of PP release (Kinzig et al., 2007, Zipf et al., 1981). The magnitude of PP secretion is proportional to caloric intake and postprandial circulating PP levels then remain elevated for up to 6 hours (Zipf et al., 1983, Berntson et al., 1993). PP secretion is largely dependent on a vagal cholinergic mechanism (Schwartz et al., 1978). Vagal afferent connections from the GI tract to the NTS and the AP of the brainstem relay to the DMN indicating the presence of food in the stomach and intestines while vagal efferents from the DMN projecting to the pancreas signal to stimulate PP release (Strodel et al., 1984). PP release can, however, also be stimulated by CCK, ghrelin, motilin, secretin as well as in response to adrenergic activation during hypoglycaemia or exercise (Adrian et al., 1986).

PP is involved in a range of functions including inhibition of food intake, pancreatic secretions, gastric acid secretions, gallbladder motility and delay of gastric emptying (Martella et al., 1997). Lesions to the AP have been shown to ablate the ability of exogenous PP to inhibit pancreatic secretions (Deng et al., 2001).

Amino acid sequence Peptides 1 5 10 15 20 25 30 35 hNPY Y P S K P D N P G E D A P A E D M A R Y Y S A L R H Y I N L I T R Q R Y NH2 hPYY Y P I K P E A P G E D A S P E E L N R Y Y A S L R H Y L N L V T R Q R Y NH2 hPP A P L E P V Y P G D N A T P E Q M A Q Y A A D L R R Y I N M L T R P R Y NH2 Table 1.2 Amino acid sequence (single letter codes in Appendix A) of Y receptor ligands. Yellow are strongly conserved amino acids throughout all species; grey are highly conserved positions

PP has high affinity for the Y4 receptor (Y4r) and its effects on food intake are mediated via its activation of the Y4r (Asakawa et al., 1999). Y4r have been observed in the brainstem and hypothalamus including the AP and the ARC (Ueno et al., 1999, Jesudason et al., 2007). Similarly, peripheral administration of PP at a dose which significantly reduces food intake, shows neuronal activation in the lateral aspect of the ARC, the DMH, VMN, and LHA (Asakawa et al., 2003). However, the vagus nerve may also be involved since vagotomy abolishes the anorectic effects of PP in rats (Asakawa et al., 2003). The effect of PP on appetite can thus be mediated through directs actions of circulating hormone at the brainstem or hypothalamus, through vagal afferents or through a combination of both. The anorexigenic mechanism of PP appears to occur via a decrease orexigenic NPY, orexin and ghrelin expression, while increasing the expression of anorexigenic urocortin and BDNF in the hypothalamus (Sainsbury et al., 2010).

Increased basal levels of PP (usually caused by pancreatic endocrine tumours) have often been associated with weight loss (Mentlein et al., 1993a, Helou et al., 2008, Ballantyne et al., 1993). Exogenous administration of PP reduces food intake in both humans and mice (McFadden et al., 1992, Zhang et al., 1993). Infusion of PP which results in supraphysiological circulating levels reduced acute food intake by 20% compared to saline control, and 24 hour food intake by 25% (Batterham et al., 2003b). A lower infusion of PP which resulted in circulating levels of PP to within the pathophysiological range of pancreatic tumours reduced acute food intake by 10% but had no effect on 24 hour food intake (Savage et al., 1987). Over-expression of PP in mice produced a hypophagic and thin phenotype, suggesting that chronic exposure to PP does not lead to attenuation of the anorectic effect. The administration of anti-PP antiserum restores this phenotype relative to wild type controls (Batterham et al., 2003a).

Obese subjects demonstrate a blunted postprandial PP response (Broberger et al., 1997) while subjects with anorexia nervosa have presented with exaggerated postprandial PP responses (Abbott et al., 2005a, Abbott et al., 2005b, Gustafson et al., 1997). Chronic PP administration to leptin deficient ob/ob mice significantly reduced body weight gain and also resulted in improvements in blood glucose and lipid profiles (Asakawa et al., 2003). Paradoxically, Prader-Willi patients who are obese and suffer uncontrollable hyperphagia have high basal levels of PP. However, they still exhibit the blunted PP response to feeding

(Halatchev et al., 2004, Koda et al., 2005) and infusion of PP has been shown to significantly reduce acute food intake (Vrang et al., 2006).

Chronic PP administration also demonstrated an increase in oxygen consumption as well as an increase in the discharge rate of sympathetic efferent nerves innervating the adrenal medulla and BAT (Asakawa et al., 2003). This suggests that PP may have a role in energy expenditure. Combined with the lack of any adverse effects in human studies, PP may have potential in the regulation of food intake and the treatment of obesity.

1.2.2.4.3 Peptide YY (PYY)

PYY is another amidated 36 amino acid PP-fold peptide hormone released post-prandially from enteroendocrine L-cells in the distal gut (Adrian et al., 1985). PYY is cleaved by the membrane-bound enzyme dipeptidyl peptidase-IV (DPPIV) at the N-terminal tyrosine- proline to generate PYY3-36, the major circulating form of PYY (Vrang et al., 2006). PYY3-36 binds selectively to the Y2r whereas full length PYY is a Y1r- and Y5r-preferring ligand. PYY is released within 15 minutes of nutrient ingestion and peaks 1-2 hours after, and remains elevated for a period of several hours (Adrian et al., 1985). Caloric load, consistency and nutrient composition of food have all been seen to affect the resulting levels of circulating PYY (Batterham et al., 2006). Vasoactive intestinal peptide (VIP), CCK and vagal impulses have also been suggested to regulate PYY secretion (le Roux et al., 2008a, Nannipieri et al., 2013, Pfluger et al., 2007).

Before 2002, the main role of PYY3-36 was thought to be in the regulation of gastric motility and gastric secretion (Guo et al., 2006). It was described as a mediator of the ‘ileal brake’ which slowed the transit of a meal from the stomach into the small intestine in response to nutrient sensing. In 2002, Batterham et al. demonstrated that PYY3-36 binds specifically to the Y2r in the CNS to cause inhibition of food intake in rodents as well as in both obese and normal weight humans (Batterham et al., 2002, Sloth et al., 2007).

The anorexigenic mechanism of PYY3-36 is thought to act through activation of autoinhibitory Y2r found on orexigenic NPY-expressing neurons of the medial ARC to inhibit intracellular cAMP accumulation. This inhibits the orexigenic output of these neurons and thus reduces

46 appetite (Boey et al., 2006, Batterham et al., 2007, Bell et al., 1983). The NTS region of the brainstem has high expression of Y2r mRNA (Gustafson et al., 1997) thus this area may be important in relaying satiety signals from vagal afferents in the gut via projections to the hypothalamus. Indeed neuronal activation is seen in the NTS and AP following intravenous

(IV) infusion of PYY3-36 (Halatchev et al., 2004, Lijnen et al., 2010). However, this area also has an incomplete blood brain which means that PYY3-36 action in the hypothalamus may not be entirely transmitted through vagal signals.

In rodents, peripheral administration of PYY3-36 acutely inhibits food intake, and chronically significantly reduces body weight and adiposity in normal and obese rodents (Vrang et al., 2006). Moreover, PYY-null mice are hyperphagic and have increased adiposity suggesting a physiological role for PYY as a regulator of energy intake (Batterham et al., 2006, Boey et al., 2006, Zhang et al., 2012). These effects are both reversed with exogenous administration of

PYY3-36.

IV infusion of PYY3-36 also acutely reduces food intake in humans. Healthy volunteers infused with physiological doses of PYY3-36 (to mimic post prandial levels), reduced their food intake at a buffet meal by 35%. Food consumption is further significantly decreased over the next 12 hours following the infusion (Batterham et al., 2002). However, supra-physiological doses of PYY3-36 are associated with nausea (le Roux et al., 2008a).

PYY3-36 has been implicated in the long term regulation of body weight (Speakman et al.,

2011, Sumithran et al., 2011) with high levels of PYY3-36 found in patients suffering from anorexia nervosa (Misra et al., 2006) and frequently low levels measured in obese subjects

(Pfluger et al., 2007). It has been suggested in several studies that PYY3-36 may increase energy expenditure and there have been several correlations made between postprandial

PYY3-36 levels and postprandial thermogenesis, resting metabolic rate and 24 hour respiratory quotient (Guo et al., 2006, Sloth et al., 2007). PYY may also have a role for in glucose homeostasis as PYY knock-out mice are hyperinsulinaemic (Boey et al., 2006). The insulin-lowering effect of PYY after glucose stimulation has been suggested to be the result of Y1r agonism by full length PYY, as exhibited in vitro using isolated rat and mouse islets (Nieuwenhuizen et al., 1994, Chandarana et al., 2013). Inhibition of insulin secretion by islets is not reflected by PYY3-36, which is a selective Y2r agonist. This is consistent with the

47 lack of Y2r present in islets. However, there is evidence in rodent in vivo studies demonstrating that PYY3-36 improves glucose tolerance (Pittner et al., 2004, van den Hoek et al., 2004). This is suggested be an indirect effect of PYY3-36, and occurs via increased hepato-portal active GLP-1 plasma levels (Chandarana et al., 2013). PYY3-36 may also have a role in improving insulin sensitivity as it increases glucose disposal rate under hyperinsulinaemic conditions (van den Hoek et al., 2004)

Interestingly, as well as the homeostatic areas of the hypothalamus described so far, functional magnetic resonance imaging showed increased neuronal activity with PYY3-36 in the caudolateral orbital frontal cortex, an area of the brain involved in the hedonic aspect of feeding (Batterham et al., 2007). This fits well with the satiety effect of PYY which may include the increase in the reward aspect of food intake following a period of fasting.

1.2.2.5 Proglucagon-derived products

The proglucagon gene was discovered in 1983 (Bell et al., 1983). Pre-proglucagon is expressed in pancreatic α-cells, intestinal L-cells and the NTS of the brainstem (Larsen et al., 1997a) and then undergoes tissue specific post-translational processing depending on its location (Mojsov et al., 1986) (Fig 1.3). In the intestine and CNS, pre-proglucagon is cleaved by prohormone convertase 1 (PC1 – also known as PC3) into glicentin, glicentin-related pancreatic peptide (GRPP), oxyntomodulin (OXM), glucagon-like peptide 1 and 2 (GLP-1 and GLP-2) (Tang-Christensen et al., 2001b, Bonic and Mackin, 2003). In the pancreas, preproglucagon is ultimately processed by PC2 to produce glucagon, GRPP and major proglucagon fragment (MPGF) (Patzelt and Schiltz, 1984, Rouille et al., 1994).

Figure 1.3 A diagrammatic representation of the tissue-specific processing of preproglucagon in the pancreas and the CNS/intestine.

1.2.2.5.1 Glucagon

Glucagon is a 29 amino acid peptide released from the α-cells of the islets of Langerhans in the pancreas. Glucagon opposes the actions of insulin on blood glucose levels, by increasing plasma glucose levels through de novo synthesis (gluconeogenensis), and/or the release of glucose from storage as glycogen (glycogenolysis). Glucagon binds to the glucagon receptor (GCGr), which is a G-protein coupled receptor (GPCR) linked to the activation of cAMP (Jelinek et al., 1993). High affinity glucagon binding sites have been identified in the liver, kidney, intestinal smooth muscle, brain, adipose tissue and islet β-cells (Moens et al., 1996). Glucagon acts primarily at the liver to stimulate gluconeogenesis and glycogenolysis (Ikezawa et al., 1998). However, the activation of GCGr on pancreatic β-cells is linked to an increase in insulin release. This allows newly available circulating glucose to be taken up by insulin-dependent tissues, such as liver and muscle.

The release of glucagon is stimulated by the direct effect of glucose on α-cells (i.e. in the post-prandial state). This occurs through glucose metabolism in the cell where ATP production causes opening of ATP-dependent potassium channels in the cell membrane,

49 depolarisation of the cell, and subsequent exocytosis of vesicles containing glucagon (Olsen et al., 2005, Salehi et al., 2006). Although this would seem paradoxical, the effect of glucose to mediate glucagon release from α-cells is overridden by the incretin effect of glucagon via β-cells. The release of insulin as well as GABA from β-cells in response to glucose in turn leads to inhibition of glucagon release from α-cells (Rorsman et al., 1989, Wendt et al., 2004, Xu et al., 2006). In parallel, hypoglycaemia is avoided through autoregulatory pathways where GCGr activation in α-cells stimulates further glucagon secretion (Ma et al., 2005). There also exists an indirect autoinhibitory pathway where L-glutamate released from α-cells stimulates GABA release from β-cells and thereby causes a decrease in glucagon secretion (Wendt et al., 2004, Uehara et al., 2004).

Although the primary function of glucagon is the regulation of hepatic glucose metabolism, glucagon has also been associated with a role in the regulation of energy expenditure. A study in rats where chronic glucagon administration caused a 20% reduction of body weight with no change in food intake (Chan et al., 1984) strongly suggests a role for glucagon in regulating energy expenditure, although the mechanisms are not fully characterised. The role of glucagon in causing an increase in energy expenditure has also recently been demonstrated in humans (Tan et al., 2013).

A possible mechanism for glucagon’s effect on body weight could involve effects on fatty acid oxidation and lipogenesis. Fasting, and the subsequent decrease in insulin and increase in glucagon levels, leads to increased fatty acid beta oxidation and decreased lipogenesis (Pegorier et al., 1989, Longuet et al., 2008). The expression levels of genes related to fatty acid oxidation are increased during fasting, increasing the potential for the hepatocytes to oxidise fat. The effects of fasting are all reversed in GCGr-null mice.

The actions of glucagon on increased energy expenditure make it an attractive target for anti obesity therapies. However this should be balanced with its effect on glucose homeostasis so as not to promote hyperglycaemia while still utilising its ability to promote weight loss.

1.2.2.5.2 Glucagon-like peptide 1 (GLP-1)

GLP-1(7-36) amide (GLP-1) is a 30 amino acid peptide. It is secreted from enteroendocrine L- cells of the intestinal tract as two equally bioactive forms of GLP-1 into the circulation (Herrmann et al., 1995, Orskov et al., 1993). GLP-1 (7-37) and GLP-1 (7-36)-amide are both released in response to the ingestion of nutrients (Orskov et al., 1996). GLP-1 has a very short active half-life of less than 5 minutes due to degradation by the enzyme DPPIV (Kieffer et al., 1995, Deacon et al., 1995).

GLP-1 binds to and activates the GPCR GLP-1 receptor (GLP-1r). GLP-1r is coupled to adenylate cyclase and thereby stimulates cAMP production through a Gs alpha subunit. GLP- 1r are expressed in the β-cells and α-cells of the pancreatic islets (Thorens, 1992) as well as in the lung in rodents (not in humans) (Richter et al., 1990), brain (Alvarez et al., 1996, Goke et al., 1995), heart (Wei and Mojsov, 1995), stomach (Schepp et al., 1994, Schmidtler et al., 1994), kidney and intestine (Bullock et al., 1996).

GLP-1 is an incretin, which stimulates glucose-dependent insulin secretion from the pancreas and inhibits glucagon secretion (Drucker, 2001). It binds to GLP-1 receptors on the β-cells to stimulate insulin release (Kreymann et al., 1987). GLP-1r knockout mice show normal feeding behaviour but reduced glucose tolerance and fasting hyperglycaemia (Scrocchi et al., 1996). Antagonism of the GLP-1r by exendin9-39 (Ex9-39) abolishes the incretin effect. GLP-1 also potentiates all steps of insulin biosynthesis (MacDonald et al., 2002). Activation of the GLP-1r stimulates cAMP and subsequent Protein Kinase A (PKA) production which leads to insulin biosynthesis and has a pro-mitotic effect on β cells (Hui et al., 2003). GLP-1r stimulation also causes an increase in intracellular calcium, which leads to insulin exocytosis (Gromada et al., 1997).

As mentioned, GLP-1 is rapidly degraded by DPPIV. This enzyme cleaves the two N-terminal amino acids to produce GLP-1(9-36). GLP1(9-39) was initially shown to have antagonistic actions at the GLP-1r (Knudsen and Pridal, 1996) but is now believed to have weak partial insulinotropic agonist activities at the GLP-1r (Beinborn et al., 2005). It also has extrapancreatic insulin-like actions in the heart, vasculature, and liver, which appear to be mediated independently of the GLP-1r (Tomas and Habener, 2010, Beinborn et al., 2005).

Post-prandially released GLP-1 promotes satiety as does exogenously administered GLP-1 (Turton et al., 1996). GLP-1 may increase satiety in two different ways. The first is via inhibition of gastric emptying and secretion of gastric juices (Wettergren et al., 1993, Holst, 1994, Nauck et al., 1997, Naslund et al., 1999). The second is through central appetite regulation. Intracerebroventricular (ICV) GLP-1 potently reduces food intake in fasted rats (Tang-Christensen et al., 1996, Tang-Christensen et al., 1998). Part of the anorectic effect of GLP-1 may be through nausea and conditioned taste averson (Seeley et al., 2000, Liang et al., 2013); however as low doses of centrally administered GLP-1 still cause a significant anorectic effect without causing taste aversion or altered locomotor behaviour (McMahon and Wellman, 1998), this cannot be the only mechanism. ICV GLP-1 also causes a shift in the proportion of carbohydrate to fat utilised as energy substrate (Tachibana et al., 2007).

The central effectors of GLP-1 action are the brainstem, hypothalamus and vagus. GLP-1r have been detected in the brain in regions important in the regulation of energy homeostasis including the ARC, and PVN of the hypothalamus, and the AP and NTS of the brainstem (Shughrue et al., 1996, Larsen et al., 1997b, Merchenthaler et al., 1999). Animals with lesions in the ARC are no longer responsive to the anorectic effects of GLP-1 (Tang- Christensen et al., 1998), nor are those who have had a vagotomy (Abbott et al., 2005a). GLP-1 release and receptor expression in the brainstem and hypothalamus could mean that GLP-1 plays a role as a neurotransmitter as well as a circulating hormone (Calvo et al., 1995, Jin et al., 1988, Kreymann et al., 1989, Yoshimoto et al., 1989).

The effect of GLP-1 on overall energy expenditure is not fully understood. Some studies show no effect (Abu-Hamdah et al., 2009), increased EE (Osaka et al., 2005), or reduced EE in line with weight loss (Tomas and Habener, 2010). Lockie et al. (van Can et al., 2013) suggested increased BAT thermogenesis, caused by central GLP-1r agonism, as the mechanism whereby pharmacological GLP-1r activation controls energy balance, and also mediates the increased EE seen with oxyntomodulin.

1.2.2.5.3 Oxyntomodulin (OXM)

OXM is a 37 amino acid peptide comprising the entire glucagon structure with an additional C-terminal octapeptide extension (Bataille et al., 1981). Radioligand competition binding assays reveal that OXM binds to both the GLP-1 receptor and the glucagon receptor (Baggio et al., 2004a, Kosinski et al., 2012) though with lower affinity than either cognate ligand. OXM is degraded through cleavage by the enzymes DPPIV and NEP (Zhu et al., 2003, Druce et al., 2009); this degradation explains its rapid removal from the human circulation (Druce et al., 2009). Proglucagon undergoes similar processing in the caudal part of the NTS and OXM is also produced here; this leads to the suggestion that OXM may act as a neurotransmitter in the NTS (Vrang et al., 2007).

Physiological release causes an increase in pancreatic endocrine secretions in rats and in human volunteers and also leads to increased gastric acid secretion and delayed gastric emptying (Biedzinski et al., 1987, Schjoldager et al., 1989). OXM also has a significant role in the regulation of energy homeostasis. Commensurate with it being a satiety signal, OXM is released post prandially from the intestinal L cells in a proportion to caloric intake (Ghatei et al., 1983, Stanley et al., 2004). Central and peripheral administration of OXM to rats cause acute and chronic decreases in food intake, body weight gain and adiposity (Baggio et al., 2004a, Dakin et al., 2001, Dakin et al., 2002). Acute administration of OXM to humans also demonsrated a reduction of FI. Additionally, OXM causes an increase in energy expenditure. Rats given OXM twice per day for one week lost more weight than a pair-fed group, and chronic administration in man increased activity-related energy expenditure as measured by an Actiheart monitor (Cohen et al., 2003, Wynne et al., 2005, Wynne et al., 2006, Dakin et al., 2002).

Some of the anorectic effect of OXM occurs through the GLP-1r: OXM does not reduce food intake in GLP-1r knockout mice and central administration of the GLP-1 antagonist Exendin(9-39) prevented the anorectic effects of ICV OXM (Baggio et al., 2004a). OXM and GLP-1 reduce food intake to the same extent when administered centrally at equal doses, despite the lower affinity of OXM for the GLP-1r than GLP-1 (Fehmann et al., 1994, Dakin et al., 2001). However, the anorectic effect of OXM is not exclusively mediated via the same pathway as GLP-1. Central Ex(9-39) blocks the anorectic effect of peripherally administered

OXM, but not peripherally administered GLP-1 (Dakin et al., 2004). Furthermore, MEMRI scans have shown that the hypothalamic neuronal activity patterns exhibited by OXM and GLP-1 in mice differ considerably (Chaudhri et al., 2006, Parkinson et al., 2009). While these differences may be simply due to different brain penetration, it is plausible that there is engagement of additional receptor(s) and/or that OXM and GLP-1 have different effects on the GLP1r signalling pathway (Baggio et al., 2004a, Sowden et al., 2007).

The differential effect of OXM compared with GLP-1 could be mediated through the GCGr. OXM can functionally activate the GCGr and cause hepatic glycogenolysis (Kosinski et al., 2012). An interesting programme of investigations has been undertaken by the group lead by Pocai. By changing OXM at position 3 from Gln to Glu (OXM-Glu3), they created a version of OXM that is a pure GLP-1r agonist (Santoprete et al., 2011) with no GCGr activity. OXM stimulates liver ketogenesis, while OXM-Glu3 does not, suggesting this action occurs via the GCGr. OXM causes increased EE via the GCGr. Chronic OXM in DIO mice causes greater weight loss compared to those given OXM-Glu3, despite similar decreases in food intake, and administration of the GCGr angatonist, Cpd A, reduces the weight loss effect of OXM to that of OXM-Glu3 (Kosinski et al., 2012). In humans, mimicking the dual agnoism of OXM by co-infusion of glucagon and GLP-1 caused an increase in EE compared to GLP-1 alone (Tan et al., 2013).

The effect of OXM on glucose homeostasis reflects its actions at both the GCG and GLP-1 receptors. OXM reduces glucose excursion during a glucose tolerance test in mice (Parlevliet et al., 2008, Du et al., 2012). However, the dose of OXM to completely suppress glucose excursion is ten-times higher than when the pure GLP-1 agonist, OXM-Glu3, is used (Du et al., 2012). In a hyperglycaemic clamp, a higher glucose infusion rate was required to maintain glucose levels when OXM-Glu3 was infused compared to an OXM infusion. The ratio of GCG and GLP-1 receptor activation is important in determining the effects of OXM- like compounds. Balanced agonism shows the best weight loss and glucose lowering effects in rodents (Day et al., 2009, Day et al., 2012).

1.3 CURRENT STRATEGIES IN ANTI-OBESITY THERAPEUTICS

1.3.1 LIFESTYLE MODIFICATIONS

First line treatment for obesity is lifestyle modification with caloric restriction and increased physical activity. However, meta-analyses suggest that neither diet or exercise, either alone or in combination, are particularly efficacious at causing weight loss (Dombrowski et al., 2014). Exercise alone causes minimal weight loss over 12 months. Calorie restriction alone caused 5% weight loss over 6 months, reduced to 3% by the end of two years. There is no difference between types of calorie-restricted diet (Sacks et al., 2009). A combination of diet and exercise caused a significant reduction in weight of 8.5% at 6 months; however by 48 months, this had fallen to 4% (Franz et al., 2007). This lack of success is mostly due to poor compliance to treatment (Makris and Foster, 2011). Diet and exercise are therefore often recommended only as an aid for weight maintenance once a healthy BMI has been achieved (Slentz et al., 2004).

1.3.2 SURGICAL

Bariatric surgery is currently the most successful, long-term treatment for obesity (Vines and Schiesser, 2014). The Swedish Obesity Study shows weight loss of 18% up to twenty years after the procedure (Sjostrom, 2013). However, surgical intervention is not without risk. Generally, surgery carries with it high risks of infection, anaesthesia complications and post- surgical complications such as malnutrition/malabsorption (Gong et al., 2008). Surgery related death can occur in up to 0.3% of bariatric surgery operations (Flum et al., 2009). In addition, given the size of the obesity problem, it is not economically possible to use bariatric surgery to treat all cases (Avenell et al., 2004; Bessesen, 2008).

There are a number of different surgical options depending on the level of obesity, co- morbidities, patient preference and the weight loss required. These can generally be divided into two categories. There are procedures which are purely restrictive, such as gastric banding and vertical sleeve gastrectomy (VSG) (Fig 1.4 (B)), and those that also bypass part

55 of the gut, including Roux–en-Y gastric bypass (RYGB) and biliopancreatic diversion (BPD) with or without duodenal switch (DS).

The most commonly used bariatric surgery is RYGB (Fig 1.4 (C)). This contains both restrictive and bypass elements. The stomach is sectioned to produce a small proximal pouch which is attached to the distal limb of the jejunum. This bypasses 95% of the stomach, the duodenum and some of the jejunum. The remaining stomach and intestine is then anastomosed to the jejunum, so digestive juices can be channelled here. RYGB causes an average of 34% weight loss after one year (Hatoum and Kaplan, 2013).

The reduction in appetite seen after bariatric surgery, although not completely understood, can perhaps be partially attributed to the increased circulating levels of PYY3-36 compared to pre-surgery levels (Nannipieri et al., 2013). Following RYGB there are a number of changes that may contribute to weight loss:

 Reduced food intake due to gastric pouch  Increased circulating levels of anorexigenic gut hormones GLP-1, insulin and PYY, and decreased levels of orexigenic ghrelin (Morinigo et al., 2004; Korner et al., 2005)  Changes in taste (Tichansky et al., 2006) and brain-hedonic responses to food after RYGB (Scholtz et al., 2014).  Increase in energy expenditure following RYGB surgery in rats (Bueter et al., 2010), and in humans (Faria et al., 2012, Faria et al., 2014, Wilms et al., 2013).

There is little true malnutrition, as measured by albumin – the malabsorption occurs more from vitamins/micronutrients.

Figure 1.4 Simplified diagrams of (A) a normal uninterrupted GI tract, (B) removal of the majority of the stomach in a VSG procedure without any bypass of the gut, and (C) RYGB that removes an even greater proportion of the stomach and bypass of the duodenum and part of the jejunum.

1.3.3 DRUG TREATMENT

According to the FDA (FDA, 2007), an anti-obesity medication is considered effective for weight management if after 1 year of treatment either of the following occurs:

1) there is a statistically significant reduction in body weight of ≥5% compared to placebo (or a significant weight loss of 5-10% according to the EMA (EMA, 2014)). 2) if at least 35% of study participants lose ≥5% of their body weight from baseline

The EMA also requires that, of the responders in the treatment arm of a study, response is >10% weight loss at the end of a 12-month period (EMA, 2014).

The following sections describe the different classes of drugs which have been used or are currently in use for the treatment of obesity.

1.3.4 CURRENT AND PREVIOUSLY LICENSED PHARMACOLOGICAL TREATMENTS FOR OBESITY

At present, Orlistat (Xenical - POM & Alli - OTC, Roche pharmaceuticals, Switzerland) is the only approved anti-obesity drug in the UK. Orlistat is a pancreatic lipase inhibitor that prevents the hydrolysis, metabolism and absorption of dietary fat (triglycerides) (Davidson et al., 1999). Its efficacy is limited (Padwal and Majumdar, 2007, Avenell et al., 2004, Neovius and Narbro, 2008): over one year it causes 3kg more weight loss than placebo controls (Davidson et al., 1999, Sjostrom et al., 1998, Hill et al., 1999, Leung et al., 2003), and this weight is regained once treatment with Orlistat is stopped (Sjostrom et al., 1998, Svendsen et al., 2008, Rossner et al., 2000, Rucker et al., 2007). It is also associated with significant side effects including steatorrhoea, bloating, oily spotting, faecal urgency and increased defecation (Guerciolini, 1997, Leung et al., 2003). The EMA is currently reviewing reports of possible liver toxicity linked with orlistat (EMA, 2011). There is an associated decrease in absorption of fat-soluble vitamins, like vitamin E and β-carotene (Davidson et al., 1999), but levels rarely fall outside of the normal limits (Bray and Tartaglia, 2000).

Sibutramine (Reductil® (Abbott)) is a monoamine reuptake inhibitor. This causes a 9.3% loss of body weight from baseline (Bray et al., 1999). However, the EMA recommended its suspension from sale in 2010 due to concerns over the increased incidence of cardiovascular events (Williams, 2010). Rimonabant (Acomplia® (Sanofi-Aventis)) is an antagonist for the cannabinoid receptor, CB1. A meta-analysis of rimonabant trials showed a 10% weight reduction after 1 year compared with the placebo group (Christensen et al., 2007). However, rimonabant had its licence withdrawn in 2009 due to psychiatric adverse reactions including depression, anxiety, irritability, aggression and suicide.

Serotoninin (also called 5-hydroxytryptamine (5-HT)) is a neurotransmitter involved in mediating singals related to hunger. Locaserin (Belviq®) is a specific 5-HT2C receptor agonist approved by the FDA for use in the treatment of obesity in July 2012 as a complement to lifestyle and behavioural modification (Nigro et al., 2013). Trials showed it caused 3% body weight loss when compared with placebo (Smith et al., 2010). However it did not meet the first FDA benchmark (a statistically significant reduction in body weight of ≥5% compared to placebo), and the EMA rejected its use on safety grounds. Compounds that stimulate the release of serotonin and/or inhibit its reuptake (such as fenfluramine (L-isomer) and dexfenfluramine (D-isomer)) have also been investigated as obesity therapies. Fenfluramine was combined with phentermine (fen-phen) to maximise appetite suppression. Both fen and dexfen were removed from the US market in 1997 following reports of valvulopathy (Connolly et al., 1997) and pulmonary hypertension (Abenhaim et al., 1996).

Sympathomimetics (which include phentermine, diethlypropion, benzphetamine, phendimetrazine), which increase the release of catecholamines and reduce their reuptake, have been licensed as therapy for obesity in the US for many years. However, they can only be prescribed for short term use as they may cause addiction and be open to abuse. Phentermine does not appear to enhance release of dopamine (Rothman and Baumann, 2009), therefore its addiction potential is believed to be less. Qsymia (Vivus) is a combination of phentermine and topiramate (phen-tpm) approved in July 2012 by the FDA. Topiramate is an anticonvulsant used in seizure disorders and prevention of migraines. Its exact mechanism of action is unknown but properties which may contribute to its antiepileptic and antimigraine effects include the modification of voltage-dependent sodium and calcium channels, enhancement of GABA receptors, antagonism of AMPA/KA receptors,

59 and inhibition of carbonic anhydrase. These properties may also influence its effects as a weight loss drug by impeding lipogenesis through the inhibition of carbonic anhydrase (Scozzafava et al., 2013), reduction in compulsive or addictive food craving through AMPA/KA receptor antagonism and decreased nightmares and sleep deprivation-induced feeding may be achieved through GABA receptor activation (Guardia et al., 2011). The phenteramine/toparimate combination is expected to produce about 9-10% body weight loss. The FDA expressed safety concerns in five areas of phen-tpm use: teratogenicity, cardiovascular, cognitive, psychiatric and metabolic acidosis, and the EMA rejected its use due to a lack of safety data (Woloshin and Schwartz, 2014).

The limited options for the management of obesity and the rapidly increasing incidence of obesity means there remains a clear unmet clinical need for safe and effective treatments.

Most of the proposed treatments are centrally acting drugs that interact with appetite regulation and hedonic CNS pathways, discussed in more detail below.

1.3.5 DRUG IN DEVELOPMENT FOR OBESITY

1.3.5.1 Small Molecules

Contrave (Orexigen Therapeutics) contains sustained release formulations of naltrexone (an opioid receptor antagonist) and bupropion (a catecholamine reuptake inhibitor). It is currently used as an antidepressant and an aid in smoking cessation. It was noted to also reduce food intake (Morton et al., 2006). The effects of naltrexone-bupropion (NB) in reward pathways may partly explain the increased ability of participants to control eating and avoid responding to food cravings (Greenway et al., 2010). Treatment with NB may aid compliance to a mild hypocaloric diet as demonstrated in the latest phase 3 trial (Apovian et al., 2013).

The anti-epileptic drug, zonisamide is being developed as a weight loss therapy It inhibits carbonic anhydrase (Scozzafava et al., 2013) which is implicated in de novo lipogenesis. High dose of 400mg zonisamide produces a moderate weight loss of 3.3kg compared to placebo after one year. However, there is a high incidence of gastrointestinal, nervous system and

60 neuropsychiatric side effects (Gadde et al., 2012). Despite these serious side effects, current patents exist for its use in combination with a sympathomimetic, a 5-HT2C receptor agonist, or a CB1 receptor antagonist (Scozzafava et al., 2013). Clinical trials for Orexigen Therapeutic’s new drug, Empatic (sustained-release formulations of both bupropion and zonisamide) have shown promising phase 2b results with weight loss (9.9% Empatic 360mg and 7.7% empatic 120mg vs 1.7% placebo) and no serious side effects (Orexigen, 2009).

Beloranib (made by Zafgen) is an analogue of the antimicrobial agent fumagillin and is a potent and selective inhibitor of MetAP2. MetAP2 is an essential enzyme in cell growth and has been used as an anti-angiogenic, anti-cancer agent. It was shown to prevent diet- induced obesity through adipocyte hypotrophy (Lijnen et al., 2010). Clinical trials have shown dose-dependent weight loss with beloranib treatment but only the highest dose produced sustained weight loss over 26 days (Hughes et al., 2013). The highest dose used in this study was over 20-times lower than that used in oncology trials, and was therefore not associated with anti-angiogenic effects.

1.3.5.2 Biologics

A class of drugs called biologics, which includes antibodies, proteins and peptides, offer a greater degree of specificity by nature of the molecules. As this class of drugs are based on naturally occurring peptides that target physiological pathways, they are less likely to have side effects. With their high specificity and low toxicity profile, biologicals offer viable alternatives to small molecule therapeutics.

1.3.5.2.1 GLP-1 Agonists

As previously mentioned, GLP-1 is an incretin that stimulates insulin release and improves glucose tolerance. It also acts as a satiety signal and can cause weight loss. There are currently five GLP-1 receptor agonists licensed for the treatment of diabetes, based on the native GLP-1 peptide. These include exenatide (Byetta®) and exenatide extended-release (Bydureon®); liraglutide (Victoza®), Lixisenatide (Lyxumia®) and albiglutide (Tanzeum®).

Exenatide (Byetta - twice daily, or Bydureon - once weekly (Amylin Pharmaceuticals)) is a stable and potent GLP-1 agonist based on a naturally occurring GLP-1 analgoue, exendin. Although licensed for the treatment of T2DM, a mean weight loss of 1.4kg versus placebo was found with exenatide (Amori et al., 2007). Liraglutide (Novo Nordisk®) is a once-daily diabetes therapy. It is 97% homologous to native human GLP-1, changed by substituting arginine for lysine at position 34 and attaching palmitic acid (C-16 fatty acid) with a glutamic acid spacer on the remaining lysine residue at position 26 of the peptide precursor. These changes improve its stability. Liraglutide causes a dose dependent decrease in BW which is sustained for 2 years with continued use (Astrup et al., 2012). Recently, it has also been shown to help with weight maintenance after initial loss of BW using a low calorie diet (Wadden et al., 2013). Novo Nordisk filed for regulatory approval of high dose liraglutide (3mg) purely for obesity treatment in December 2013.

Albiglutide and Lixisenatide both received their licenses as diabetes treatments in 2013. Lixisenatide (des-Pro38-ex4-Lys39-44) is a once daily agent based on ex-4 but it is superior to Byetta at reducing post-prandial plasma glucose levels with less adverse effects and equally good as competitors (e.g. Byetta, Bydureon, liraglutide) at regulating HbA1c, fasting plasma glucose and body weight (Fisch et al., 1996). Albiglutide is a once weekly GLP-1 receptor agonist generated by the genetic fusion of a DPP-4-resistant GLP-1 dimer to human albumin (Baggio et al., 2004b). Weight loss seen with albiglutide was 0.2kg after 2 years use compared with placebo control (Ahren et al., 2014).

There are many other GLP-1 agonists in development for the treatment of obesity but none have yet been approved by regulatory bodies. Indeed, Roche®’s DPPIV-resistant GLP-1 analogue, taspoglutide, (substitution of Ala2 for the non-natural AIB) was withdrawn from late-stage development due to serious adverse events during trials (UKMi, 2010).

1.3.5.2.2 MC4r Agonists

A new potential peptide target for the treatment of obesity is agonism of the MC4r. In humans, MC4r mutations are associated with obesity and preclinical studies demonstrate reduction in both food intake and body weight with MC4r stimulation (Kievit et al., 2013).

RM-493 (Rhythm®) is a small peptide MC4r agonist in phase 2 clinical trials as a treatment for obesity and diabetes that reduces body fat and insulin resistance and improves cardiovascular function. Trials using RM-493 for the treatment of obesity in individuals with a genetic deficiency in the MC4r pathway has also been initialised to restore function in this pathway and improve weight regulation.

1.3.5.2.3 Leptin

The native hormone leptin is an effective treatment for the small number of subjects with obesity due to leptin deficiency (Farooqi et al., 2002). However, the trend for obese and overweight individuals to be leptin-resistance (English et al., 2002) has led to the unpopularity of leptin-based therapies. Nevertheless, the potential for leptin as an obesity treatment is still being explored in the face of understanding of the molecular mechanisms of leptin resistance (Jung and Kim, 2013). An example is Fc-leptin where leptin is attached on the end of an Fc fragment of an immunoglobulin gamma chain to extend the circulating half-life of the peptide (Lo et al., 2005). While this strategy improves the pharmacokinetics of leptin as a drug, it still relies on the ability of the peptide to produce an anorexigenic effect in sensitive individuals. Another leptin-based therapy is its coadministration with amylin, a peptide co-secreted with insulin from pancreatic β-cells. Amylin appears to restore leptin responsiveness in obese rats and humans causing an additional 10% weight loss compared to each peptide given individually (Roth et al., 2008, Trevaskis et al., 2008).

1.3.5.2.4 FGF-21

FGF-21 is a metabolic regulator of glucose and lipid metabolism and is a protein that belongs to the “hormone-like” subgroup of fibroblast growth factors (FGFs) . FGF-21 reduces body weight and blood glucose levels in several animal models, including diabetic rhesus monkeys (Nygaard et al., 2013). FGFs regulate a diverse range of metabolic pathways (Long and Kharitonenkov, 2011). This presents a challenge in drug development as non-specific, global activation of FGF receptors is undesirable. The native FGF-21 protein also demonstrates a poor pharmacokinetic profile. However, as the FGF-21 receptor requires a co-receptor

(βKlotho), selectivity can be acquired. A human monoclonal antibody (mimAb1) has been developed that only activates the FGFR1c/βKlotho complex (Foltz et al., 2012), and in obese monkeys, injection of mimAb1 led to FGF21-like metabolic effects, including decreases in body weight, plasma insulin, triglycerides, and glucose.

1.4 DEVELOPMENT OF GUT HORMONES AS THERAPEUTIC AGENTS

The results of GLP-1, PYY, and OXM administration in humans have made these peptides, and their receptors, potential targets for the development of therapeutic agents in the treatment of obesity (Small and Bloom, 2005, Boggiano et al., 2005, Ueno et al., 2008, Vincent and le Roux, 2008, Tan and Bloom, 2013a). However, there are limitations in using native hormones as obesity therapies; the problems, and means of overcoming them, will be discussed below.

1.4.1 CONFERRING ENZYME RESISTANCE IN GUT PEPTIDES

Gut hormones have short half-lives due to enzymatic degradation (Medeiros and Turner, 1994). The circulating half-life of GLP-1 is approximately 4 minutes in man (Kreymann et al., 1987), 12 minutes for OXM (Schjoldager et al., 1988), 7minutes for PP (Adrian et al., 1978), and PYY is approximately 8 minutes in rats (Lluis et al., 1989), just to name a few. This characteristic limits the use of native hormones as anti-obesity agents. Several strategies have been utilised to increase the half-life of peptides.

Specific amino acid substitutions can alter a peptides susceptibility to proteolytic degradation. Exendin-4, a GLP-1r agonist, shares over 50% sequence homology with GLP-1. However there are changes, notably at the N terminus (His1-Gly2), that render Exendin-4 resitant to degradation by DPPIV. This means exendin-4 has a circulatory half-life of

64 approximately 8 hours (Nielsen et al., 2004) compared to the half-life of GLP-1 that is less than 10 minutes (Deacon et al., 1995). This has allowed exendin-4 to be developed and marketed as a therapeutic agent (Chen and Drucker, 1997).

Addition of acyl side chains can increase the half-life of peptides in vivo by promoting binding to albumin which restricts access to the N-terminal by DPPIV, allowing the molecule to escape renal filtration (Knudsen et al., 2000), and slowing the release of the peptide from the extracellular SC compartment (Garber, 2005). However, potential disadvantages in the addition of larger side chains are solubility issues and the possible decrease in central penetration across the BBB.

The GLP-1 analogue, liraglutide, differs from GLP-1 by a single amino acid exchange (Lys34 → Arg34) as well as the addition of an acyl side chain (Degn et al., 2004). These modifications do not alter the potency of the ligand but increase its plasma half-life to 10-12 hours in humans (Chang et al., 2003, Juhl et al., 2002, Knudsen et al., 2000).

Other methods of developing resistance to degradation include conjugation to biotin (Chaturvedi et al., 1984), albumin (Burnham, 1994), or polyethylene glycol (PEGylation) (Veronese, 2001, Lee et al., 2005) Dependent on the site and degree of conjugation, this has the potential to enhance the pharmacological profile while retaining the biological profile.

PEGylation of PYY3-36, for instance, reduced its affinity to the receptor and therefore its bioactivity. However, reversible PEGylation of PYY3-36 ensured release of active PYY3-36 from the PEG group over an extended time period thereby prolonging its biological effect (Lee et al., 2005, Shechter et al., 2005). Merck have also developed a PEGylated OXM which retains the anorectic and glucose-lowering effects of OXM (Bianchi et al., 2013). However PEG-OXM has no affinity to the GCGr and is therefore a pure GLP-1 agonist. A conjugated peptide most recently licensed for the treatment of T2DM is albiglutide (Tanzeum™) (GSK, 2014). It is a DPPIV-resistant GLP-1 dimer fused to albumin (Baggio et al., 2004b) and therefore has an extended half-life (Matthews et al., 2008) which enables less-frequent (once-weekly) dosing (Ahren et al., 2014).

There are also some GLP-1/GCG receptor co-agonists in development for treatment of obesity. Thiakis Ltd developed a degradation-resistant OXM analogue (TKS1225) (Cooke and

Bloom, 2006). Trials by Wyeth Pharmaceuticals, who purchased TKS1225, showed this compound caused significant weight loss and improved glycaemic control. Oxyntomdulin analogues have also been developed by Zealand Pharma.

1.4.2 CONTROLLED RELEASE AND CONJUGATION OF DRUGS

An alternative to altering the half-life of a peptide is to modify its release following administration. A system which allows for a controlled release rate in order to attain and maintain the desired therapeutic level of the drug can bypass the need for a long circulatory half-life and also avoid peak and trough levels following drug administration. For PYY to be developed as a drug, for example, a slow release system is essential in order to avoid nausea due to the burst release of PYY3-36 to supra physiological level (le Roux et al., 2008b, Degen et al., 2005). The theoretical ideal profile of a slow-release analogue of PYY compared with the typical sharp increase of PYY3-36 after intravenous, intranasal or subcutaneous administration is presented in figure 1.2.

Figure 1.5 Challenges of formulation. Giving a supra-physiological dose of PYY3-36 in a bolus can cause nausea (red). A sustained-delivery formulation would avoid peak and trough levels (blue). 40pmol/l = post-prandial levels of PYY(3-36) and also levels found to reduce food intake by 30% by Batterham et al, 2002 (Batterham et al., 2002, Adrian et al., 1985). 70pmol/l = plasma levels of PYY(3-36) at which nausea was felt after infusion (Degen et al., 2005).

An example of the use of slow release formulations to improve drug pharmacokinetics is insulin, which in the presence of zinc ions assembles as stable hexamers (Brange and Langkjoer, 1993, Brange and Langkjaer, 1997) and forms microcrystalline precipitates which have a ‘sustained-release’. Other examples include Bydureon, a slow release preparation of exendin-4, where the peptide is encapsulated into bio degradable microspheres and then slowly released from these complexes, allowing a once a weekly injection (Neumiller, 2009) (Madsbad et al., 2011) (Buse et al., 2010).

1.4.3 TACHYPHYLAXIS

Gut hormones, as well as other endogenous peptides are considered less likely than small chemical entities to have non-specific side effects as they often have greater receptor selectivity (Muller, 2006). Tachyphylaxis (a decrease in the response to a drug due to previous exposure to that drug), which is a potential risk for any drug development programme, is also considered to be a much lower risk for gut hormones as they are repeatedly released daily throughout life (Savini, 1964). However, this remains a controversial matter as some evidence demonstrates that repeated dosing of PYY, for example, may cause tachyphylaxis (Chelikani et al., 2007) whilst other research did not observe such an effect (Pittner et al., 2004).

1.5 AIMS AND HYPOTHESES

The overall aim of this thesis is to assess key points in the development of a peptide-based anti-obesity agent on its route to clinical trials.

This thesis hypothesises that a synthetic peptide analogue based on the structure of gut hormones may prove to be a valuable therapeutic tool for the treatment of obesity.

To address this hypothesis, the susceptibility of an endogenous gut hormone to degradative enzymes and tissue preparations were determined and vulnerable positions in the molecule identified. Rational modifications to the amino acid sequence of the gut hormones were designed and then assessed to establish the receptor binding and potencies of the resulting analogues. This includes in vivo effects on longevity and efficacy at reducing food intake in rodents following acute peripheral administration and effects of chronic administration of analogues on food intake and body weight in rodent models of obesity.

This thesis also hypothesised that the immunogenicity of a novel drug can be identified and techniques employed to reduce this unwanted side effect without great negative consequences on its potency and pharmacokinetics. To test this hypothesis, the studies described in this chapter aimed to identify the immunogenic region of the peptide analogue and different methods to solve or reduce the immunogenicity were explored.

CHAPTER 2

MATERIALS AND METHODS

2.1 PEPTIDES

Human PYY and human PP were purchased from Bachem Ltd. (Merseyside, UK). Synthetic human sequence PYY and PP were used in all experiments where stated. Peptide analogues were custom synthesised by Bachem Ltd. or BIOMOL International LP.

2.1.2 CUSTOM SYNTHESIS OF PEPTIDES

All peptides were synthesised by and purchased from Bachem Ltd or BIOMOL International LP. Peptide synthesis was completed using an automated Fluorenyl-Methoxy-Carbonyl (Fmoc) solid phase peptide synthesis strategy (SPSS) with each amino acid sequentially added from the C to the N terminus. The Fmoc-SPSS method uses repeated cycles of coupling and deprotection to create peptides of up to 100 amino acids.

Briefly, the starting block is a single amino acid joined by covalent bonds to a resin (TentaGel® resin). The N-terminal amine of the amino acid attached to the resin is then deprotected (removal of the Fmoc group) by the addition of piperidine in dimethylformamide (DMF). After deprotection, the resin is washed to remove the Fmoc groups, thus allowing the next amino acid to be coupled when added in excess. Subsequent amino acids are added in the same deprotection-coupling fashion. While each amino acid has the Fmoc to protect the reactive amine group, other side groups are protected as follows: t-butyl ethers are used for the protection of hydroxyl side chains of tyrosine, serine, and threonine; t-butyl esters protect the side chains of aspartic acid and glutamic acid residues; histidine and lysine side chains were protected as their N-tert-butyoxycarbonyl (t- Boc) derivatives, cysteine as its S-trityl derivative, and the guanidine moiety of arginine protected as its Pbf derivative.

Once the addition of amino acids is completed, the peptide is cleaved from the resin and the side chain protecting groups are removed by treatment with aqueous trifluoroacetic acid (TFA) (95%) containing triisobutylsilane (TIS) (5%) as a scavenger. TFA and scavengers were removed by evaporation and trituration. Peptides were purified by reverse phase preparative high performance liquid chromatography (HPLC) followed by lyophilisation.

Where used, the polyethylene glycol (PEG) group was N-terminally coupled to the peptide via a linker methyl group. The type of PEG used was PEG12 (i.e. the PEG moiety had a carbon chain of n=12 in the central core group – molecular weight 570Da) and reverse phase HPLC was used to purify the PEGylated peptide from the non-PEGylated peptide after the reaction.

The purified products were subsequently analysed by reverse phase HPLC and by matrix- assisted laser desorption ionisation mass spectroscopy (MALDI-MS) to confirm the correct molecular weight and to assess purity. All peptides supplied had a purity of >95%. The amino acid sequences of the peptides discussed in this document are shown in Appendix B.

2.2 ANIMAL STUDIES

All animal procedures performed were approved by the British Home Office under the Animals (Scientific Procedures) Act 1986 (Project licence number 70/6402 and 70/7236).

2.2.1 ANIMALS

2.2.1.1 C57BL/6 mice

Adult male C57BL/6 mice (Harlan, UK) weighing 20-45 g were maintained in individual cages under controlled temperatures of 21-23°C and controlled light cycles, each lasting 12 hours, with lights on at 0700. Animals had ad-libitum access to water at all times, and ad libitum access to standard chow diet (pelleted rodent chow, RM1 diet SDS, UK Ltd., Witham UK) outside of study periods. Prior to commencement of studies, animals were regularly handled and received at least two subcutaneous (SC) or intraperitoneal (IP) saline injections to acclimatise them to the study procedures and to minimise any non-specific stress effects.

2.2.1.2 Diet-induced obese (DIO) C57BL/6 mice

Adult male C57BL/6 mice (Harlan, UK) aged 8 weeks old were group housed under controlled temperatures of 21-23°C and controlled light cycles, each lasting 12 hours, with lights on at 0700. For approximately 8 weeks prior to the start of the study, animals had ad- libitum access to water at all times, ad libitum access to high fat diet (energy content: 60% from fat, 20% from protein, 20% from carbohydrate – Research Diets Inc., NJ, USA) and remained on this diet until the end of the study. Prior to commencement of studies, animals were singly housed for two weeks and the individual cages maintained under the same conditions. Animals were regularly handled and acclimatised to receive SC or IP saline injections to minimise any non-specific stress effects at the time of the study. At the start of the study, the mice had a mean body weight of 38g (36-40g), which is approximately 35% more than the average weight of a mouse at 15 weeks old fed a standard chow diet (www.harlan.com).

2.2.1.3 Wistar rats

Adult male Wistar rats (Charles River Ltd., Margate, Kent, UK) weighing 300-500 g were maintained in individual cages under controlled temperatures of 21-23°C and controlled light cycles, each lasting 12 hours, with lights on at 0700. Animals had ad-libitum access to water at all times, and ad libitum access to standard chow diet (pelleted rodent chow, RM1 diet SDS, UK Ltd., Witham UK) outside of study periods. Prior to commencement of studies, animals were regularly handled and received at least two subcutaneous (SC) or intraperitoneal (IP) saline injections to acclimatise them to the study procedures and to minimise any non-specific stress effects.

2.3 ACUTE FEEDING STUDIES

Anorectic compounds were tested under a fasted paradigm. This model was used to eliminate the natural nocturnal feeding habits of mice and to allow them to consume their food during daylight hours in which the experimental procedure is conducted.

Individually-housed male C57BL/6 mice (Charles River Ltd., Kent UK) weighing between 24- 45g were used for acute feeding studies. Mice were fasted from 16:00 on the day immediately before an acute feeding study. Animals were then randomised into groups of approximately equal mean body weight. Mice received a single SC injection (in the flank region of the animal) of vehicle (0.9% w/v NaCl) or peptide using a 0.5ml insulin syringe with a 29 gauge needle during the early light phase (0800-0900) after which the animals were returned to their home cages with free access to a known amount of food. Remaining food was reweighed at 1, 2, 4, 8, and 24hours post-injection by using a balance accurate to 0.01g and a visual inspection of the cage made for any small pieces of food. All studies were conducted under condition in which all external disturbances were minimised. A maximum injection volume of 50l was used and injection volume adjusted by body weight.

2.4 CHRONIC FEEDING STUDIES

2.4.1 CHRONIC ADMINISTRATION TO DIO MICE

DIO mice were randomised into groups of approximately equal mean body weight. A single daily SC injection of either vehicle or peptide in a maximum volume of 50µl, adjusted by body weight was administered at 15:00-16:00 daily. DIO mice had ad libitum access to high fat diet for the duration of the study (28-29 days). Group sizes ranged from 7-14 mice/treatment depending on study. Body and food weights of each mouse were measured immediately prior to injection.

2.4.2 CHRONIC ADMINISTRATION TO RATS

Male Wistar rats weighing 300-350g were randomised into groups with approximately equal mean body weight. Rats had ad libitum access to pelleted rodent chow (RM1 diet). They were administered with a single daily SC injection in the flank region of the animal (15:00- 16:00), for up to 28 days of either vehicle or peptide. Group sizes ranged from 6-14 rats/treatment depending on study. The body and food weights of each rat were measured immediately prior to injection.

2.5 RAT PHARMACOKINETICS

To determine the clearance of peptides in plasma following SC administration, group housed male Wistar rats (Charles River Ltd) were used, with no more than 5 rats per cage, and each animal weighing 200-300g. Rats had free access to water and standard chow diet (pelleted rodent chow, RM1 diet SDS) at all times. On the study day, each rat received a single SC injection of known amount of peptide (1-4mg) dissolved in 20µl of either 0.9% w/v

NaCl or ZnCl2 solution in sterile water for injections, which contained 1mol of ZnCl2 for every 1mol of peptide (1:1 molar ratio). Different formulations were used, depending on the aim of the study. A group size of 3 or more animals was used for each study group. Peptide was injected at time=0 and a 200µl blood sample from each rat was taken through superficial venesection of the lateral tail vein at time points such as 0.5, 3, 24, 48, 96hours, 7 and 10 days following peptide administration. Blood was collected into heparinsed microtubes (10% v/v heparin in 0.9% w/v saline) to prevent clotting. Each tube also contained 4µl of a stock solution (10x stock solution: 1 tablet of S8820 SIGMAFAST™ Protease Inhibitor Tablets in 10ml distilled water (dH2O)) to prevent post-sampling degradation of the injected peptide. Blood samples were stored on ice immediately after collection for a maximum of 15minutes before they were centrifuged at 6000g for 10minutes at 4°C (Sigma Laboratory centrifuges 3 K18, Rotor No. 19777-H). After centrifugation, the upper plasma layer was separated and kept at -20°C until analysed for plasma peptide concentrations as determined by radioimmunoassay (described in sections 2.13.2. and 2.13.3).

2.6 PRODUCTION OF HUMAN Y2 AND HUMAN Y4 RECEPTOR OVEREXPRESSING CELL LINES

2.6.1 PRODUCTION OF COMPETENT BACTERIA

E. coli bacteria were made competent to allow entry of DNA (plasmids) through treatment with cations (Mandel and Higa, 1992, Hanahan, 1996, Wood, 1983).

Materials LB (Appendix C) Tetracycline (Sigma, Poole, Dorset, UK) 10 mg/ml in absolute ethanol TFB I (Appendix C) TFB II (Appendix C) XL1-Blue Escherichia coli (Stratergene Ltd)

Method

100ml LB supplemented with 5μg/ml tetracycline (LBtet) was inoculated with a colony of XL1-Blue and incubated overnight, with vigorous shaking, at 37°C. One millilitre of this culture was inoculated into 100 ml of fresh LBtet (pre-warmed to 37°C) and incubated at

37°C, with vigorous shaking, until the bacteria were in log phase growth (OD550 = 0.4-0.5) as actively growing cells in early log phase are more susceptible to transformation. The bacteria were recovered by centrifugation at 800g for 15 minutes. The bacteria were resuspended in 40 ml ice cold TFB I and incubated on ice for 10 minutes. The bacteria were recovered as before and resuspended in 4 ml TFB II and incubated on ice for 15 minutes. They were aliquoted into 50μl aliquots, frozen in a dry ice/ethanol bath and stored at -70°C.

2.6.2 TRANSFORMATION OF COMPETENT BACTERIA

Transformation of competent bacteria increases efficiency of DNA entry into the bacteria. Incubation on ice of the plasmid with the competent bacteria is believed to allow attachment of the plasmid to the bacterial cell wall. Subsequent heat shock allows efficient transport of the plasmid into the interior of the bacteria. Finally, incubation allows expression of the resistance genes before exposure to the antibiotic.

Materials LB LB(amp) agar plates Competent bacteria (section 2.6.1) human Y2 receptor (cDNA.org, Missouri S&T cDNA Resource Center, University of Missouri- Rolla) (Appendix D) human Y4 receptor (cDNA.org, Missouri S&T cDNA Resource Center, University of Missouri- Rolla) (Appendix D)

Method

An aliquot of competent bacteria was thawed on ice and 10ng of plasmid (5μl) added and the mixture incubated on ice for 20 minutes. The reaction was then incubated at 42°C for two minutes followed by incubation on ice for two minutes. 200μl of LB was added and the reaction incubated at 37°C for 30 minutes. Simultaneously, agar plates (supplemented with ampicillin) were dried and the transformed bacteria added to the plate. The bacteria were streaked over the surface of the agar, the plates inverted and incubated at 37°C overnight.

2.6.3 SMALL SCALE PREPARATION OF PLASMID

Individual colonies of bacteria were isolated and prepared on a small scale to allow several clones to be analysed simultaneously. To obtain cDNA from the bacteria, the cell wall was disrupted and contaminating proteins, genomic DNA and RNA removed (as most genomic DNA in bacteria is anchored to the cell wall). This is done through treatment with alkaline

SDS followed by precipitation of bacterial debris with potassium acetate. The RNA is removed by treatment with RNase A.

Materials

LBAmp GTE (Appendix C) Alkaline SDS (Appendix C) 5M potassium acetate pH 4.6 Phenol/chloroform (VWR) Propan-2-ol 0.3M sodium chloride Absolute ethanol

Method

2ml of LB supplemented with 0.05 mg/ml ampicillin (LBAmp) were inoculated with a single bacteria colony and incubated overnight at 37°C with vigorous shaking. 1.5ml of the culture was centrifuged for 2 minutes at 12300g to pellet the bacteria. The supernatant was discarded and the pellet resuspended in 100μl GTE. 200μl of alkaline SDS was added and the sample incubated on ice for 5 minutes before the addition of 150μl 5M potassium acetate and further 5 minute incubation on ice. The precipitated bacterial debris was removed by centrifugation for 5 minutes at 12300g at room temperature and 350μl of supernatant was transferred to a clean tube. An equal volume of phenol/chloroform was added and the sample mixed. The phases were separated by centrifugation for 3 minutes at 12300g and the aqueous phase (top layer) transferred to a fresh tube. The DNA was precipitated by the addition of 0.6 volumes of propan-2-ol and incubated at room temperature for 10 minutes. The DNA was pelleted by centrifugation for 7 minutes at 12300g at room temperature and the pellet resuspended in 100μl 0.3M sodium chloride and 250μl of ice cold absolute ethanol and the solution incubated at -20°C for at least one hour. The DNA was recovered by centrifugation for 7 minutes at 12300g at room temperature and the pellet dissolved in 10μl GDW.

2.6.4 RESTRICTION ENDONUCLEASE DIGESTION OF PLASMID DNA

In order to select which clones contained the correct plasmid, DNA was digested with restriction endonuclease enzymes and analysed by gel electrophoresis. Clones containing the correct size inserts were chosen for large scale preparation.

Materials Plasmid DNA Restriction endonucleases: EcoR I and Xho I (New England Biolabs, Hitchin, Hertfordshire, UK) 10x restriction buffer (as supplied) 10x BSA

Method

In separate reaction tubes, the products of small scale preparation of plasmid were diluted in GDW, BSA and restriction buffer added to give a final concentration of 1x BSA and 1x restriction buffer. Restriction endonucleases were added to five times excess however the volume of enzyme added was kept below 10% of the final volume. The reaction was incubated for a minimum of one hour at 37°C.

2.6.5 ELECTROPHORESIS OF DNA FRAGMENTS

After restriction digestion, the DNA was size fractionated by electrophoresis on an agarose gel to confirm if the correct size insert was present.

Materials Agarose, type II-A medium EEO 50x TAE (Appendix C) Ethidium bromide (10mg/ml)

DNA marker (1KB ladder, Invitrogen) Gel loading buffer

Method

A 0.7% (w/v) agarose gel was prepared by dissolving the agarose in 1x TAE using a microwave oven. The gel was cooled to 45°C and ethidium bromide added to achieve a final concentration of 0.5μg/ml and allowed to set in a mould. 3μl of loading buffer (appendix C) was added to 10μl of restriction enzyme digest, 1μl of DNA marker was added to 9μl GDW and treated the same way. The samples were loaded onto the gel and electrophoresed at 1.5V/cm. The DNA was visualised by illumination with UV light (300nm).

2.6.6 LARGE SCALE PLASMID PURIFICATION

Following small scale preparation of the plasmid, the size of the insert was confirmed by restriction digest (2.6.4). A large scale preparation of the plasmid is then carried out prior to purification using a caesium chloride gradient. This step is a larger scale procedure of the previously described method (section 2.6.3.) but it is followed by a caesium chloride gradient to further purify the plasmid.

Materials

LBAmp GTE: lysis solution for large scale preparation (Appendix C) Lysozyme (Sigma) Alkaline SDS 5M potassium acetate pH 4.6 Propan-2-ol 1x TE (Appendix C) DNase free RNaseA (Sigma) Phenol/Chloroform 2M sodium acetate, pH 5.2

Method

A small quantity of bacteria containing the plasmid with the correct insert size was inoculated into 500 ml LBAmp and incubated at 37°C overnight with vigorous shaking. The bacteria were recovered by centrifugation for 8 minutes at 3000g (HS-4 rota in RC-5B super speed centrifuge, Du Pont) at 4°C. The pellet was resuspended in 25 ml GTE supplemented with 2mg/ml of lysozyme and the samples incubated at room temperature for 5 minutes. Alkaline SDS (50ml) was added, the sample mixed until clear and incubated on ice for 5 minutes. Then 38ml of 5M potassium acetate was added, mixed and incubated on ice for 10 minutes. Bacterial debris was removed by centrifugation for 15 minutes 9000g (HS-4 rota in RC-5B super speed centrifuge) at 4°C. The supernatant was filtered through nylon gauze to remove cell debris and transferred to a clean tube. 0.6 volumes of propan-2-ol was then added and samples incubated on ice for 15 minutes. As before the DNA was recovered by centrifugation for 15 minutes (9000g, 4°C). The pellet was dissolved in 10ml 1x TE and RNase A added to a concentration of 0.1mg/ml and the reaction incubated at 37°C for 30 minutes. Addition of an equal volume of phenol/chloroform terminated the reaction, the mixture mixed and the phases separated by centrifugation for 20 minutes at 10000g and 4°C. The aqueous phase (top layer) was removed and 0.1 volume of 2M sodium acetate, pH 5.2 and one volume propan-2-ol was added. The reaction was then incubated at -20°C for at least one hour.

2.6.7 CAESIUM CHLORIDE GRADIENT PURIFICATION

This procedure was used to purify large quantities of plasmid. Under high centrifugal force, a solution of caesium chloride (CsCl) molecules will dissociate, and the heavy Cs+ atoms are forced towards the bottom (outer) end of the tube, thus forming a shallow density gradient. DNA molecules in this gradient will migrate to a point where they have equal density (the neutral buoyancy or isopycnic point). When nucleic acids bind ethidium bromide (EtBr), it affects their density as EtBr binds by intercalation into the DNA thereby causing the DNA helix to partially unwind. Binding occurs to a higher degree in linear genomic DNA or nicked plasmid DNA than with closed circular supercoiled plasmid DNA. Therefore, different states

80 of DNA form separate bands in the CsCl gradient. EtBr however damages DNA and is therefore immediately and thoroughly removed once purification is complete.

Materials TES (Appendix C) Caesium chloride Ethidium bromide (10mg/ml) Caesium chloride saturated propan-2-ol (Appendix C) 1x TE (Appendix C) Paraffin 2M sodium acetate, pH 5.2 Absolute ethanol

Method

The DNA obtained from section 2.7.6. was recovered by centrifugation at 4°C for 20 minutes at 24000g (HB4 rotor in RC5B super speed centrifuge, Du Pont) and dissolved in 8.25ml TES. To the solution of DNA, 8.4g of CsCl and 150μl EtBr was added and the solution mixed. The sample was loaded into a polyallomer centrifuge tube (Ultracrimp, Du Pont), overlaid with paraffin, crimp sealed and centrifuged for 16 hours at 20°C and 185500g (A1256 rotor, OTD- 55B centrifuge, Du Pont). After centrifugation, the DNA bands were visualised by UV light and the band containing the closed circular DNA removed using a 20G needle and a 2ml syringe. The EtBr was removed from the plasmid by repeated extraction with an equal volume of caesium chloride saturated propan-2-ol, until both phases were colourless. The DNA was precipitated by addition of two volumes of GDW and six volumes of room temperature absolute ethanol. The DNA was recovered by centrifugation for 15 minutes at room temperature and 24000g, the supernatant containing CsCl was removed and the pellet dissolved in 0.4ml GDW. The DNA was ethanol precipitated by adding 0.1 volume of 2M sodium acetate, 2.5 volumes of absolute ethanol and the mixture incubated at -20°C for at least one hour. The DNA was recovered by centrifugation for 10 minutes at 12300g, dissolved in 0.1ml GDW and quantified spectrophotometrically (section 2.6.8.).

2.6.8 QUANTIFICATION OF DNA BY SPECTROPHOTOMETER

The DNA was diluted 1:1000 in GDW and 1ml placed into a quartz cuvette. The absorbance was measured at 260 and 280nm (UV-160 spectrophotometer, Shimadzu, Kyoto, Japan) where the ratio A260:A280 was checked to be 2:1 (DNA:RNA). The concentration of DNA was calculated using the following formula: concentration (μg/ml) = (A260 x dilution factor) x 50.

2.6.9 POLYETHYLENIMINE (PEI) MEDIATED IN VITRO GENE TRANSFER

Stable, over-expressing cell lines for hY2 and hY4 were created by transfection of plasmids containing cDNA for hY2 and hY4 receptors into HEK 293 cells using the PEI transfection method. Cationic polymers like PEI can tightly associate with negatively charged nucleic acids. The DNA:PEI complex is positively charged and can come into close association with the negatively charged cellular plasma membrane. The DNA:PEI complex is then taken up into cells by pinocytosis across the cell membrane in the presence of glucose. This mixture produces small stable particles which is an advantage in their uptake into cells.

2.6.10 MAINTENANCE OF CELLS

Materials

HEK-293 cells (ATCC® CRL-1573™) (ATCC, LGC Standards, Middlesex, UK) Dulbecco’s modified medium (DMEM) without sodium pyruvate containing 4.5g/l glucose (Invitrogen) Foetal bovine serum (FBS) (Invitrogen) 100x antibiotic (10,000 units/ml of penicillin and 10,000 µg/ml of streptomycin) (Invitrogen) Non-enzymic cell dissociation buffer (Sigma)

Method

HEK 293 cells were cultured in DMEM media supplemented with 10% FBS and final concentration of 100units/ml penicillin and 100 µg/ml streptomycin (standard media). Cell media was changed every 2-3 days and the cells sub-cultured when 70% confluent using a non-enzymatic cell dissociation buffer. Briefly, the medium was aspirated from the flask and 2 3ml cell dissociation buffer at room temperature added to a 175cm flask of cells and incubated at room temperature until they detached from the flask. Fresh medium was added to the flask to quench the dissociation reaction and cells recovered by centrifugation for five minutes at 100g and the supernatant discarded. The pelleted cells were resuspended in fresh media and transferred to a new flask at a dilution of 1:5.

2.6.11 TRANSFECTION OF CELLS

Materials 0.1M 25kDa PEI (Aldrich Chemical Company) pH 7 (Appendix C) GDW 10% w/v glucose (sterile filtered) Plasmid DNA (pcDNA3.1+ human NPY2R or pcDNA3.1+ human NPY4R) G418 antibiotic (50mg/ml Sigma)

Method

Prior to transfection, HEK-293 cells were plated at a density of 20000 cells/well of a 6-well plate (Nunc) in standard DMEM media (as described in 2.6.10). Transfections were carried out when the cells were 40% confluent. The plasmid DNA was transfected at a concentration of 6μg DNA/well in 5% glucose. PEI:DNA complexes were made with PEI

(average MW ~ 25kDa) according to the method described by (Abdallah et al., 1995, Abdallah et al., 1998). PEI is a cationic polymer in which every third atom is an amine nitrogen residue. PEI was added to the DNA at different ratios of PEI nitrogen to DNA phosphate (N:P ratio). The cells were transfected with N:P ratios of 6, 9 and 12 nitrogen equivalents of PEI. A volume of 1μl of 0.1M PEI (prepared as described in Appendix C) 83 contains 100nmol amine nitrogen while 10μg DNA contains 30nmol of DNA phosphate. Thus 6μg DNA (18nmol DNA phosphate) needs 108nmol amine nitrogen for a N:P ratio of 6 (or 1.08μl of 0.1M PEI). To prepare the transfection mix, PEI solution was added slowly to the DNA in glucose solution, vortexed for 30 seconds and allowed to stand at room temperature for 10 minutes before use. Prior to transfection, the media on the cells was replaced and the DNA/glucose/PEI mix was slowly added to each well and gently swirled. Cells were incubated at 37°C, 5% CO2 for 3 hours, after which the media was removed and replaced with fresh media and the cells allowed to grow for 2 days. At least 6 wells were transfected with the plasmid DNA for each receptor and 2 control wells with just PEI in glucose. Forty eight hours later, media was supplemented with 0.8mg/ml G418 (geneticin), and media replaced every 48 hours with fresh G418 antibiotic until all control cells were dead (typically 10 days after the start of treatment). G418-resistant cells were transferred from the wells to 25cm2 flasks (1 well to 1 flask), and then maintained as described in 2.7.10. The population of mixed clones in each flask was tested for receptor expression by measuring change in specific binding of radiolabelled PYY3-36 (for hY2r cell lines) or PP (for hY4r cell lines). Mixed clones with the highest specific binding of radiolabelled ligand were selected for use in receptor binding studies.

2.7 RECEPTOR BINDING ASSAYS

Cell membrane suspensions were prepared from both hY2r and hY4r over-expressing HEK- 293 cell lines. The subsequent binding studies tested the specific binding of peptides to the receptors on these membrane preparations.

2.7.1 PREPARATION OF MEMBRANES FROM CELLS

2.7.1.1 Maintenance of cells

HEK-293 cells (ATCC® CRL-1573™) (ATCC, LGC Standards, Middlesex, UK) transfected with either hY2r or hY4r plasmid/cDNA were maintained in standard DMEM media and were passaged as described in section 2.6.10.

2.7.1.2 Receptor purification

Materials 1x 0.01M phosphate buffered saline (PBS) (10x in Appendix C) 1mM HEPES buffer (Appendix C) Homogenisation buffer (Appendix C)

Method

2 From a minimum of 30 x 175cm flasks, culture medium was removed and the cells detached by scraping into ice cold 0.01M PBS). Cells were centrifuged at 100g for 5 minutes (Sigma Laboratory Centrifuges 3, K18, rotor No. 19777-H), the supernatants discarded and the cell pellets put onto ice. Pellets were resuspended in 2 ml of ice-cold PBS and cells osmotically lysed by dropwise addition to 1mM HEPES solution containing protease inhibitors and left to stir for 5minutes. The solution was centrifuged at 3000rpm (Beckman J2-21, rotor JS-13.1) for 20 minutes at 4°C and the supernatant discarded. 20ml of homogenisation buffer was added and the cells homogenised for 20s with an Ultra Turrax homogeniser (IKA Labortechnik) and then put onto ice for 1minute. Mechanical homogenisation was repeated a second time before the homogenates were further centrifuged at 2500rpm (Beckman J2-21, rotor JS-13.1) for 20 minutes at 4°C. Membrane homogenates present in the resultant supernatant were collected and pelleted through centrifugation at 100,000g (Sorvall OTD 55B ultra-centrifuge, DuPont) for 60 minutes at 4°C. Finally, pellets were resuspended to a final protein concentration of 1-2mg/ml and stored at -80°C. Protein concentration was measured by Bradford assay (section 2.7.1.3.).

2.7.1.3 Bradford Assay 0.1ml cell membrane solution (purified from cells over-expressing hY2r or hY4r) was added to 3ml of Bradford reagent (Sigma). A standard curve with known concentrations of bovine serum albumin (BSA) (Sigma) (0.1ml 0, 0.25, 0.5, 1.0, 1.5 mg/ml solution) was added to 3ml of Bradford reagent. All solutions were incubated at room temperature for 45 minutes. A sample of each solution was added to a cuvette and absorbance was measured at 595nm using a spectrophotometer (UV-160, Shimadzu, Kyoto, Japan). The standard curve was used to determine the unknown concentration of protein in the membrane samples.

2.7.2 IODINATION OF PEPTIDES

Iodination of a peptide enables it to be used as a tracer in displacement assays. It involves the introduction of the radioactive isotope of iodine (125I) into certain amino acids (usually tyrosine) in a peptide. Iodination takes place at the positions adjacent to hydroxyl groups on the tyrosine molecule which indicates that mono- or di-substitutions can occur.

Radioactive 125I can be incorporated into peptides either by enzymatic or chemical oxidation. In the chemical oxidation (or direct iodogen) method, sodium iodide (Na125I) is converted to its corresponding reactive iodine form, which then spontaneously incorporates into tyrosine hydroxyl groups. While necessary for iodine activation, oxidising reagents (such as chloramine T) are potentially damaging to proteins.

3nmols (15μg) of peptide in 10μl of 0.2M phosphate buffer pH 7.2 was reacted with 37MBq of Na125I (GE healthcare) and 23nM (10μg) of the milder oxidising reagent 1,3,4,6,- tetrachloro-3α, 6α-diphenylglycoluril (Iodogen, Pierce Chemical Co, Perbio Science UK Ltd., Cramlington, UK) for 4 minutes at 22°C. The 125I-peptide was purified by reverse-phase HPLC using an AcN/ H2O /0.05% TFA gradient.

2.7.3 Y1, Y2 AND Y4 RECEPTOR BINDING STUDIES

Cell membranes from hY2, mY2, or hY4 receptor overexpressing HEK-293 cell lines, or the SK-N-MC cell line (source of hY1 receptor), were used for these studies. The assays were performed in 1.5ml siliconised microtubes (Sigma), with a final volume of 500μl. 125I-PYY in 50µl assay buffer (appendix C) with an activity of 1kBq was incubated with 10μl of the membrane suspension (at protein concentration 1-2μg/ml as determined by Bradford assay in section 2.7.1.3). Microtubes contained increasing concentrations of non-radiolabelled competing peptide. Each peptide concentration was assayed in duplicate or triplicate. Once the competing non-radiolabelled and tracer radiolabelled ligands were added, microtubes were each vortexed and incubated at room temperature for 90 minutes. After incubation, microtubes were centrifuged at 15781g (Sigma Laboratory Centrifuge 3 K18) for 3 minutes at 4°C, the supernatant removed and discarded, and the pelleted receptors washed with 500μl of fresh assay buffer to wash the pellet of any unbound radio-ligand. The tubes were centrifuged as before and the supernatant was again discarded and the pellets measured for γ-radiation for 240 seconds (Gamma counter, NE 1600, NE Technology Ltd, Reading, UK).

The half-maximal inhibition concentrations (IC50) were calculated and compared for each peptide tested. IC50 values were calculated using the Prism 5.01 program (GraphPad Software Inc. San Diego, USA) using a three-parameter logistic equation for the non-linear regression fit: Y=Bottom + (Top-Bottom)/1+10^(LogEC50-X).

2.8 PROTEOLYTIC DEGRADATION OF PEPTIDES

The general model of enzymatic cleavage (Fig 2.1) states that amino acid residues in a substrate undergoing cleavage are designated P1, P2, P3, P4 etc. in the N-terminal direction from the cleaved bond. The residues in C-terminal direction from the cleaved bond are likewise designated P1', P2', P3', P4' etc (Schechter and Berger, 2012).

Figure 2.1 Schematic diagram depicting the nomenclature for enzyme cleavage sites within a substrate. Positions Pn to Pm’ are counted from the bond where enzymatic cleavage occurs between P1 and P1’ (Schechter and Berger, 2012).

2.8.1 LIVER MICROSOMES PREPARATION

The preparation of rat liver microsomes (RLM) by homogenisation and differential centrifugation was based on the method of Leadbeater and Davies (Leadbeater and Davies, 1964). RLM were prepared from 250-300g male Wistar rats. The rats were humanely killed by CO2 asphyxiation, the liver removed and snap-frozen in liquid nitrogen and stored at - 80°C. After collecting livers from 20 rats, they were thawed and rinsed in 0.10 M Tris-HCl buffer, pH 7.5, then homogenized using an Ultra Turrax homogeniser (IKA Labortechnik). The homogenates were centrifuged at 10,000 g (Beckman J2-21, rotor JS-13.1) for 20 min to spin down the nuclei, mitochondria and unbroken cells. The microsomal and soluble fractions of the liver were in the supernatant. The preparation was further purified by centrifuging the supernatant at 10,000 g (Beckman J2-21, rotor JS-13.1) for 20 min. The resultant supernatant (S-10 fraction) was then centrifuged at 100,000g (Sorvall OTD 55B ultra-centrifuge, DuPont, St. Neots, Cambridgeshire, UK; rotor A-841) for 60min to spin down the microsomes. After decanting the supernatant the microsomal pellet was resuspended in fresh buffer and stored at -80°C until use. The microsomes were stored in aliquots so that each assay was carried out with microsomes which had been frozen and thawed only once.

2.8.1.1 Biuret assay

RLM was quantified for protein content using a BSA (Sigma) standard curve at 0, 1, 2, 3, 4 and 5mg of protein/500μl with dH2O. RLM was assayed at 20μl and 200μl and volumes adjusted to 500μl with dH2O. Biuret reagent (3ml – Sigma) was then added to all tubes and incubated at room temperature for 30 min. Absorbance of each sample at 540nm was measured using a spectrophotometer (UV-160, Shimadzu) using dH2O as the zero reference. The absorbance values of the membrane samples were converted to mg protein by linear regression analysis of the standard curve and interpolation of the unknown samples to be quantified.

2.8.2 LIVER MICROSOME HYDROLYSES

Digest buffer (0.10 M Tris-HCl buffer, pH 7.5) with a total volume of 110μl containing 2nmol of peptide and 1mg/ml (10ug/reaction) of RLM preparation (prepared as described in section 2.8.1) was incubated at 37°C. The reaction was terminated by addition of 5μl of 10% v/v trifluoroacetic acid (TFA) and the RLM removed by centrifugation at 21,000 g for 1minute. The incubation time and RLM concentration were selected based on studies that demonstrated differences in susceptibility to degradation between peptides.

The reactions were analysed using reverse phase HPLC (Jasco HPLC system: solvent delivery system PU-2080 plus, autosampler AS-2057 plus, degaser DG2080-53, dynamic mixer 2080- 32 UV detector uv-2075. Column: Phenomenex Gemini C18 column containing 5μm particles, 250 mm x 4.6 mm). The samples were loaded onto the column and eluted using an acetonitrile (AcN)/water gradient, both acidified with 0.05% v/v TFA. The gradient characteristics were 15-60% AcN over 35 minutes as shown in Fig 2.2. The eluted peptides were detected spectrophotometrically by the UV detector set to read at a wavelength of 214 nm as this is the point where the peptide bond absorb, minimising any change in absorbance caused by quantity of any one amino acid. Percentage degradation was calculated by comparing area under peak of the original peptide in the presence and absence of RLM. Percentage degradation values were calculated from a minimum of three experiments and chromatograms shown are representative examples.

100

% Acetonitrile

2 0 5 40 50 55 Time (min)

Figure 2.2 Graphical representation of the AcN gradient in water. Peptides eluted in the linear gradient between 5 and 40 minutes. The flow rate was set at 1ml/min.

2.8.3 RENAL BRUSH BORDER (RBB) MEMBRANE PREPARATION

Rat RBB membranes were prepared based on the method described by Booth and Kenny (Booth and Kenny, 1974). Briefly, rats were killed by decapitation and kidneys removed and snap-frozen in liquid nitrogen and stored at -70°C. Kidneys were placed in cold 0.5M sucrose solution and the renal cortices dissected out and weighed. The cortices were finely sliced and gently homogenised at a ratio of 1g tissue to 6ml 0.5M sucrose using a 15ml hand- homogeniser (Jencons, UK). Tissue was then further homogenized using a motorised homogeniser (Ultra Turrax, IKA Labortechnik) with a tight fitting Teflon coated pestle

(Jencons) at 1000rpm. Following this, 1M MgCl2 was added to the homogenate to achieve a final concentration of 10mM MgCl2. This solution was stirred in an ice bath for 15 minutes to remove subcellular structures from the brush border cells through their aggregation by bivalent metal ions. The subcellular structures were removed by centrifugation at 1500g (Beckman J2-21, rotor JS-13.1) at 4°C for 20 minutes. The resulting supernatant containing RBB was collected and subsequently centrifuged at 15000g (Beckman J2-21, rotor JS-13.1) for 12 min at 4°C to remove the sucrose. The supernatant containing sucrose was discarded and the RBB pellet resuspended in fresh buffer of 100μl of 300mM mannitol in 12mM Tris-Cl

(pH7.4), with 10mM MgCl2. This suspension was first centrifuged at 2200g at 4°C (Sigma Laboratory Centrifuge 3 K18), the supernatant collected and centrifuged a second time for 12min at 15000g at 4°C. The resultant RBB pellet was then resuspended in 300mM mannitol in 12mM HEPES (pH7.4). Protein content was measured by Biuret assay as described in 2.8.1.1.

2.8.4 RENAL BRUSH BORDER MEMBRANE HYDROLYSES

Digest buffer (12mM HEPES, 300mM Mannitol pH7.4) containing 2nmol of peptide and 1mg/ml of RBB membrane preparation (prepared as described in section 2.8.3) was incubated at 37°C. The reaction was terminated at 30minutes by addition of 5μl of 10% trifluoroacetic acid (TFA). The incubation time and RBB concentration were selected based on studies that demonstrated differences in susceptibility to degradation between peptides. The reactions were subsequently analysed using reverse phase HPLC using the same column and AcN gradient profile to analyse the RLM digests (section 2.8.2). Again, the eluted peptides were detected spectrophotometrically by the UV detector set to 214nm and percentage degradation was calculated by comparing area under peak of the original peptide in the presence and absence of RBB. Percentage degradation values were calculated from a minimum of three experiments and chromatograms shown are representative examples.

2.8.5 DIPEPTIDYL-PEPTIDASE IV (DPPIV) HYDROLYSES

Digest buffer (100 mM TRIS-HCl, pH8) containing 2 nmol of peptide and 10-50 mU of porcine kidney DPPIV (Sigma) was incubated at 37°C. The reaction was terminated at 120 minutes by addition of 5μl of 10% TFA. The reactions were initially analysed using reverse phase HPLC (section 2.8.2).

Samples were centrifuged at 12500xg for 5 minutes at room temperature prior to HPLC analysis. The samples were applied to a C18 column and eluted using a linear AcN/water gradient acidified with 0.05% TFA. The gradient characteristics were 31.5-33.5% AcN over 45

91 minutes. The eluted peptides were detected spectrophotometrically at a wavelength of 214 nm. Percentage degradation of peptide was unable to be calculated by comparing area under peaks of reactions with and without enzyme as metabolites and parent compound eluted at the same retention time and there were no additional degradation peaks detected. Therefore, analysis by MALDI-TOF was used for these hydrolyses.

2.8.5.1 Matrix-assisted laser desorption/ionization-Time of flight (MALDI- TOF) analysis of DPPIV proteolytic breakdown products

MALDI-TOF mass spectrometry (MS) analysis of digests was performed at the Proteomic facility by the Advanced Biotechnology Centre (ABC, Imperial College, London, UK). Digests were performed as described in section 2.8.5, except 5nmol of peptide was digested by 25mU of DPP IV in a reaction volume of 340 µl for 240 minutes. Digests were dried down using a SpeedVac sample Concentrator (Savant SPD2010, Thermo Electron Co., MA, USA) and spotted on an α-cyano-4-hydroxycinnamic acid (HCCA) matrix for MALDI TOF analysis using an Ultra Flex MALDI-TOF (Germany). In MALDI-TOF MS, the peptides are ionised on a plate and the ions are then accelerated through a vacuum tube by an electrical field towards a detector. Smaller fragments are accelerated to higher velocities than heavier ones, and the “time of flight” (TOF) is proportional to the mass:charge ratio (m/z). The charge of the ionised proteins is often +1, making the m/z value equal to the mass value. If the molecule obtains a +2 or +3 charge, the resulting value will be incorrect and care is needed to analyse this data. Assuming monoisotopic mass, theoretical masses for peptide breakdown products were calculated using the program FindPept (http://web.expasy.org/findpept/) with a mass tolerance of ± 5 Da, and were compared to the results of masses identified by MALDI-TOF MS.

2.8.6 HUMAN TRYPSIN HYDROLYSES

Trypsin is a highly active and specific protease which cleaves the C-terminal to lysine and arginine residues of a protein to generate peptides that fall within the mass range of 500- 5000 Da.

The trypsin protease assay was carried out as the DPPIV assay protocol above, except for the digest buffer used (10 mM TRIS-HCl, 10mM CaCl2 pH7), with 0.1mU of human trypsin (Sigma) in a volume of 120μl. The assay was incubated for 15 minutes before the reaction was terminated with 5μl of 10% TFA. The incubation time and enzyme concentration were selected based on studies that demonstrated differences in susceptibility to degradation between peptides. The reactions were subsequently analysed using reverse phase HPLC using the same column and AcN gradient profile to analyse the RLM digests (section 2.8.2). Eluted peptides were detected spectrophotometrically by the UV detector at 214nm and percentage degradation was calculated by comparing area under peak of the original peptide in the presence and absence of trypsin. Percentage degradation values were calculated from a minimum of three experiments and chromatograms shown are representative examples.

Digests were also sent for a MALDI TOF analysis by the Proteomic facility at the ABC (Imperial College, London, UK) as described in section 2.8.5.1.

2.8.7 HUMAN RECOMBINANT NEPRILYSIN HYDROLYSES

The NEP protease assay was completed as the trypsin digest protocol above, except for digest buffer used (25 mM TRIS-HCl, 0.1M NaCl pH8), with 200ng of human recombinant NEP (R&D systems, Germany) in a volume of 130μl. The assay was incubated for 10-120 minutes before the reaction was terminated with 5μl of 10% TFA. The reactions were subsequently analysed using reverse phase HPLC as described in section 2.8.6 with a slight change to the gradient (15-55% AcN in 35mins). Eluted peptides were detected spectrophotometrically by the UV detector at 214nm and percentage degradation was calculated by comparing area under peak of the original peptide in the presence and

93 absence of NEP. Percentage degradation values were calculated from a minimum of three experiments and chromatograms shown are representative examples.

2.9 RADIOIMMUNOASSAY (RIA)

All RIAs used (including iodination of peptides, production of antibodies, arranging synthesis of custom peptides) were derived and maintained by Professor MA Ghatei (Professor of Regulatory Peptides, Section of Investigative Medicine, Division of Diabetes, Endocrinology & Metabolism, Imperial College London).

2.9.1 PRODUCTION OF PP-x AND PYY2-36(αLTX-4H) POLYCLONAL ANTIBODY

PP analogues with mid-section modifications and the PYY analogue, PYY2-36(αLTX-4H) did not cross-react with antibodies raised to either human PP or porcine PYY respectively, even when the concentration of competing standard peptide was increased 10-fold. Therefore new antibodies were raised against PP-x (sequence detailed in appendix B) and PYY2-

36(αLTX-4H). Sheep (Scotland Blood Transfusion Service, Penicuik, Scotland) were inoculated with 200µg PP-x or PYY2-36(αLTX-4H) conjugated to BSA (Sigma) as the carrier protein using carbodiimide (Sigma) as the coupling agent. Booster inoculations were conducted fortnightly and test bleeds were done 2 weeks after the third booster injection and every month thereafter. Each bleed was tested by incubating the sera (neat) with 125I-PP-x (for 125 sheep inoculated with PP-x) or I-PYY2-36(αLTX-4H) (for sheep inoculated with PYY2-36(αLTX- 4H)) for 2 hours, to test for excess binding of label as with the immunogenicity protocol described in section 2.10.

The unbound radiolabelled peptide was separated from the bound radiolabelled peptide by dextran-coated charcoal separation. Following centrifugation at 1000 x g for 20 minutes at 4°C, free label is contained in the charcoal pellet and label bound to the antibody remains in

94 the supernatant. Both the bound and free fractions of label are counted in a γ-counter (model NE1600, Thermo Electron Corporation) and the amount of bound label is calculated as a percentage of the total counts.

A population of sheep plasma was chosen for the highest levels and most specific antibodies produced. A titre test of the chosen plasma was then conducted to determine the correct titre of antibody to use for the RIA.

2.9.2 PYY AND PYY ANALOGUES RIA

A specific and sensitive RIA (principles of which are described in Appendix E) was used to determine the levels of PYY and PYY analogues in plasma. Plasma samples from animals injected with PYY3-36, PYY2-36(4H), PYY2-36(αLTX-4H) or PYY2-36(αLTX-4H)-PEG were assayed using the conditions described in Table 2.1. All peptides cross reacted 100% with the antibody used.

RIA component Animals injected Animals injected Animals injected with

with PYY3-36 with PYY2-36(4H) PYY2-36(αLTX-4H)

Antibody Anti-porcine PYY Anti-porcine PYY Anti-PYY2-36(αLTX-4H) Antibody titre 1:70000 1:47000 1:230000 125 125 125 Radiolabel I-PYY1-36 I-PYY2-36(4H) I-PYY2-36(αLTX-4H) Peptide for PYY1-36 PYY2-36(4H) PYY2-36(αLTX-4H) standard curve Concentration of 1pmol/ml 2pmol/ml 2pmol/ml standard curve Method of 2° antibody 2° antibody Dextran-coated separation charcoal

Table 2.1 Components of the PYY and PYY analogues radioimmunoassay used.

The anti-PYY antibody was produced in sheep against purified porcine PYY (differs from the human sequence by 2 amino acids at positions 3 and 18) coupled to bovine serum albumin

(BSA) by glutaraldehyde and used at a final dilution of 1:7000 to measure PYY3-36, or at a final dilution of 1:47000 to measure PYY2-36(4H). This antibody cross reacts to PYY and PYY2-

36(4H) and has very poor cross-reactivity with the PYY analogue PYY2-36(αLTX-4H).

As described in section 2.9.1, the anti-PYY2-36(αLTX-4H) antibody was produced in sheep against purified PYY2-36(αLTX-4H) coupled to BSA by carbodiimide and used at a final dilution of 1:230000. This antibody cross reacts to PYY2-36(αLTX-4H) and has poor cross-reactivity with native PYY and PYY2-36(4H). Both anti-PYY and anti-PYY2-36(αLTX-4H) antibodies do not cross-react with other known gastrointestinal hormones.

A radiolabelled form of PYY was prepared by the direct iodination method and purified by reverse phase high-pressure liquid chromatography.

Assays were performed in 0.06M phosphate EDTA buffer (0.05M Na2HPO4.2H2O, 0.6mM

KH2PO4, 0.01M disodium-EDTA.2H2O, 8mM NaN3), at pH7.4 with 0.3% BSA. All samples were assayed in duplicate in a final volume of 700µl per tube.

Standard curves for each assay were prepared in assay buffer at the concentrations given in the table above and added in duplicate at volumes of 1, 2, 3, 5, 10, 15, 20, 30, 50 and 100μl. A sample volume of 1, 3, 5, 10, 50, or 100μl of plasma was used. The assays were incubated for 72 hours at 4°C before the separation of free- and antibody-bound radiolabel.

Free label was separated from bound using either 2° antibody precipitation or charcoal adsorption. For 2° antibody separation, samples were pre incubated for 1hour with 100µl of 0.1% goat anti-rabbit 2° antibody at room temperature. Immediately prior to centrifugation, 500µl of 0.01% Triton-X-100 (Sigma) was added to each tube, and then spun at 1500g, for 20minutes at 4°C. For charcoal separation, 250µl of dextran-coated charcoal solution (2.4g activated charcoal, 0.24g dextran, 100ml 0.06M phosphate EDTA buffer pH7.4 with gelatin) was added to each tube. The assay was then centrifuged also at 1500g, for 20minutes at 4°C.

Bound and free label were separated and both the pellet and supernatant counted for 180 seconds in a γ-counter (model NE1600, Thermo Electron Corporation). Peptide

96 concentrations in the samples were calculated using a non-linear plot (RIA Software, Thermo Electron Corporation) and results calculated in terms of the standard.

Noncompartmental analysis of drug exposure was calculated using the trapezoidal rule to establish the area under curve (AUC) using GraphPad Prism 5 software.

2.9.3 PP AND PP ANALOGUES RIA

PP plasma levels were measured using established methods (Adrian et al., 1976). The anti-PP antibody was produced in rabbits against human PP coupled to bovine serum albumin (BSA) by glutaraldehyde and used at a final dilution of 1:50000 to measure PP. This antibody cross reacts to PP and has very poor cross-reactivity with PP analogues.

RIA component Animals injected with Animals injected with PP PP analogues Antibody Anti-PP Anti-PP-x Antibody titre 1:50000 1:13000 Radiolabel 125I-PP 125I-PP-x Peptide for standard PP PP analogue curve Concentration of 1pmol/ml 0.25pmol/ml standard curve Method of Dextran-coated Dextran-coated separation charcoal charcoal

Table 2.2 Components of the PP and PP analogues radioimmunoassay used.

The anti-PP analogue antibody was produced in sheep against purified PP-x coupled to BSA by carbodiimide and used at a final dilution of 1:13000. This antibody cross reacts to PP analogues which have poor cross-reactivity to the anti-PP antibody, and therefore has poor cross-reactivity with native PP. Both anti-PP and anti-PP-x antibodies do not cross-react with other known gastrointestinal hormones.

A radiolabelled form of PP and PP-x was prepared by the direct iodination method and purified by reverse phase high-pressure liquid chromatography.

PP RIAs were performed in 0.06M phosphate EDTA buffer (pH7.4) with 0.3% BSA and 0.002% Tween-20. All samples were assayed in duplicate in a final volume of 700µl per tube.

Standard curves for each assay were prepared in assay buffer at the concentrations given in the table above (Table 2.2) and added at the same volumes as the PYY RIA (section 2.9.2). Sample volumes of 1, 3, 5, 10, 50, or 100μl of plasma were used. The assays were incubated for 72 hours at 4°C before the separation of free- and antibody-bound radiolabel by charcoal adsorption.

Bound and free label were separated and both the pellet and supernatant counted for 180 seconds in a γ-counter (model NE1600, Thermo Electron Corporation). Peptide concentrations in the samples were calculated using a non-linear plot (RIA Software, Thermo Electron Corporation) and results calculated in terms of the standard.

Noncompartmental analysis of drug exposure was calculated using the trapezoidal rule to establish the area under curve (AUC) using GraphPad Prism 5 software.

2.10 DETECTION OF IMMUNOGENICITY OF PYY ANALOGUES

The presence of anti-analogue (anti-PYY2-36(4H), anti-PYY2-36(αLTX-4H)) or anti-PYY antibodies were measured using a Radioimmunoprecipitation Assay (RIPA). Briefly, 20µl plasma of animals treated with PYY2-36(4H) or anti-PYY2-36(αLTX-4H) was incubated with 125 125 125 either I-PYY2-36(4H), I-PYY2-36(αLTX-4H), or I-PYY1-36 respectively. This mixture was incubated overnight at 4°C and the free label separated from the bound through dextran- coated charcoal separation (unbound radiolabel is found in the charcoal pellet following centrifugation at 1500g for 20minutes at 4°C). Both the bound and free fractions of label are counted in a γ-counter and the amount of bound label is calculated as a percentage of the total counts (bound + unbound label). Positive samples were identified as those with ≥4-fold percentage binding compared to control groups.

2.10.1 ADA COMPETITION ASSAY WITH PYY ANALOGUES

125 “Zero” binding (Z) was defined as the binding of I-PYY2-36(αLTX-4H) to plasma containing anti-drug (PYY2-36(αLTX-4H)) antibodies (ADAs) in the absence of any competing non- radiolabelled PYY2-36(αLTX-4H) peptide. Binding sites on the ABs would therefore solely be 125 occupied by I-PYY2-36(αLTX-4H).

Known and increasing amounts of non-radiolabelled PYY2-36(αLTX-4H), fragments of PYY2-

36(αLTX-4H), or analogues of PYY2-36(αLTX-4H) were then used to compete with a constant 125 quantity of I-PYY2-36(αLTX-4H) peptide. This should dose-dependently decrease the amount of binding of radiolabelled peptide if the non-radiolabelled peptide and fragments compete at the same binding sites.

The difference of this decrease in binding (drop from Z) was compared across all known doses of non-radiolabelled PYY2-36(αLTX-4H), against the different fragments/analogues of

PYY2-36(αLTX-4H) synthesised, amongst plasma samples which tested positive for PYY2-

36(αLTX-4H) ABs. It is then expressed as a ratio compared with binding of full length PYY2-

36(αLTX-4H) at the same concentration (equation below). If the ratio is close to 1, it suggests 125 silimilar displacement of I-PYY2-36(αLTX-4H) compared to the full length peptide. 125 Fragments which do not compete as well with I-PYY2-36(αLTX-4H) binding for the ADAs will have a ratio <1.

Ratio = (Z binding – binding of ᵡ pmol non-radiolabelled peptide/fragment)

(Z binding – binding of ᵡ pmol non-radiolabelled PYY2-36(αLTX-4H))

Figure 2.3 Equation to measure relative displacement strength of various PYY analogues or fragments against radiolabelled drug.

2.11 STATISTICAL ANALYSIS

For animals studies, power calculations were used to determine the minimum sample size needed to reject the null hypothesis, assuming the null hypothesis is false (i.e. to be able to detect an effect, if the effect exists). (https://www.dssresearch.com/KnowledgeCenter/toolkitcalculators/samplesizecalculators. aspx). Factors which influence this include variance and magnitude of the effect. Assuming standard deviation is constant, experiments where a big effect was expected (e.g. using high doses of drug or making big changes to a peptide), sample size was reduced to limit the use of animals.

All data are expressed as the mean value ± standard error of the mean (SEM). In acute feeding studies, food intake was compared by one-way ANOVA followed by Dunnett’s post- hoc test (to determine the significance in the difference of food intake between the groups being tested and the saline group) or Bonferroni’s or Tukey’s post-hoc tests (to determine the significance in the difference of food intake between the different treatment groups). In pharmacokinetic studies, plasma levels of peptide hormone were compared by one-way ANOVA followed by Dunnett’s or Bonferroni’s post-hoc tests.

Continuous or longitudinal data was analysed using the generalized estimating equation (GEE) with the Mann-Whitney U adjustment or using a two-way ANOVA followed by a Tukey’s post-hoc test. GEE data analysis was performed using STATA 9 (StataCorp LP, College Station, TX) and all other statistical analyses used GraphPad Prism Version 5.01 (GraphPad Software Inc. San Diego, USA).

In all cases, p<0.05 was considered statistically significant.

100

CHAPTER 3

DEVELOPING ANALOGUES OF PANCREATIC POLYPEPTIDE AS A PHARMACOTHERAPY FOR OBESITY

101

3.1 INTRODUCTION

Satiety factors such as gut hormones, which are released in response to a stimulus (e.g. food intake), are transient signal molecules released in response to food intake and are required to be cleared quickly so as to modify behaviour for relatively short periods of time. Short half-life and limited efficacy and potency, are properties which have to be considered/modified when developing gut hormones as potential therapeutics.

There are several methods by which gut hormones can be modified in order to address their limitations as drug candidates. Coupling hormones to larger molecules such as albumin or fatty acid side chains has been successfully applied to increase half-life, for example the GLP-1r agonists albiglutide and liraglutide. Large side chains can extend the molecules half- life in circulation by reducing the rate of renal filtration through steric hindrance, or by reducing the metabolic clearance rate. Steric hindrance may also make a hormone less available to the active site of an enzyme and thus reduce its susceptibility to enzyme degradation.

Another method of enhancing the longevity of gut hormones is through substitution of amino acid residues (as in the case of taspoglutide) or by the addition of short sequences (e.g. lixisenatide) to reduce the peptides susceptibility to proteolytic degradation. Modifying the amino acid sequence can also result in an improvement of plasma circulatory half-life by increasing the affinity of the peptide to large plasma proteins such as serum albumin and lipoprotein.

For the gut hormones to physiologically exhibit their effects, they have to both bind to and activate their respective receptor. All of the gut hormones bind to GPCRs and any change of conformation to the ligand can alter its interaction with the receptor. The sensitivity of the receptor to changes in ligand structure cannot be overestimated. Small changes, such as the loss of the C-terminal amide group of PP, are enough to almost completely stop the molecule binding to the Y4r (Gehlert et al., 1996). On the other hand, there are examples where large changes, such as the addition of a fatty acid moiety to GLP-1 as with liraglutide, have very little effect on receptor affinity (Knudsen et al., 2000).

102

As well as affecting receptor affinity, changes to a ligand can also affect the molecule’s ability to activate the receptor, as with the conversion of the potent GLP-1r agonist exendin- 4 to the N-terminal truncated form, exendin-9-39, which is a GLP-1r antagonist (Schirra et al., 1998). In addition, as seen in plieotropically coupled receptors such as GLP-1r (Koole et al., 2013), there may be ligand-directed stimulus bias. Although most GLP-1r agonists preferentially activate cAMP over other signalling pathways in vitro, the degree of bias between the pathways varies between ligands with truncated GLP-1 and ex-4 having a greater bias towards cAMP than full length GLP-1 and OXM (Koole et al., 2010, Koole et al., 2012a, Koole et al., 2012b).

Receptor specificity is another feature which may be altered when making changes to a ligand. This is exemplified by the change in receptor preference by PYY1-36 (which binds preferentially to the Y1r) compared with PYY3-36 (which binds with greater affinity to the Y2r) (Grandt et al., 1992). In contrast, the C-terminal fragment, PYY30-36, has a much lower affinity at the Y2 receptor, but maintains much of the biological activity of PYY3-36, as shown in the bioassay using contractile effects in Y2r-enriched rat vas deferens (Fournier et al., 1994, Barden et al., 1994). The demonstrates how ligand activity can also be affected by peptide modification.

Increasing half-life and improving efficacy and potency may reduce the dose and frequency of drug administration, which may improve complicance with the dosing regimen, increase the attractiveness of the therapeutic option (where a choice is available), help tolerability, and reduce the cost of drug manufacturing. With improved pharmacokinetic profiles of the peptide analogue (gradual increase in systemic exposure with no abrupt peaks), side effects such as nausea may also be reduced

In this chapter I will describe the investigations I completed on pancreatic polypeptide (PP) to exemplify the design of an analogue with an extended circulatory half-life. PP is a member of the PP-fold family of peptides and is released post-prandially from pancreatic F cells (Adrian et al., 1976, Adrian, 1978). When PP is administered to healthy adults via a 90minute IV infusion, there is a 16-fold rise in circulating PP compared to basal levels, which significantly reduces food intake over 24 hours compared to a saline control (Batterham et al., 2003b, Jesudason et al., 2007). This anorectic effect suggests that PP may have potential

103 as an obesity treatment. It is a relatively small peptide, and is susceptible to a number of degradative enzymes and other eliminating mechanisms; PP consequently has a short circulating half-life of around 7 minutes (Adrian et al., 1978). This short half-life limits the use of native PP as an economically viable and tolerable obesity treatment.

Previous studies on other gut hormones including GLP-1, oxyntomodulin and PYY3-36 have shown that small regulatory peptides are susceptible to degradation by a number of membrane bound ectopeptidases and other membrane bound enzymes, limiting their circulating half-life (Kenny, 1986, Roques et al., 1993). As the PP family of peptides show strong sequence homology and are thought to be derived from a common ancestor, it is likely that the peptidases that break down other members of the PP-fold family will also degrade PP (Ekblad and Sundler, 2002, Blomqvist et al., 1992, Larhammar, 1996a, Larhammar, 1996b).

Therefore, the enzymes investigated in this chapter are those relevant to the degradation of PYY and other gut hormones such as those contained in preparations of rat liver microsomes (RLM), rat kidney brush border (KBB) membrane, trypsin, DPPIV, neprilysin, and meprin.

3.1.1 HEPATIC METABOLISM

The liver acts as the waste management system of the body, breaking down waste and detoxifying compounds. The “first pass effect” of hepatic metabolism mostly reduces the availability of many orally administered drugs. Most of the venous system of the GI tract enters the hepatic portal vein, and subsequently the liver. Consequently, after absorption in the intestine, most drugs pass through the liver and are degraded. Subcutaneously administered drugs initially enter systemic circulation but eventually reach the portal vein to undergo hepatic metabolism.

The liver eliminates most drugs by metabolism, biliary excretion or both. Rane et al. (Rane et al., 1977) discovered the potential for an in vitro-in vivo hepatic clearance prediction. Models for prediction of hepatic clearance include the use of liver microsomes, isolated hepatocytes, recombinant (heterozygously expressed) cytochrome P450 (CYP) isozymes, 104 liver slices and ex vivo liver perfusion preparations. All methods have their limitations; for example, recombinant heterozygously expressed CYPs are not predictive of intrinsic clearance because of contributions of several CYPs in the metabolism of different drugs and because metabolic rates differ with the expression system used. Liver slices are not useful in kinetic predictions due to a lack of uniform diffusion of compounds into all the cells within the slice. The in vitro methods using hepatic microsomes or hepatocytes are the most widely used approaches for estimating hepatic clearance.

Houston et al. (Houston, 1994) found that intrinsic hepatic clearance is a pure measure of enzyme activity towards a drug and is independent of hepatic blood flow and protein binding. They also demonstrated that it was possible to predict in vivo metabolic clearance over four orders of magnitude using in vitro techniques. A good correlation was found between in vitro and in vivo results with most compounds being within 3-5-fold range of intrinsic to in vivo clearance values. However, there was a tendency for the in vivo metabolic clearance to be higher than the estimated in vitro intrinsic metabolic clearance. These differences could be due to possible factors from in vivo contributions from other organs, presence of active transport mechanisms and inter-individual differences (Ito et al., 1998, Chaturvedi et al., 2001).

Investigating hepatic metabolism may therefore be a good predictor of in vivo metabolism when considering the design of proteolytic-resistant PP analogues. Although there is evidence to suggest that hepatic metabolism is not involved in the initial clearance of PP- fold peptides (Boden et al., 1980, Hagopian et al., 1983, Cohen et al., 1984, Beckh et al., 1992), it may have a role in the secondary phase of PP metabolism (e.g. secondary to enzymatic degradation at the site of administration) or may play a part in the degradation of PP analogues.

105

3.1.2 RENAL METABOLISM

The exact mechanisms of PP clearance are unknown. PYY, another member of the PP-fold family, has been more widely studied and is broken down by a number of enzymes and peptidases located in the kidney (Medeiros and Turner, 1994, Medeiros Mdos and Turner, 1994, Mentlein et al., 1993b). As PP and PYY have a high degree of sequence homology, it is therefore likely that the kidneys are also responsible for the clearance of PP. The kidney is thought to play a major role in the degradation of a number of other peptide hormone (Bastl et al., 1977, Ruiz-Grande et al., 1990) and enzymes located in the brush border, in particular metallopeptidases, are central to this route of peptide degradation. Enzymes including aminopeptidase N, aminopeptidase A, DPPIV, and neprilysin are all located within membranes of renal brush border (RBB) (Stephenson and Kenny, 1987, Kenny and Stephenson, 1988, Vanneste et al., 1988, Hupe-Sodmann et al., 1995, Medeiros and Turner, 1995, Bertenshaw et al., 2001). Interestingly, it has been suggested that the levels of certain enzymes within the RBB are dependent upon nutritional status. For example, starvation increases the levels of DPPIV in rats (Ihara et al., 2000). A consequence of this would be a decrease in circulating levels of GLP-1 which would result in a reduced satiety signal. This suggests a mechanism by which nutritional status regulates circulating gut peptide levels.

The kidneys may therefore be a key site for the breakdown of PP, and the reabsorption of amino acids, and preparations of RBB may be a useful tool (Kenny and Stephenson, 1988) to investigate the breakdown of PP and PP analogues.

3.1.3 DPPIV DEGRADATION

DPPIV is a member of the serine family of peptidases which acts to cleave the first two amino acids at the N-terminus of small (0.5kDa) to medium sized peptides (5kDa) (Mentlein et al., 1993b, Mentlein, 1999, Drucker et al., 1997). It is a membrane bound enzyme targeting peptides comprised of less than 80 amino acids and with a N-terminal sequence of either X-Pro, X-Ala, X-Ser or X-Gly (where ‘X’ is any amino acid except proline), and a hydrophobic amino acid such as tyrosine at the P3 position (Bongers et al., 1992, Proost et

106 al., 1999, Lambeir et al., 2002). DPPIV is an ectopeptidase highly expressed in endothelial cells and also present in plasma (De Meester et al., 1999, Ohkubo et al., 1994).

DPPIV has a number of hormone substrates, including NPY, PYY and GLP-1 (Mentlein et al., 1993b, Mentlein et al., 1993a). DPPIV is generally considered a peptidase which inactivates peptides by degrading them as in the case of GLP-1. However, it does also activate peptides: when PYY1-36 is cleaved by DPPIV, it forms PYY3-36, a highly active hormone, with high affinity for the Y2 receptor (Grandt et al., 1993) and a potent anorectic satiety factor.

The importance of DPPIV in degrading GLP-1 is highlighted by the emergence of DPPIV inhibitors such as Sitagliptin (Januvia, MSD) and Vildagliptin (Galvus, Norvartis) which prolong the half-life of endogenously released GLP-1 and are now approved drugs for treating T2DM (Davidson et al., 2008). They act to inhibit the breakdown of GLP-1, resulting with increased circulating levels and lowered blood glucose levels. The long lasting GLP-1 analogue exenatide is a GLP-1r agonist which is DPPIV resistant (first three amino acid sequence of His-Gly-Glu), and has therefore significantly increased plasma half life (Nielsen et al., 2004, Green et al., 2003). The sequence of PP (first three amino acid sequence is Ala- Pro-Leu) suggests it is a likely candidate for DPPIV proteolysis and this could be a mechanism by which the peptide is inactivated (as with PYY1-36 and NPY). The role of DPPIV degradation in PP will be important to establish when designing PP analogues with a longer half-life.

3.1.4 NEPRILYSIN DEGRADATION

Neprilysin (NEP) is a 94kDa metallopeptidase enzyme with a zinc core. It is usually found bound to membranes, and is expressed throughout the body. NEP was originally discovered in purified kidney brush border (Gee et al., 1985, Roques et al., 1993, Hupe-Sodmann et al., 1995), and the enzyme is found at a high concentration in the kidneys, brain, and lungs. NEP has a wide number of substrates, including GLP-1, Neurokinin A and B, Neurotensin, Oxytocin and Gastrin (Erdos and Skidgel, 1989). NEP cleaves peptide chains on the amino side of hydrophobic residues. This has been demonstrated in GLP-1, in which a number of peptide bonds are cleaved by NEP. Notably, the Glu27-Phe28 and the Trp31-Leu32 bonds of GLP-1 were cleaved most readily (Hupe-Sodmann et al., 1995).

107

Another GLP-1 receptor agonist, Exendin-4, does not possess some NEP target sites present in GLP-1, yet, despite having some potential NEP degradation sites, it remains a poor substrate for the enzyme and this partly explains its extended half-life (Hupe-Sodmann et al., 1995, Goke et al., 1993). In addition, its larger size of 39 amino acids, and its greater propensity at forming a stable α-helix may contribute to it being a poor substrate for NEP. This demonstrates that properties of peptides other than sequence can influence a peptides susceptibility to degradation; properties such as tertiary structure which can block access of the enzyme to the substrate.

However, substituting amino acids susceptible to attack by peptidases may increase peptide hormone bioactivity. PYY and NPY have also been shown to be cleaved by NEP, suggesting that PP may also be processed by NEP (Medeiros Mdos and Turner, 1994, Medeiros and Turner, 1994, Medeiros Mdos and Turner, 1996). Establishing the importance of NEP in the clearance of PP may help the development of PP analogues with a long half-life.

3.1.5 TRYPSIN DEGRADATION

Parenteral routes of drug administration (IV, IM or SC) generally avoid the first-pass metabolism of the liver because it bypasses the GI tract. It introduces drugs directly across the body’s barrier defences and into systemic circulation. Unlike the IV route, IM and SC require absorption of the drug from the injection site and are therefore slower than IV administration. Absorption of drugs from an aqueous solution is fast, whereas absorption from depot preparations (as when the drug precipitates at the site of injection) is slow.

Skin consists of 3 layers: the epidermis, dermis and hypodermis (Fig 3.1). The location of subcutaneous administration is between the dermis (a matrix of connective tissue, blood vessels, nerves and lyphatics) and hypodermis (an insulating layer of connective tissue containing many adipose cells). Metabolism of a drug at this level can constitute first-pass metabolism which may result in sub-therapeutic doses reaching the systemic circulation. The metabolic activity of the skin enzymes may mean that different doses of the drug are delivered resulting in toxicity or apparent decreased effectiveness. Particular regard must therefore be paid to drugs of narrow therapeutic index.

108

Figure 3.1 Diagrammatic representation of the 3 layers which comprise the skin.

Despite enzyme activity within the skin approaching and sometimes exceeding that of the corresponding hepatic enzyme (Bashir S J and Maibach H. 1999) Cutaneous metabolism of xenobiotics. In RL Bronaugh and HI Maibach (Eds), Percutaneous absorption, Dekker, New York, 1999, pp65-85), first-pass effects of skin normally play a minor part in systemic drug metabolism (Benet, 1978) (Martin R et al. 1987 Int J Pharmaceutics). Nevertheless, if a subcutaneous depot is likely to form as a result of a controlled-release drug formulation, consideration should be made for the potential for enzymatic modification during this process. Indeed, slow release could lead to extensive first-pass metabolism whereas instant release could cause saturation of enzyme systems resulting in the availability of large amounts of drug going into systemic circulation.

Viable skin possesses enzyme activity that can hydrolyse peptides such as insulin which is rapidly degraded in the dermal side of rat skin. This degradation is strongly inhibited by PMSF (a serine protease inhibitor of enzymes such as trypsin and chymotrypsin) and phenanthroline (inhibitor of metalloenzymes such as aminopeptidases and endopeptidases) (Ogiso et al., 1997). Both inhibitors completely inhibit the proteolytic enzymes in skin and viable tissues where the peptides are metabolized, thus effectively abolishing degradation in the skin (Ogiso et al., 2000). Enzyme activities of cathepsin-B, collagenase-like peptidases, and DPPIV were also found to be high in rat subcutaneous tissue (Komada et al., 1985).

109

Trypsin cleaves peptide chains predominantly at the carboxyl side of the basic amino acids arginine or lysine, except when either is followed by proline (Ortiz et al., 2007) although there is evidence to suggest that cleavage occurs even with proline (Rodriguez et al., 2008).

Due to the ubiquity of trypsin in cells (cytoplasm, nucleus and nucleolar) (Hodges et al., 1973), extracellular matrix (Didangelos et al., 2010), and the nature of its characteristic cleavage sequence of amino acids, trypsin was an ideal enzyme to investigate when designing a long-acting PP analogue for subcutaneous administration.

3.1.6 DESIGN OF ENZYME-RESISTANT PEPTIDE ANALOGUES

A number of hormone analogues which are more resistant to enzymatic breakdown than the native forms have already been developed. One example is the GLP-1 analogue exendin- 4 that has a significantly increased half life compared to native GLP-1 (Goke et al., 1993). This is, at least, partly due to increased resistance to peptidases including NEP and DPPIV (Hupe-Sodmann et al., 1995). The oxyntomodulin analogue, OXM6421, has a longer half life than native OXM following peripheral administration (Druce et al., 2009, Liu et al., 2010), which correlates with the removal of sites susceptible to cleavage by peptidases including NEP and DPPIV. Using enzyme resistant analogues of gut hormones such as PP would allow a decrease in the frequency of administration while still maintaining receptor specificity and therefore reducing the risk of off-target effects.

110

3.1.7 HYPOTHESIS AND AIMS

The breakdown of native human PP will be investigated using hepatic microsomes and RBB membrane preparations, and a number of purified recombinant degradative enzymes to establish which amino acid sites are targeted.

Once sites identified for susceptibility to proteolytic enzymes are established, PP analogues with rational amino acids substitutions at these sites will be tested for their ability to withstand enzymatic degradation. Using efficacy studies, capability of receptor affinity, and measurement of peptide levels following a single administration, an assessment of the physiological relevance of analogues with increased resistance to degradation will be made.

3.1.7.1 Hypothesis

The substitution of amino acids at key sites targeted by hepatic microsomes, RBB membrane preparations, DPPIV, trypsin and neprilysin will produce PP analogues that are resistant to enzymatic degradation, maintain greater levels in circulation, and possess longer duration of action (bioefficacy) following a single administration.

3.1.7.2 Aims

1) To determine the effects on native PP of incubation with rat liver microsomes (RLM) and renal brush border (RBB) membrane, trypsin, DPPIV and neprilysin, using high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectroscopy.

2) To establish if amino acid substitutions can prevent degradation

3) To investigate if the changes made improve biological actions of the peptide by: a. Binding affinity at the human and mouse Y4 receptor b. Acute feeding studies in C57BL/6 mice c. Pharmacokinetic profile in vivo

111

3.2 RESULTS

3.2.1 CHARACTERISATION OF PP DEGRADATION BY TISSUE ENZYME PREPARATIONS

3.2.1.1 Investigation into RBB-mediated breakdown of PP (HPLC)

HPLC analysis of PP in RBB buffer alone showed a single peak with a retention time of 25minutes (RT=25m). Incubation of PP with RBB membrane in the same digest buffer at 37oC over a range of times from 15- to 60mins demonstrated a time dependent decrease in the parent peptide peak (Fig 3.2). The peak area of the parent molecule was reduced by 8% after 15 minutes incubation with RBB, 32% at 30 minutes and 67% after a 60 minute incubation with RBB at 37oC (Fig 3.2). In addition to the reduction in size of the primary PP peak, additional peaks with earlier retention times were observed. Five major UV-absorbing peaks were observed at retention times of 7.6, 8.5, 9.3, 11 and 14.7 minutes (Fig 3.2).

Figure 3.2 HPLC chromatogram of PP incubated in the presence of RBB at 37oC after various incubation times.

112

3.2.1.2 Identification of RBB target sites on PP (MALDI)

Analysis of PP incubated with RBB using MALDI-TOF MS indicated that there were a number of PP breakdown products detectable following a 10 minute or 60 minute incubation with RLM. At 10 minutes incubation of PP with RBB, shown in Fig 3.3(A), peaks with mass to charge ratios of 3259, 4015 and 4183 M/Z were detected. These masses are consistent with full length PP and the PP fragments 6-33/10-36 and PP3-36.

At 60 minutes incubation of PP with RBB, shown in Fig 3.3(B), peaks with mass to charge ratios of 1867, 3097, 3259, 3853, 4015 and 4183 M/Z were detected. These masses are consistent with full length PP and the PP fragments 23-36, 3-29/5-31, 6-33/10-36, 3-35 and PP3-36.

113

Observed Theoretical Δ mass PP

Mass (m/z) mass (Da) (Da) fragment A4183 4183 0 1-36 4015 4015 0 3-36 3853 3851 2 3-35 6-33 3259 3259 0 10-36 3096 -1 3-29 3097 3098 1 5-31 1867 1867 0 23-36

Peptide D fragment 1-36 3-36 3-35 6-33 10-36 3-29 5-31 23-36

Figure 3.3 MALDI MS analysis of PP in the presence of RBB for (A) 10mins or (B) 60mins at 37°C. (C) table (Table 3.1) of identified peaks correlated with PP fragments of known mass; (D) schematic diagram of PP fragments lined up against the corresponding PP sequence 114

3.2.1.3 HPLC analysis of PP degradation by RLM

HPLC analysis of the control sample PP reconstituted in RLM buffer showed a single peak with a retention time of 25minutes (RT=25m). Incubation of PP with RLM preparation in RLM buffer at 37oC over a range of times from 15-60minutes demonstrated a time dependent decrease in the parent peptide peak with the retention time of 25 minutes (Fig 3.4(A)). The peak area of the parent molecule was reduced by 8% after 15 minutes incubation with RLM, 10% at 30 minutes and 35% after a 60 minute incubation with RLM at 37oC (Fig 3.4 (C) and (D)). In addition to the reduction in size of the primary PP peak, additional peaks with earlier retention times were observed. Two major UV-absorbing peaks were observed at retention times of 16.5 and 21 minutes (Figure 2.1).

Figure 3.4 HPLC chromatogram of PP incubated in the presence of RLM at 37oC after (A) 0mins, (B) 15mins, (C) 30mins, and (D) 60mins incubation.

115

B 10 minutes

Observed Theoretical Δ mass Peak PP fragment A Mass (m/z) mass (Da) (Da) 1 4184 4183 1 1-36 2 4060 4052 8 1-35(2Met-O) 3 4022 4020 2 1-35 4 2758 2757 1 6-29 5 2601 2597 4 15-35 6 2094 2094 0 3-21 7 2068 2070 -2 14-30

C Peptide Peak fragment 1 1-36 2 1-35(2M-O) 3 1-35 4 6-29 5 15-35 6 3-21 7 14-30

Figure 3.5 MALDI MS analysis of PP in the presence of RLM for 10mins at 37°C. (A) MALDI spectra; (B) table (Table 3.2) of identified peaks correlated with PP fragments of known mass; (C) schematic diagram 116

of PP fragments lined up against the corresponding PP sequence MALDI-TOF analysis of PP incubated with RLM showed fewer peaks at 60 minutes compared to 10 minutes incubation. At 60 minutes, peaks with mass to charge ratios of 2030, 2068, 2105, 4016, and 4185 M/Z were detected (Fig 3.6(A)). These masses are consistent with PP and the PP fragments PP1-19, PP14-30, PP8-26 and PP1-35/3-36 (Fig 3.6 (B)).

The heaviest fragment (fragment 1 for both time points) corresponds to the molecular weight of full length PP(1-36) as shown in Fig 3.5 and 3.6. PP1-36 was present at both the 10 and 60 minute time points but at different peak intensities (100% and 20% respectively).

Peak 2 from both 10 and 60min PP digests corresponds to PP2-36 and is present at both time points however this peak exhibited the greatest mass difference (>50 Da) when compared to its theoretical mass. Due to this large mass difference, peak 2 from both time points could also correspond to PP1-35(2Met-O) where both Met residues are oxidised (expected mass of 4052 Da).

PP1-35 and PP3-36 are candidates for peak 3 from the 60 minute sample. PP1-35 also represents peak 3 from the 10 minute time point where it is observed with increased peak intensity.

Peak 4 of the 10 minute sample has 3 possible candidates: PP4-27, PP6-29, and PP15-36. They all have similar mass (±2 Da) however PP6-29 has the least difference compared to the m/z value (1 Da). The m/z value for peak 4 in the 10 minute sample has no equivalent in the 60 minute time point. Peak 5 from the 10 minute sample corresponds to PP15-35 and is also unique to this time point.

Peak 6 from the 10 minute sample corresponds to PP3-21 which matches exactly the theoretical mass. However, it is also similar to PP8-26 (mass difference 10 Da) which is present as peak 4 of the 60 minute sample. Peak 7 (10 minutes) and 5 (60 minutes) are common in both time points and corresponds to the mass of PP14-30. In both sample spectras, it is present in relatively similar peak intensities.

117

B 60 minutes

Observed Theoretical Δ mass Peak PP fragment A Mass (m/z) mass (Da) (Da) 1 4185 4183 2 1-36 2 4055 4052 3 1-35(2Met-O) 3 4016 4015 1 3-36 4 2106 2104 2 8-26 5 2068 2070 -2 14-30 C 6 2030 2028 2 1-19 Peptide Peak fragment 1 1-36 2 1-35(2M-O) 1-35 3 2-36 4 8-26 5 14-30 6 1-19

Figure 3.6 MALDI MS analysis of PP in the presence of RLM for 60mins at 37°C. (A) MALDI chromatogram; (B) table (Table 3.3) of identified peaks correlated with PP fragments of known mass; (C) schematic diagram of PP fragments lined up against the corresponding PP sequence 118

3.2.1.5 Identification of RLM target sites on PP – mass spectrometry of HPLC fractions

In an attempt to further characterise the breakdown products of PP after incubation with RLM, the digests were chromatographed by HPLC and discrete fractions (RT: 16-17m, 21- 22m, and 24-25m) collected and analysed by MALDI-TOF MS. Incubation of PP at 37C with reaction buffer alone for 60 minutes produced a single peak with an identical elution time (RT=25m) to that of freshly dissolved PP was detected. This single peak (RT=25m) was still observed when PP was incubated with RLM preparation for 60 minutes (Fig 3.7). This time point was chosen as there was still what appeared to be native PP present (represented by the peak at RT=25m) but also the appearance of extra peaks representing breakdown products by RLM incubation. RLM was also incubated in the absence of PP to serve as a control for the detection of peaks not associated with the peptide.

0.20 RLM alone PP - RLM 60mins PP + RLM 60mins 0.15 2) RT=21-22m 3) RT=24-25m 0.10 AU 1) RT=16-17m PP 0.05

0.00 5 10 15 20 25 30 Time

Figure 3.7 HPLC chromatogram of PP incubated with RLM at 37oC for 60mins

MALDI-TOF MS analysis of the RT=16-17m sample showed the presence of 4 major peaks corresponding to PP25-32/PP26-33 (equal mass), PP29-36, PP15-21, and PP6-11 (Table 3.4, Fig 3.8).

119

A B

(1) RT=16-17m

Observed Theoretical Δ mass PP APeak Mass (m/z) mass (Da) (Da) fragment 25-32 1 1067 1066 1 26-33 2 1051 1050 1 29-36 3 839 840 -1 15-21 4 666 664 2 6-11

PP Peak fragment 25-32 1 26-33 2 29-36 3 15-21 4 6-11

Figure 3.8 PP in the presence of RLM for 60mins at 37°C. After HPLC separation, this fraction (RT=16- 17m) was collected and MALDI MS analysis performed. (A) MALDI chromatogram; (B) table (Table 3.4) of identified peaks correlated with PP fragments of known mass; (C) schematic diagram of PP fragments lined up against the corresponding PP sequence 120

The fragments detected in sample 1 are schematically shown in Fig 3.8(C). Two are fragments of the C-terminal (PP25-32/PP26-33 (equal mass) and PP29-36), while two other fragments are from the N-terminal). Fragments PP25-32 and PP26-33 are indistinguishable by MALDI-TOF MS therefore, either or both of these species could be the result of PP degradation by RLM. Both fragments are the result of cleavage on either side of Arg25, Arg26 and Arg33. Incidentally, PP29-36 is part of the PP molecule just after the classic PP- fold conformation of the two stabilised helices (Fig 3.9).

Figure 3.9 Diagram of the PP sequence showing the tertiary structure with the stabilising bonds (represented by the dashed lines) between known residues. Pro2-Pro8 comprises the polyproline- like helix and Glu15-Thr32 makes up the α-helix. Gly9-Pro14 encompasses the β-turn. The grey suares indicate fragments identified by MALDI-MS from RLM digests of PP.

121

The second sample consisted of fractions RT=21-22m (Fig 3.7). Mass analysis of this sample showed the presence of 8 major peaks (Table 3.5, Fig 3.10).

The fragments detected in sample 2 are shown in the schematic diagram Fig 3.10(C) Peak 1 (PP8-31) corresponds to a mid-section of the PP molecule that encompasses the β-turn and α-helix (just after the polyproline-like helix). However this peak could also correspond to PP1-25(1Met-O) where Met17 is oxidised. Peak 2 (PP1-24) corresponds to the N-terminal region of PP after cleavage at the amino side of Arg25. This peak could also correspond to PP3-25(1Met-O) where Met17 is oxidised.

Peak 3 (PP14-26) is a section just after the β-turn of the PP molecule (induced by Pro14) to Arg26. Peak 4 has an m/z value which is similar to both fragments PP1-13 and PP17-27. PP1- 13 consists of the N-terminal region of PP in which the polyproline-like helix exists, while PP17-27 is made up of the mid-section α-helix.

Peak 5 has the highest signal intensity (Fig 3.10(A)) and also has two candidates: PP21-31 and PP24-33. Both fragments are C-terminal sections and are just variations of from the middle to the end of the α-helix.

Peaks 3-5 have the greatest signal intensity. Peaks 4 and 5 each have two possible candidates contributing to that signal whereas peak 3 has only one possible sequence (PP14-26). This could indicate PP14-26 as the main breakdown product of PP at RT=21-22m.

Peak 6 is PP29-36, a C-terminal fragment that was also found in the sample 1 (RT=16-17m). This was not expected as it is unusual for a fragment with same molecular weight to elute in two different HPLC fractions at different retention times. However, it could be due to the high concentration of peptide that was needed to run on the HPLC to obtain adequate signal for subsequent MALDI-TOF MS.

Peak 7 has two candidates: PP15-20 and PP17-23. Both peptide fragments occur at the beginning of the α-helix just after the β-turn. Finally peak 8 is a pentapeptide of PP19-23. With the frequency of fragments around position 23 of the PP molecule (both peak 7 and 8 of sample 2), this could either be a secondary breakdown product (the case with the shorter fragments) or could indicate a susceptibility to degradation at this point.

122

(2) RT=21-22m Observed Theoretical Δ mass A B PP fragment Peak Mass (m/z) mass (Da) (Da) 2739 -4 8-31 1 2735 A 2734 1 1-25(1Met-O) 2562 5 1-24 2 2567 2566 1 3-25(1Met-O) 3 1554 1549 5 14-26 1343 8 1-13 4 1351 1358 -7 17-27 -1 21-31 5 1335 1336 -1 24-33 6 1052 1050 5 29-36 -1 15-20 7 768 769 -1 17-23 C 8 567 567 0 19-23 PP Peak fragment 1 8-31 1-25(M-O) 2 1-24 3-25(M-O) 3 14-26 4 1-13 17-27 5 21-31 24-33 6 29-36 7 15-20 17-23 8 19-23 Figure 3.10 MALDI MS analysis of fraction RT=21-22m of PP in the presence of RLM for 60mins at 37°C. 123 (A) MALDI chromatogram; (B) table (Table 3.5) of identified peaks correlated with PP fragments of known mass; (C) schematic diagram of PP fragments lined up against the corresponding PP sequence The final sample consisted of fractions RT=24-25m (fig 3.7) which contained the peak for native PP. Mass analysis of this sample showed the presence of 5 major peaks (Table 3.6, Fig 3.11(A)) demonstrating that some PP breakdown products have equal RT as PP1-36 by HPLC separation.

The fragments detected in sample 3 are shown in the schematic diagram Fig 3.11(C). Peak 1, with an m/z value of 4056 does not closely correspond to any peptide fragment of PP (> ±5

Da). The nearest fragments from the MALDI spectra output correspond to PP2-36(desNH2) (Δ mass: -21 Da) and PP1-35 (Δ mass: +36 Da). However if the oxidation of methionine residues are taken into account, the fragment PP1-35(2Met-O) is a close match (a difference of -4Da) to peak 1.

Peak 2 is identified as PP24-34. Peak 3 has a similar m/z value to 4 PP fragments: PP21-31, and PP24-33 (C-terminal fragments), and PP13-24 and PP10-21 (mid-section fragments). As peak 3 has the highest intensity, it is possible that all 4 fragments contribute to this signal and MALDI MS cannot resolve the difference between these different species of fragments.

Peak 4 could correspond to PP10-20(1Met-O) which has a difference of 1Da, or PP5-16. Finally peak 5 only has a similar molecular weight to that of PP18-27 which comprises the α- helix of the PP molecule (entire α-helix spans position 15-32).

124

(3) RT=24-25m Δ A B Observed Theoretical mass PP fragment Mass (m/z) mass (Da) Peak (Da) 1 4056 4052 4 1-35(2MetO) 2 1433 1433 0 24-34 21-31 A 1336 1 24-33 3 1337 1337 0 13-24 1338 -1 10-21 1283 1 10-20(1MetO) 4 1284 1287 -3 5-16 5 1227 1226 1 18-27

C PP Peak fragment 1A1-35(2M-O) 2 24-34 3 21-31 24-33 13-24 10-21 4 10-20(M-O) 5-16 5 18-27

Figure 3.11 MALDI MS analysis of fraction RT=24-25m of PP in the presence of RLM for 60mins at 37°C. (A) MALDI chromatogram; (B) table (Table 3.6) of identified peaks correlated with PP fragments of 125 known mass; (C) schematic diagram of PP fragments lined up against the corresponding PP sequence 3.2.2 CHARACTERISATION OF PP DEGRADATION BY INDIVIDUAL RECOMBINANT ENZYMES

3.2.2.1 Investigations into the breakdown of PP by DPPIV

3.2.2.1.1 Breakdown of PP by DPPIV

Whole intact PP eluted at 21.8 min (RT=21.8m) on the 32-35% AcN over 45 min gradient used to examine DPPIV degradation. Following the incubation of PP at 37°C with DPPIV buffer alone for 120 minutes, a single peak with a retention time identical to that of freshly dissolved PP was detected (Fig 3.12). A peak at RT=21.8m was also observed when PP was incubated in the presence of DPPIV, and no secondary peaks were observed. There was no change in peak area between PP incubated in the presence or absence of DPPIV therefore percentage degradation could not be calculated.

0.15

no DPPIV 10mU DPPIV 20mU DPPIV 0.10 50mU DPPIV

0.05

18 19 20 21 22 23 24 25 Time (mins)

Figure 3.12 HPLC analysis of PP with 10, 20 and 50mU DPPIV for 2h at 37°C run on a shallow acetonitrile/water/0.05% TFA gradient of 31-35% acetonitrile over 45mins.

126

MALDI-ToF analysis of PP incubated with DPPIV indicated that the peptide eluting at the same position as native PP was not intact. Following incubation of PP alone at 37°C for 120mins, the single peak observed had a mass to charge ratio of 4188 m/z (Fig 3.13(A)) consistent with the molecular mass of full length native PP1-36 within the error of ± 5 Da. After a 120mins incubation of PP with 10mU DPPIV, the single peak detected by HPLC had a mass to charge ratio of 4014 m/z (Fig 3.13(B)) which is consistent with the theoretical mass of PP3-36.

A B

PP1-36 PP3-36

Figure 3.13 MALDI-TOF MS analysis of (A) PP alone and, (B) PP + 10mU DPPIV for 2h at 37°C

127

3.2.2.1.2 Effect of PP and DPPIV fragment PP3-36 on acute food intake in mice

PP and PP3-36 at 50nmol/kg both significantly reduced food intake in mice at 0-1 and 1-2h post injection (0-1h: saline: 0.71 ± 0.07g vs. PP: 0.19 ± 0.05g p < 0.01; vs. PP3-36: 0.41 ± 0.04g p < 0.05. 1-2h: p < 0.01 vs. saline for both peptides). Both peptides additionally reduced food intake during the 2-4h time period although this did not reach significance. Only PP significantly reduced food intake in the 4-8h interval (p < 0.01 compared with saline) (Fig 3.14). Over the entire 8h period, both PP and PP3-36 significantly reduced food intake (saline: 2.92 ± 0.29g vs. PP: 0.93 ± 0.10g p < 0.01; vs. PP3-36: 1.71 ± 0.29g p < 0.05). There were no significant changes in food intake at any other time point between PP and PP3-36

vs. saline control and between PP vs. PP3-36 at any time point studied (Fig 3.14).

1.4 4.0 saline 0-8 hour 1.2 3.5 PP (50nmol/kg) 1.0 PP3-36 (50nmol/kg) 3.0 2.5 0.8 2.0 * 0.6 ** * 1.5 Food intake (g) intake Food

0.4 (g) intake Food 1.0 ** *** 0.2 ** 0.5

0.0 0.0 0-1 1-2 2-4 4-8 PP saline Time (hours) PP3-36

Figure 3.14 The effect of SC administration of PP and PP3-36 (50nmol/kg) on food intake in C57/BL6 mice fasted overnight A) 0-1, 1-2, 2-4, 4-8 hour intervals post injection, B) 0-8 hours post injection. PP and PP3-36 compared to saline (*) using oneway ANOVA with Bonferroni post hoc test *p≤0.05, **p≤0.01,or *** p≤ 0.001 (n=7).

128

3.2.2.1.3 Effect of N-terminal modifications on DPPIV-mediated degradation

DPPIV-resistant analogues of PP were designed through N-terminal extension (PP-Ala0) and N-terminal truncation (PP2-36) to eliminate the DPPIV target sequence of proline at position 2 of the molecule. After a 120 minute incubation of each analogue with 10mU DPPIV, the single peaks detected (as determined by MALDI-TOF) had a mass to charge ratio of 4255 m/z which is consistent with the theoretical mass of PP-Ala0 (Fig 3.15(A)) and 4112 m/z (Fig 3.15(B)) which is consistent with the theoretical mass of PP2-36. These mass to charge ratios are consistent with the molecular mass of the respective analogues within an error of ± 5 Da.

A B

Figure 3.15 MALDI-TOF MS analysis of (A) PP-Ala0 + 10mU DPP4, and (B) PP2-36 + 10mU DPP4 for 2h at 37°C

129

3.2.2.1.4 Effect of N-terminal modifications on Y4 receptor affinity

PP bound to the hY4r with an IC50 of 0.26 ± 0.04nM. Both N-terminal modifications of PP had similar binding affinities which were approximately 1.3-fold worse (PP-Ala0 IC50: 0.35 ±

0.05nM and PP2-36 IC50: 0.31 ± 0.06nM) than that of the endogenous ligand (Fig 3.16).

PP IC : 0.26  0.04nM 100 50 PP-Ala0 IC50: 0.35  0.05nM

PP2-36 IC50: 0.31  0.06nM 80

Binding Specific % 20

0 -14 -13 -12 -11 -10 -9 -8 -7 -6 log [Peptide] M

Figure 3.16 Binding affinities of PP and analogues to human Y4r expressed as IC50 values calculated from the mean ± SEM of 3-5 separate experiments

130

3.2.2.1.5 Effect of N-terminal modifications on acute food intake in mice

Whilst a single SC injection of PP at 150nmol/kg did not result in a significant reduction of food intake at any time point, PP2-36 at the same dose, significantly reduced food intake at 2-4h post injection when compared with saline control (saline: 0.57 ± 0.05g vs. PP2-36: 0.19 ± 0.03g, at 2-4h p < 0.001).

PP2-36 significantly reduces food intake compared to the saline control over a 24h period (saline: 6.16 ± 0.22g vs. PP2-36: 4.98 ± 0.22g, at 0-24h p < 0.05). There were no significant changes in food intake at any time point between PP vs. saline control and PP vs. PP2-36 (Fig 3.17).

0-24 hour 6.5 1.2 saline

PP (150nmol/kg) 6.0 1.0 PP2-36 (150nmol/kg) 5.5 0.8 * 5.0 0.6 4.5 0.4

Food intake (g) intake Food 4.0 Food intake (g) intake Food *** 0.2 3.5

0.0 3.0

0-1 1-2 2-4 PP saline Time (hours) PP2-36

Figure 3.17 The effect of SC administration of PP and PP2-36 (150nmol/kg) on food intake in C57/BL6 mice fasted overnight A) 0-1, 1-2, 2-4 hour intervals post injection, B) 0-24 hours post injection. PP and PP2-36 compared to saline (*) using oneway ANOVA with Dunnett’s post hoc test *p≤0.05, **p≤0.01,or *** p≤ 0.001 (n=7).

131

PP and PP-Ala0 at 300nmol/kg both significantly reduced food intake in mice at 0-1 and 1-2h post injection (p < 0.001 vs. saline for both peptides). Both peptides additionally reduced food intake to a significant degree during the 2-4h time period (saline: 0.61 ± 0.05g vs. PP: 0.41 ± 0.04g p < 0.05; vs. PP-Ala0: 0.36 ± 0.06g p < 0.01). Only PP-Ala0 significantly reduced food intake over the whole 0-24h period (p < 0.01 compared with saline) (Fig 3.18). There were no significant changes in food intake at any other time point between PP and PP-Ala0

vs. saline control and between PP vs. PP-Ala0 at any time point studied (Fig 3.18).

1.0 saline 0-24 hour 7.0 PP (300nmol/kg) 0.8 PP-Ala0 (300nmol/kg) 6.5 *** ** 6.0 0.6

* ** 5.5 0.4

5.0 Food intake (g) intake Food *** (g) intake Food 0.2 4.5

0.0 4.0 0-1 1-2 2-4 PP saline Time (hours) PP-Ala0

Figure 3.18 The effect of SC administration of PP and PP-Ala0 (300nmol/kg) on food intake in C57/BL6 mice fasted overnight A) 0-1, 1-2, 2-4 hour intervals post injection, B) 0-24 hours post injection. PP and PP-Ala0 compared to saline (*) using oneway ANOVA with Dunnett’s post hoc test *p≤0.05, **p≤0.01,or *** p≤ 0.001 (n=7).

132

3.2.2.2 Investigations into the breakdown of PP by Trypsin

3.2.2.2.1 Breakdown of PP by trypsin

Trypsin is a highly active and specific protease which cleaves the C-terminal to lysine and arginine residues of a protein. The sequence below highlights the predicted digest sites of PP:

Following the incubation of PP at 37°C with trypsin buffer alone for 15 minutes, a single peak with a retention time identical to that of freshly dissolved PP was detected (Fig 3.19) by HPLC when run on an AcN of 15-60% over 35 minutes. The peak at retention time 24 minutes (RT=24m) was not observed when PP was incubated with 0.1mU of trypsin for 15 minutes. Instead, two major peaks were observed at the earlier retention times of 14 and 18minutes (called PPF1 and PPF2 respectively (Fig 3.19). These peaks were collected and analysed by MALDI-ToF mass spectrometry.

1.0 PPF2

0.8

PPF1 0.6

AU PP 0.4

0.2

0.0 5 10 15 20 25 Time (min)

Figure 3.19 HPLC analysis of PP with 0.1mU trypsin for 15mins at 37°C run on an acetonitrile/water/0.05% TFA gradient of 15-60% acetonitrile over 35mins.

133

MS analysis of PPF1 identified a peak with a mass to charge ratio of (1) 1642 and (2) 1165 m/z (Fig 3.20). A mass of 1462 is consistent with the molecular mass of the fragment PP25- 36. Several potential fragments of PP: 25-36, 27-35, 26-34, and 28-36 have a molecular mass of 1165 ± 5 Da (Table 3.7). The presence of trypsin-sensitive Arg residues at position 25, 26, 33, and 35 confirm that these fragments are plausible degradation products.

PPF1

Observed Theoretical Δ mass PP Peak Mass (m/z) mass (Da) (Da) fragment 1 1642 1639 3 25-36 27-35 1163 -2 26-34 2 1165 28-36 1166 1 16-25

Figure 3.20 MALDI MS analysis of fraction PPF1 of PP in the presence of trypsin for 15mins at 37°C and Table 3.7 of identified peaks correlated with PP fragments of known mass.

134

MS analysis of PPF2 showed four peaks with mass to charge ratio of (1) 2875, (2) 1958, (3) 1481 and (4) 1165 m/z (Fig 3.21). These peaks were consistent with the molecular mass of fragments PP1-26 for peak 1, PP2-19 and PP16-31 for peak 2, PP26-36 for peak 3, and fragments PP16-25, 26-34, 27-35, and 28-36 for peak 4. All candidate fragments were within the error of ± 5 Da (Table 3.8)

PPF2 Peak Observed Theoretical Δ mass PP Mass (m/z) mass (Da) (Da) fragment 1 2875 2874 1 1-26 2-19 2 1958 1957 1 16-31 3 1481 1483 -2 26-36 1166 -1 16-25 26-34 4 1165 1163 2 27-35 28-36

Figure 3.21 MALDI MS analysis of fraction PPF2 of PP in the presence of trypsin for 15mins at 37°C and Table 3.8 of identified peaks correlated with PP fragments of known mass.

135

3.2.2.2.2 Effect of modifications at trypsin-target sites on Y4 receptor affinity

PP bound to the hY4r with an IC50 of 0.23 ± 0.03nM and PP-Lys25 had an IC50 which was approximately 1.5-fold worse (PP-Lys25 IC50: 0.36 ± 0.03nM) than that of the endogenous ligand (Table 3.9). However, substituting the native Arg25 with Ala25 produced an analogue which bound 3-fold worse at the hY4r.

Similar substitutions at position 26 with positive amino acids (Lys26 and His26) bound with affinities 2-fold worse to the hY4r (PP-Lys26 IC50: 0.47 ± 0.04nM and PP-His26 IC50: 0.49 ±

0.01nM) as did the hydrophobic substitution of PP-Ala26 (IC50: 0.45 ± 0.03nM) (Table 3.9).

Double substitutions at position 25-26 from the native Arg-Arg to Gln25,26 or His25,26 decrease binding affinity 5 fold (PP-Gln25,26 IC50: 1.10 ± 0.01nM and PP-His25,26 IC50: 1.21 ± 0.14nM).

Substitutions at position 33 from native Arg to a similarly positive amino acid Lys33 decreased the binding affinity 2-fold however, an Ala33 substitution, decreased binding affinity 38-times (PP-Lys33 IC50: 0.42 ± 0.09nM and PP-Ala33 IC50: 8.80 ± 0.70nM).

Finally, substitutions at position 35 with either Lys35 or His35 only had a 2.5-3-fold decrease in binding affinity to the hY4r (PP-Lys35 IC50: 0.57 ± 0.01nM, PP-His35 IC50: 0.72 ± 0.17nM), while an Ala35 substitution has a 10-fold decrease in binding affinity. Combination substitutions at positions 26 and 35 decreased binding affinity by 28.5-fold (PP-Ala26,His35

IC50: 6.60 ± 0.40nM).

136

hY4r SEM IC (analogue) Peptide 50 IC [nM] [nM] IC (PP) 50 50 PP 0.23 0.03 1 Lys25 0.36 0.03 1.5

Ala25 0.69 0.02 3 Lys26 0.47 0.04 2

His26 0.49 0.01 2 Ala26 0.45 0.03 2

Gln25,26 1.10 0.01 5 His25,26 1.21 0.14 5

Lys33 0.42 0.09 2 Ala33 8.80 0.70 38 Lys35 0.57 0.01 2.5 His35 0.72 0.17 3 Ala35 2.20 0.19 10 Ala26,His35 6.60 0.40 29

Table 3.9 Binding affinities of PP and analogues to human Y4r expressed as IC50 values calculated from the mean ± SEM of 3-5 separate experiments. Affinity ratios were then used to demonstrate relative affinity to the Y4r compared to native PP.

3.2.2.2.3 Breakdown of PP analogues by trypsin

As demonstrated in section 3.4.1, PP can be degraded by trypsin in an in vitro setting. Analogues with substitutions at the residues known to be possible cleavage points by trypsin were made. Incubation of PP-Lys25 (Fig 3.22(B)) and PP-Lys26 (Fig 3.22(C)) with 0.1mU of trypsin for 15 minutes resulted in the complete degradation of the peptide into two detectable fragments as determined by HPLC. However, substitution of both position 25 and 26 (as with PP-Gln25,26) results in 100% retention of the original peak of the compound not treated with trypsin (Fig 3.22(D)).

137

PP alone PP-Lys25 PP + trypsin PP-Lys25 + trypsin A B

5 10 15 20 25 5 10 15 20 25 30 Time (mins) Time (mins)

PP-Gln25,26 PP-Lys26 PP-Gln25,26 + trypsin PP-Lys26 + trypsin D C

5 10 15 20 25 30 5 10 15 20 25 30 35 Time (mins) Time (mins)

Figure 3.22 HPLC analysis of (A) PP, (B) PP-Lys25, (C) PP-Lys26 and (D) PP-Gln25,26 with 0.1mU trypsin for 15mins at 37°C run on an acetonitrile/water/0.05% TFA gradient of 15-60% acetonitrile over 35mins.

138

3.2.2.2.4 Effect of modifications at trypsin-target sites on acute food intake in mice

A single SC injection of PP at 150nmol/kg did not significantly reduce food intake at 0-1 and 1-2h time points, however it did reduce food intake significantly at 2-4 and 4-8h post injection when compared with saline control (saline: 0.51 ± 0.05g vs. PP: 0.21 ± 0.05g, at 2- 4h p < 0.01 and saline: 0.77 ± 0.04g vs. PP: 0.50 ± 0.02g, at 4-8h p < 0.05). PP-Lys26 at the same dose, significantly reduced food intake at 0-1 and 2-4h post injection when compared with saline control (saline: 0.67 ± 0.05g vs. PP-Lys26: 0.32 ± 0.11g, at 0-1h p < 0.001; saline: 0.51 ± 0.05g vs. PP-Lys26: 0.31 ± 0.03g, at 2-4h p < 0.05). PP-Lys25, also at the same dose, only significantly reduced food intake at the 0-1h time point (saline: 0.67 ± 0.05g vs. PP- Lys25: 0.42 ± 0.10g, at 0-1h p < 0.05).

Overall, there were no significant differences in food intake over a 24h period compared to the saline control (Fig 3.23).

1.0 saline 6.0 0-24 hour A PP (150nmol/kg) B PP-Lys26 (150nmol/kg) 0.8 5.5 PP-Lys25 (150nmol/kg) 5.0 0.6 * * 4.5 0.4 *** * 4.0 Food intake (g) intake Food ** (g) intake Food 0.2 3.5

0.0 3.0 0-1 1-2 2-4 4-8

Time (hours)

Figure 3.23 The effect of SC administration of PP, PP-Lys25 and PP-Lys26 (150nmol/kg) on food intake in C57BL/6 mice fasted overnight. (A) Food intake at 0-1, 1-2, 2-4, 4-8 hour intervals post- injection, (B) 0-24 hours post-injection. PP, PP-Lys25 and PP-Lys26 compared to saline (*) using oneway ANOVA with Dunnett’s post hoc test *p≤0.05, **p≤0.01,or *** p≤ 0.001 (n=8-9).

139

As the receptor affinity of analogues with substitutions at position 25 or 26 decreased by 2-3 fold (with Ala substitutions in particular), acute feeding studies using doses double that of PP were conducted. A single SC injection of PP at 150nmol/kg caused a significant reduction of food intake at the 0-1 and 1-2h time point when compared with saline control (saline: 1.06 ± 0.04g vs. PP: 0.85 ± 0.08g, at 0-1h p < 0.05; saline: 0.86 ± 0.04g vs. PP: 0.11 ± 0.06g, at 1-2h p < 0.001). Both analogues at 300nmol/kg also significantly reduced food intake at 0-1 and 1-2h time points when compared with the saline control group; however PP-Ala26 reduced food intake to a greater degree in the 1-2h time point compared with PP-Ala25 (PP- Ala26: 0.18 ± 0.05g vs. PP-Ala25: 0.50 ± 0.11g, at 1-2h p < 0.05)

Over the 0-24h time period, only PP and PP-Ala26 reduced food intake significantly when compared to the saline control group. PP-Ala25 did not have a significant change in food intake compared to any of the groups, at any of the time intervals measured except when compared against PP at 0-24h (PP: 5.36 ± 0.14g vs. PP-Ala25: 6.46 ± 0.06g, at 0-24h p < 0.01) (Fig 3.24)

saline 0-24 hour 1.2 PP (150nmol/kg) 7.0 $$ PP-Ala26 (300nmol/kg) 1.0 6.5 * PP-Ala25 (300nmol/kg) * *** # 6.0 0.8 *** $$ 5.5 ** 0.6 5.0

4.5 0.4 Food intake (g) intake Food Food intake (g) intake Food 4.0 *** 0.2 3.5

0.0 3.0

0-1 1-2 PP saline Time (hours) PP-Ala26PP-Ala25

Figure 3.24 The effect of SC administration of PP (150nmol/kg), PP-Ala25 and PP-Ala26 (300nmol/kg) on food intake in C57BL/6 mice fasted overnight. (A) Food intake at 0-1 and 1-2 hour intervals, (B) 0-24 hours post-injection. PP, PP-Ala25 and PP-Ala26 compared to saline (*) using one- way ANOVA with Bonferroni’s post hoc test *p≤0.05, **p≤0.01,or *** p≤ 0.001 (n=8-9).

140

To assess the effect of the type substitution at position 26, PP-His26 was tested against PP but again at double the dose as the receptor binding affinity was decreased by 2-3 fold. A single SC injection of PP at 150nmol/kg caused a significant reduction of food intake at the 0-1 and 1-2h time point when compared with saline control (saline: 0.84 ± 0.06g vs. PP: 0.53 ± 0.04g, at 0-1h p < 0.001; saline: 0.37 ± 0.07g vs. PP: 0.05 ± 0.04g, at 1-2h p < 0.001). PP- His26 at 300nmol/kg also significantly reduced food intake at 0-1 and 1-2h time points when compared with the saline control group (saline: 0.84 ± 0.06g vs. PP-His26: 0.64 ± 0.05g, at 0- 1h p < 0.05; saline: 0.37 ± 0.07g vs. PP-His26: 0.04 ± 0.04g, at 1-2h p < 0.001).

Over the 0-24h time period, both PP and PP-His26 reduced food intake significantly when compared to the saline control group (saline: 5.78 ± 0.18g vs. PP: 5.25 ± 0.17g and PP-His26: 5.03 ± 0.16g at 0-24h p < 0.001) (Fig 3.25).

6.0 B 1.0 saline A PP (150nmol/kg) PP-His26 (300nmol/kg) 0.8 5.5 *** *

0.6 *** 5.0

0.4

Food intake (g) intake Food Food intake (g) intake Food 4.5 0.2 ***

0.0 4.0 0-1 1-2 0-24 hour

Time (hours)

Figure 3.25 The effect of SC administration of PP (150nmol/kg) and PP-His26 (300nmol/kg) on food intake in C57BL/6 mice fasted overnight. (A) Food intake at 0-1 and 1-2 hour intervals, (B) 0-24 hours post-injection. PP and PP-His26 compared to saline (*) using one-way ANOVA with Bonferroni’s post hoc test *p≤0.05, **p≤0.01,or *** p≤ 0.001 (n=9).

141

To assess joint substitutions at position 25-26, PP-His25,26 was tested against PP. The effect of substitution at position 35 was also assessed. A single SC injection of PP at 300nmol/kg caused a significant reduction of food intake at 1-2h time point when compared with saline control (saline: 0.46 ± 0.07g vs. PP: 0.09 ± 0.03g, at 1-2h p < 0.01). PP-His25,26 at 300nmol/kg only significantly reduced food intake at the 0-1h time point when compared with the saline control group (saline: 1.14 ± 0.08g vs. PP-his25,26: 0.82 ± 0.06g, at 0-1h p < 0.01) (Fig 3.26(A))

Over the 0-24h time period, only PP reduced food intake significantly when compared to the saline control group (saline: 6.01 ± 0.16g vs. PP: 5.36 ± 0.16g at 0-24h p < 0.05) (Fig 3.26(B)).

PP-His35 did not significantly reduce food intake at any of the time intervals measured.

1.4 6.5 saline B A PP (300nmol/kg) 1.2 PP-His35 (300nmol/kg) 6.0 1.0 PP-His25,26 (300nmol/kg) ** 5.5 * 0.8

0.6 5.0

(g) intake Food Food intake (g) intake Food 0.4 4.5 0.2 ** 0.0 4.0

0-1 1-2 0-24 hour

Time (hours)

Figure 3.26 The effect of SC administration of PP, PP-His35, and PP-His25,26 (300nmol/kg) on food intake in C57BL/6 mice fasted overnight. (A) Food intake at 0-1 and 1-2 hour intervals, (B) 0-24 hours post-injection. PP, PP-His35, and PP-His25,26 compared to saline (*) using one-way ANOVA with Bonferroni’s post hoc test *p≤0.05, **p≤0.01,or *** p≤ 0.001 (n=9).

142

To assess the effect of the type of joint substitution at position 25-26, PP-Gln25,26 was tested against PP. Alanine substitution at position 35 was also assessed. PP at 150nmol/kg did not cause a significant reduction of food intake at any of the time intervals measured. PP-Gln25,26 at 150nmol/kg only significantly reduced food intake at the 0-1h time point when compared with the saline control group (saline: 0.90 ± 0.09g vs. PP-Gln25,26: 0.52 ± 0.05g, at 0-1h p < 0.01).

Over the 0-24h time period, none of the peptides tested reduced food intake significantly when compared to the saline control group (Fig 3.27)

PP-Ala35 at 600nmol/kg (4-times higher dose than PP), did not significantly reduce food intake at any of the time intervals measured. However, it did cause a significant increase in food intake at the 1-2h time point when compared with the saline control group (saline: 0.14 ± 0.03g vs. PP-His26: 0.33 ± 0.08g, at 1-2h p < 0.05).

1.2 saline A 6.5 PP (150nmol/kg) B 1.0 PP-Gln25,26 (150nmol/kg) PP-Ala35 (600nmol/kg) 6.0 0.8 5.5 0.6 ** * 5.0 0.4 Food intake (g) intake Food Food intake (g) intake Food 0.2 4.5

0.0 4.0

0-1 1-2 0-24 hour

Time (hours)

Figure 3.27 The effect of SC administration of PP and PP-Gln25,26 (150nmol/kg), and PP-Ala35 (600nmol/kg) on food intake in C57BL/6 mice fasted overnight. (A) Food intake at 0-1 and 1-2 hour intervals, (B) 0-24 hours post-injection. PP, PP-Gln25,26, and PP-His35 compared to saline (*) using one-way ANOVA with Dunnett’s post hoc test *p≤0.05, **p≤0.01,or *** p≤ 0.001 (n=8-9).

143

To assess the effect of the type of substitution at position 35, PP-Lys35 was tested against PP. Alanine substitution at position 33 was also assessed, as was the effect of a similarly charged amino acid substitution (PP-Lys33). PP at 150nmol/kg caused a significant reduction of food intake at 0-1 and 2-4h time points (saline: 0.91 ± 0.08g vs. PP: 0.67 ± 0.05g, at 0-1h p < 0.05; saline: 0.65 ± 0.07g vs. PP: 0.24 ± 0.04g, at 2-4h p < 0.001). All PP analogues tested at 300nmol/kg significantly reduced food intake at the 0-1h time point when compared with the saline control group (saline: 0.91 ± 0.08g vs. PP-Ala33: 0.55 ± 0.05g; PP-Lys35: 0.55 ± 0.05g; PP-Lys33: 0.54 ± 0.05g at 0-1h p < 0.001). At the 2-4h time point, the only analogue that still significantly reduced food intake was PP-Ala33 (saline: 0.65 ± 0.07g vs. PP-Ala33: 0.39 ± 0.06g at 2-4h p < 0.05). As with PP-Ala35, PP-Lys35 also significantly increased food intake at the 1-2h time point when compared with the saline control group (saline: 0.09 ± 0.03g vs. PP-Lys35: 0.21 ± 0.05g, at 1-2h p < 0.05) (Fig 3.28(A)).

Over the 0-24h time period, none of the peptides tested significantly reduced, or increased, food intake significantly when compared to the saline control group (Fig 3.28(B)).

1.2 saline 6.5 A PP (150nmol/kg) B PP-Ala33 (300nmol/kg) 1.0 PP-Lys35 (300nmol/kg) 6.0 PP-Lys33 (300nmol/kg) 0.8 * 5.5 *** 0.6 * 5.0 0.4 * Food intake (g) intake Food Food intake (g) intake Food *** * 4.5 0.2

0.0 4.0

0-1 1-2 2-4 0-24 hour

Time (hours)

Figure 3.28 The effect of SC administration of PP (150nmol/kg), PP-Ala33, PP-Lys35, and PP-Lys33 (300nmol/kg) on food intake in C57BL/6 mice fasted overnight. (A) Food intake at 0-1, 1-2, 2-4 hour intervals, (B) 0-24 hours post-injection. PP, PP-Ala33, PP-Lys35, and PP-Lys33 compared to saline (*) using one-way ANOVA with Dunnett’s post hoc test *p≤0.05, **p≤0.01,or *** p≤ 0.001 (n=9).

144

3.2.2.3 Investigations into the breakdown of PP by neprilysin (NEP)

3.2.2.3.1 Breakdown of PP by NEP

Neprilysin has many targets but structural studies show that it preferentially cleaves on the amino side of hydrophobic amino acid residues from internal sites of peptides. The sequence below highlights the hydrophobic amino acids of PP:

Freshly reconstituted PP peptide produced a single peak with a retention time of 28mins (RT=28m) when analysed by HPLC. Incubation of PP at 37°C with NEP buffer alone for 120 minutes also produced a single peak only. When PP was incubated with the NEP enzyme for increasing amounts of time between 15 and 120 minutes (Fig 3.29), there is a reduction in size of the peak at RT=28m as well as the appearance of additional peaks with earlier retention times.

By comparing the area of the peak at RT=28m from a control sample of PP under the same conditions but not containing NEP enzyme, PP incubated with NEP for 15 minutes was broken down by 4%. At 45 minutes this had increased to 20% breakdown, and at 60 minutes to 25% (Fig 3.29). By 120 minutes, there was approximately 47 ± 3.5% degradation of PP.

In addition to the peak at RT=28m, five major UV-absorbing peaks could be observed following incubation of PP with 200ng of NEP for 120 minutes. These fractions had retention times of (1) 11.8, (2) 13.8, (3) 14.5, (4) 16.7 and (5) 17.5 minutes (Fig 3.29).

145

Figure 3.29 HPLC analysis of PP with 200ng of NEP for 15 (black), 45 (red), 60 (blue), and 120mins (green) at 37°C run on an acetonitrile/water/0.05% TFA gradient of 15-55% acetonitrile over 35mins.

A complete digest of PP by NEP was analysed by MALDI-MS (Fig 3.30(A)) where just 1 fragment was detected (Table 3.10) at both 30m and 120m incubations. From the analysis of whole digest, position 3 and 22 appear to be a common target sites as demonstrated by the generation of fragment 3-21.

146

A B

A Observed Theoretical Δ mass PP Mass (m/z) mass (Da) (Da) fragment 4183 4183 0 1-36 2092 2094 2 3-21

Peptide fragment 1-36 3-21

Figure 3.30 MALDI MS analysis of PP in the presence of NEP for (A) 30 and 120mins at 37°C. (B) table (Table 3.10) of identified peaks correlated with PP fragments of known mass; (C) schematic diagram of PP fragments lined up against the corresponding PP sequence

147

When individual fractions of PP digests by NEP were analysed by MALDI-MS, fraction RT=12- 15m (Fig 3.29) generated 2 major peaks: PP5-10 (647 Da) and PP12-26 (1721 Da) or PP7-22 (1727 Da). MALDI-MS analysis of fraction RT=17-19m (Fig 3.29) generated 3 major peaks: both the fragments present in RT=12-15m with the addition of fragment PP1-12 (1242 Da). Based on the predictions of target sites in the PP molecule and the fragments identified from the NEP digest of PP, position 6 could be a potential NEP substrate site as demonstrated by the appearance of fragment 5-10. Other susceptible sites of NEP degradation on the PP molecule are positions 7, 12, 22, and 27. These are in addition to those NEP target sites identified in whole digests (position 3 and 22).

148

A B Observed Theoretical Δ mass PP

A Mass (m/z) mass (Da) (Da) fragment 1721 3 12-26 1724 1727 -3 7-22 1242 1242 2 1-12 648 647 1 5-10

Peptide C fragment 12-26 7-22 1-12 5-10

Figure 3.31 MALDI MS analysis of RT=17-19m in the presence of NEP for (A) 120mins at 37°C. (B) table (Table 3.11) of identified peaks correlated with PP fragments of known mass; 149 (C) schematic diagram of PP fragments lined up against the corresponding PP sequence 3.2.2.3.2 Effect of NEP inhibition on acute food intake in mice

At a dose of 150nmol/kg, PP did not significantly reduced of food intake at any time point measured. Phosphoramidon alone also did not cause a significant reduction of food intake at any of the time intervals measured.

PP in combination with phosphoramidon significantly reduced food intake from 8-48h period when compared to saline (saline 24h: 3.47 ± 0.22g vs. PP+phosphoramidon 24h: 2.60 ± 0.18g, p>0.01). PP + phosphoramidon significantly decreased food intake compared to PP alone at 48h post-injection (PP 48h: 6.52 ± 0.14g vs. PP+phosphoramidon 48h: 5.63 ± 0.21g, p>0.01) (Fig 3.32).

7 Saline PP 150nmol/kg 6 Phosphoramidon 5mg/kg PP + Phosphoramidon 5 **$$ ##

2 ***$$$

cumulative food intake (g) intake food cumulative

1 * significance vs saline $ significance vs phosphoramidon # significance vs PP 0 ** 012 4 8 24 48 Hours

Figure 3.32 The effect of SC administration of PP (150nmol/kg), phosphoramidon (5mg/kg), and PP + phosphoradmidon on food intake in C57BL/6 mice fasted overnight. Cumulative food intake shown up to 48 hours post-injection. Significance analysed using two-way ANOVA with Bonferroni’s post hoc test *p≤0.05, **p≤0.01, or *** p≤ 0.001 (n=9).

150

3.2.2.3.3 Pharmacokinetics of PP in mice

The Cmax observed following a single SC injection of 1000nmol/kg PP resulted in a Cmax of 36769 ± 7861pmol/L at 30minutes post injection (Fig 3.33). Plasma levels of PP decreased by

23% from Cmax by 1h (28169 ± 5285pmol/L). PP plasma levels were significantly reduced at the 2, 4 and 8h compared to Cmax (2h: 7477 ± 5270pmol/L; 4h: 6528 ± 5200pmol/L; 8h: 1513

± 654pmol/L; 80-96% reduction from Cmax). Plasma levels of PP were also significantly reduced at the 2, 4 and 8h compared to levels at 1h (respectively 73, 77 and 95% reduction from plasma levels of PP at 1h).

$$$ $$$ $$$ 50000 * ## 40000 ## ##

30000

20000

PP (pmol/L) PP 10000

0 Plasma concentrationPlasma of

1hr 2hr 4hr 8hr basal 30mins * significance vs basal $ significance vs PP plasma level at 30mins # significance vs PP plasma level at 1h

Figure 3.33 Pharmacokinetic profile of a single subcutaneous injection of PP at 1000nmol/kg to male C57BL/6 mice. Basal plasma samples were collected from naïve mice. Latter plasma samples were collected at 30minutes, 1, 2 4 and 8h post injection (n=11). Data are shown as mean plasma levels of PP ± SEM as determined by RIA. LLOD: 40pmol/L. Significance analysed using one-way ANOVA with Bonferroni’s post hoc test *p≤0.05, **p≤0.01, or *** p≤ 0.001.

151

3.2.2.3.4 Effect of phosphoramidon on plasma levels of PP in mice

The NEP inhibitor, phosphoramidon, was administered at 3 different doses as a single IP injection 15minutes prior to SC administration of PP. Following the SC injection of 150nmol/kg of PP, plasma levels were measured at 45 and 90minues post-injection. Mice given PP alone (no phosphoramidon) were given an equal volume IP injection of vehicle 15 minutes prior to peptide administration.

Levels of PP after 45mins (Fig 3.34(A)) did not differ significantly between PP administered alone (25480 ± 2053pmol/L) and PP in combination with any dose of phosphoramidon (7mg/kg: 30233 ± 4752pmol/L; 20mg/kg: 24075 ± 1564pmol/L; 60mg/kg: 27140 ± 2993pmol/L). After 90mins (Fig 3.34(B)), plasma levels of PP were significantly higher when given in combination with any dose of phosphoramidon compared to PP given alone (PP no phosphoramidon: 1920 ± 134pmol/L vs PP + phosphoramidon at 7mg/kg: 5460 ± 807pmol/L; 20mg/kg: 5297 ± 461pmol/L; 60mg/kg: 5525 ± 348pmol/L). Across all doses of phosphoramidon, plasma levels of PP at 90mins were significantly lower compared to PP levels at 45mins (Fig 3.34(C)).

152

40000 8000 A B ** 30000 * * 6000

20000 4000

[PP] pmol/L 10000 [PP] pmol/L 2000

0 0 0 7 20 60 0 7 20 60 Dose of phosphoramidon (mg/kg) Dose of NEP Inhibitor (mg/kg)

40000 C 45m 20000 90m 8000 *** 6000 ** **

4000 [PP] pmol/L 2000 ***

0 0 7 20 60 Phosphoramidon (mg/kg)

Figure 3.34 Plasma levels of PP following a single subcutaneous injection of PP at 150nmol/kg to male C57BL/6 mice in combination with a single IP injection of phosphoramidon at 7mg/kg, 20mg/kg and 60mg/kg. Blood samples were collected at (A) 45 and (B) 90 minutes post-injection (n=2-4). Data is shown collectively in graph (C). Data are shown as mean plasma levels of PP ± SEM as determined by RIA. LLOD: 70pmol/L. Significance analysed using one-way ANOVA (graphs (A) and (B)) and two- way ANOVA (graph (C)), both with Bonferroni’s post hoc test *p≤0.05, **p≤0.01, or *** p≤ 0.001

153

3.2.3 EFFECT OF PP MODIFICATIONS ON RBA, NEP DIGESTS, ACUTE FEEDING AND PHARMACOKINETICS

Susceptibility of PP analogues to degradation by NEP was measured using the method previously described (section 2.8.3). Samples of each PP analogue in the presence and absence of enzyme was analysed by HPLC after incubation at 37°C for 1h. Peptide peak areas were measured and percentage degradation calculated as:

100 - peak area + NEP x 100% peak area no NEP

Initially, PP analogues with single substitutions were screened against binding affinity to the Y4r, NEP degradation, and in vivo efficacy (Table 3.12). N-terminal modifications (which included N-terminal extensions, truncation, and dimerisation) generally decreased Y4r affinity by 1.2-1.7 fold. Extension of His0 increased Y4r affinity the most (IC50 PP-His0

0.16nM vs 0.23nM PP) and the Cys0-dimer of PP reduced Y4r affinity the greatest (IC50 0.58nM). The N-terminal modification that reduced degradation by NEP the most was His0 and Cys0-dimer (5% and 0% degradation respectively), followed closely by Pro0, Thr0, Asp0, Glu0, and the N-terminal truncation Des-Ala1 (Table 3.12). PP analogues Ala0, Ala-1,0, Gly0, and Lys0 did not greatly alter PP susceptibility to NEP degradation.

In vivo bioefficacy as measured by acute feeding studies was calculated as below for each time point, where the time points measured are 0-4h, 4-8h, and 8-24h:

Ratio of food intake of Food intake (PP) analogue compared to PP = Food intake (analogue)

An average of the ratios from all 3 time points are then expressed as the mean in vivo bioefficacy compared to PP (Table 3.12).

154

In vivo bioefficacy in mice for PP analogues with N-terminal modifications was generally well-maintained but was unfavourable with His0 and Lys0 N-terminal extensions (respectively 0.5- and 0.1-times the bioefficacy of native PP). Pro0 extension increased bioefficacy 2-fold while the Cys0-dimer increased bioefficacy in mice 3-fold.

Ratio of bioefficacy IC (analogue) % NEP Analogue 50 (as measured by food intake) IC (PP) degradation 50 compared to PP over 24h Native PP 1 47 1

Des-Ala1 1.4 23 1.4 Ala0 1.6 30 1.0 Ala-1,0 1.2 33 1.0

Asp0 1.6 22 1.2 Gly0 1 33 1.5

Glu0 1.5 23 1.2 His0 0.7 5 0.5 Lys0 0.9 43 0.1 Pro0 0.9 13 2.0 Thr0 1.7 19 1.0 Cys0 (dimer) 2.5 0 3.0

Table 3.12 Position 0 (N-terminal) modifications of PP and the effects on hY4r affinity compared to native PP which has a ratio of 1. Ratios <1 bind better to the Y4r relative to native PP and ratios >1 bind have lower affinity. The potency ratio is the effect on the reduction of food intake by native PP normalised to 1. Ratios <1 have a weaker effect on the reduction of food intake compared to native PP over a 24h period while ratios >1 have a stronger effect on the reduction of food intake which is proportional to the number given (e.g. ratio of 3 is a reduction in food intake over 24h which is 3- times greater than native PP at the same dose). % NEP degradation is calculated as the above equation.

155

Another example to demonstrate that protecting against DPPIV prolongs bioefficacy in acute feeding studies, PP at 300nmol/kg and PP-Pro0 at 150nmol/kg both significantly reduced food intake in mice at 0-1, 1-2, 2-4, and 4-8h post injection (p ≤ 0.05 vs. saline for both peptides). Only PP-Cys0 dimer at 100nmol/kg significantly reduced food intake over the whole 0-24h period (p ≤ 0.001 compared with saline) (Fig 3.35).

6.5 1.2 saline B A PP (300nmol/kg) 6.0 1.0 PP-Pro0 (150nmol/lg) PP-Cys0dimer (100nmol/kg) * 5.5 0.8 ** *** 5.0 0.6 *** 4.5

0.4

*** (g) intake Food Food intake (g) intake Food * 4.0 ** 0.2 *** 3.5 ** 0.0 *** 3.0

0-1 1-2 2-4 4-8 0-24 hour

Time (hours)

Figure 3.35 The effect of SC administration of PP (300nmol/kg), PP-Pro0 (150nmol/kg) and PP-Cys0 dimer (100nmol/kg) on food intake in C57BL/6 mice fasted overnight A) 0-1, 1-2, 2-4 and 4-8 hour intervals post injection, B) 0-24 hours post injection. PP, PP-Pro0, and PP-Cys0 dimer were compared to saline (*) using oneway ANOVA with Dunnett’s post hoc test *p≤0.05, **p≤0.01,or *** p≤ 0.001 (n=9).

156

Single modifications at position 30 were also screened against Y4r affinity, NEP degradation, and in vivo efficacy (Table 3.13). Changes at position 30 were generally well tolerated at Y4r affinity with Lys30 being the most favourable substitution and Glu30 and Leu30 being the worst (1.6- and 1.8-fold worse than PP respectively). Overall, changes at position 30 alone did not alter susceptibility to degradation by NEP with the exception of Ile30 which was fully NEP-resistant (0% degradation). In vivo bioefficacy was disrupted the most by the Leu30 substitution (less than half as effective as native PP at equal doses). Conversely, bioefficacy was increased the greatest by Asn30, Lys30 and Ile30 changes (Table 3.13).

Ratio of bioefficacy IC (analogue) % NEP Analogue 50 (as measured by food intake) IC (PP) degradation 50 compared to PP over 24h Native PP 1 47 1

Asn30 1.1 32 3.0 Cys30 1 40 2.0 Glu30 1.6 20 1.0 Ile30 1 0 4.0 Leu30 1.8 43 0.4 Nleu30 1.2 39 1.1 Lys30 0.7 30 3.0 Thr30 1.3 53 1.0

Table 3.13 The effect of position 30 (C-terminal) modifications on Y4r affinity, susceptibility to degradation by NEP and in vivo bioefficacy.

157

Combined substitutions at position 0 and 30 were investigated for their susceptibility to NEP degradation and effect on food intake (Table 3.14). The combination substitutions were limited and only a few were tested. Affinity at the Y4r was mostly improved by these dual modifications with the best combination being Pro0, Lys30 which had 2.5-fold greater affinity (IC50 0.092nM PP-Pro0,Lys30 vs 0.23nM PP).

Ratio of bioefficacy IC (analogue) % Analogue 50 (as measured by food intake) IC (PP) degradation 50 compared to PP over 24h Native PP 1 47 1

Ala0, Thr30 0.7 37 0.7 Asp0, Lys30 1 37 3.0 Lys0, Glu30 1 0 1.9 Pro0, Leu30 0.7 6 1.4 Pro0, Lys30 0.4 17 1.9

Table 3.14 The effect of combined modifications at positions 0 and 30 on Y4r affinity, susceptibility to degradation by NEP and in vivo bioefficacy.

158

Single mid-section substitutions were investigated for their susceptibility to degradation by NEP, affinity at the Y4r and effect on food intake (Table 3.15). Substitutions at position 6 and 19 generally improved affinity at the Y4r. Position 6 substitutions did not affect bioactivity, but position 19 substitutions reduced bioactivity. Other single mid-section changes were well tolerated for affinity to the Y4r with the exception of the substitution of Ala33 which decreased affinity to the Y4r 38-fold.

Ratio of bioefficacy IC (analogue) % Analogue 50 (as measured by food intake) IC50 (PP) degradation compared to PP over 24h Native PP 1 47 1

Phe6 0.7 62 1.4 Glu6 0.7 28 1.4 Tyr11 1.1 65 1.0 Lys12 2.1 88 0.7

His14 1.3 56 0.2 Arg17 1.2 49 0.5 Leu17 2.4 13 1.2 Arg19 0.7 20 0.7 His19 0.5 72 0.6 Met26 1.7 56 1.0 His26 2.1 52 1.0 Ala33 38.3 45 0.1 Gln34 0.7 32 0.6 Lys35 2.5 44 0.3 HArg35 0.7 48 0.1

Table 3.15 The effect of single mid-section modifications on Y4r affinity, susceptibility to degradation by NEP and in vivo bioefficacy.

159

Global modifications incorporating changes at positions 0, 6, 11, 15, 16, 17, 18, 19, 23, 25, 26, 30 were investigated for their susceptibility to degradation by NEP, affinity at the Y4r and effect on food intake.

Combined substitutions at positions 6, 19, 23 and 30 generally improved affinity at the Y4r (Table 3.16). However, these changes only modestly decrease NEP degradation by 2-fold at the most when compared to native PP. The PP analogues that exhibit the greatest decreases in NEP degradation (PP-4 and PP-6) are also accompanied by the biggest improvement in bioefficacy compared to native PP. PP-4 and PP-6 also decreased food intake compared to saline by 24% and 12% respectively.

in vivo hY4r binding % NEP bioefficacy Position: 0 6 16 17 19 23 30 index degradation index LTX (970-977) Glu Ile Lys Gly 0 Ex4 (17-24) Glu Ala Arg Glu 0 PP-1 Phe 0.7 62 1.4 PP-2 Glu 0.7 28 1.4 PP-3 Arg 0.7 20 0.7 PP-4 Lys 0.7 30 3 PP-5 Phe Arg Glu 0.5 23 1 PP-6 Glu Arg Glu Lys 0.3 18 3.5

Table 3.16 The effect of combined substitutions at positions 6, 19, 23 and 30 (analogues PP-1 to PP- 6) on Y4r affinity, susceptibility to degradation by NEP and in vivo bioefficacy.

Substitutions at positions 6, 19, 23 and 30 (Table 3.17) were investigated in combination with changes that protect against DPPIV degradation. As previously seen, these modifications do not change affinity at the Y4r (section 3.2.2.2.3) and mildly improve bioefficacy over a 24h period (section 3.2.2.2.4). The best N-terminal extended analogues (Table 3.12) and an analogue with an N-terminal truncation (Des-Ala1 = DesA1) were then taken and modified to include substitutions at positions 6, 19, 23 and 30 (Table 3.17). All of these combined changes improve affinity at the Y4r by 2.5-10 times. PP analgoues PP-9 to PP-17 decrease degradation by NEP by 23% at least, when compared to native PP.

Despite the reduced susceptibility of all these analogues to degradation by NEP, they vary in their efficacy at reducing food intake (Table 3.17). Analogues PP-9 to PP-13 have only 160 modest improvements in the bioefficacy over 24h compared to native PP. This set of analogues have either Pro0 or Gly0, and Gly0 has a slightly weaker effect on protecting against NEP degradation and no improvement on bioefficacy. Analogues PP-9 to PP-13 also have either Ser6 or Thr6 where Thr6 has the greater improvement on bioefficacy (up to 2.5 times more effective than native PP at reducing food intake). Amongst the analogues with Thr6 substitutions, Arg19 combinations were slightly more potent than their Lys19 counterparts. Analogues PP-14 to PP-16 test the Glu6 substitution and, in combination with Pro0 and Arg19, bioefficacy increased 4.5-5 times that of the effect of native PP. Gly0 version of the Glu6, Lys19 analogue only increased bioefficacy 2-fold despite almost full protection against NEP. However, Gly0 in combination with Asp6 and Lys19 increased bioefficacy by 7 times compared to native PP.

in vivo hY4r binding % NEP bioefficacy Position: 0 6 16 17 19 23 30 index degradation index LTX (970-977) Glu Ile Lys Gly 0 Ex4 (17-24) Glu Ala Arg Glu 0 PYY Glu Glu Leu Arg Ser NPY Asp Asp Met Arg Ala PP-7 Pro 0.9 13 2 PP-8 Gly 1 33 1.5 PP-9 Pro Ser Lys Ala Lys 0.2 1 1.4 PP-10 Gly Ser Lys Ala Lys 0.2 11 1 PP-11 Pro Thr Lys Glu Lys 0.4 6 2 PP-12 Pro Thr Arg Ala Lys 0.1 2 2.5 (DesA1)PP-13 Thr Arg Ala Lys 0.2 5 2 PP-14 Pro Glu Arg Ala Lys 0.4 11 4.5 PP-15 Gly Glu Lys Ala Lys 0.2 2 2 PP-16 Pro Glu Lys Ala Lys 0.3 3 5 PP-17 Gly Asp Lys Ala Lys 0.3 7 7

Table 3.17 The effect of combined substitutions at positions 0, 6, 19, 23 and 30 (analogues PP-9 to PP-17) on Y4r affinity, susceptibility to degradation by NEP and in vivo bioefficacy.

161

Finally, modifications at position 16 and 17 were explored in combination with all the previous global changes (i.e. substitutions at -1, 0, 6, 19, 23, 30). Single changes at positions 16 and 17 alone did not improve receptor affinity or potency at reducing food intake in acute feeding studies in mice in spite of a reduction in the susceptibility to NEP degradation. However, in the presence of combined modifications at positions -1, 0, 6, 19, 23, and 30, (analogues PP-19 to PP-24; Table 3.18) Y4r affinity increased 2-5 fold and all analogues tested were completely resistant to degradation by NEP. Bioefficacy seen in mouse acute feeding studies also increased by at least 4-times compared to native PP

in vivo hY4r binding % NEP bioefficacy Position: 0 6 16 17 19 23 30 index degradation index LTX (970-977) Glu Ile Lys Gly 0 Ex4 (17-24) Glu Ala Arg Glu 0 PYY Glu Glu Leu Arg Ser 0 NPY Asp Asp Met Arg Ala 0 PP-18 Leu 2.4 13 1.2 (DesA1)PP-19 Ile Glu Lys Glu Lys 0.3 2 5 PP-20 Gly Glu Lys Lys Ala Lys 0.2 0 4 PP-21 Pro Thr Leu Lys Glu Lys 0.4 0 5.5 PP-22 Gly Thr Lys Lys Glu Lys 0.3 0 6.5 PP-23 Pro His Glu Lys Glu Lys 0.3 0 7 PP-24 Pro Lys Glu Leu Lys Glu Lys 0.5 0 5.5

Table 3.18 The effect of combined substitutions at positions 0, 6, 16, 17, 19, 23 and 30 (analogues PP-19 to PP-24) on Y4r affinity, susceptibility to degradation by NEP and in vivo bioefficacy.

162

3.2.4 INVESTIGATION OF PLASMA LEVELS OF PP ANALOGUES IN MICE

3.2.4.1 Effect of phosphoramidon on plasma levels of a DPPIV-resistant PP analogue in mice

Following a single SC injection of 150nmol/kg of the DPPIV-resistant PP analogue, PP-Ala0, plasma levels were measured at 45 and 90minues post-injection. PP-Ala0 was also co- administered with the NEP inhibitor, phosphoramidon, at 20mg/kg, and plasma levels also measured at 45 and 90 minutes. Phosphoramidon was given as single IP injection 15minutes prior to SC administration of PP-Ala0. Mice given PP-Ala0 alone were given an equal volume IP injection of vehicle 15 minutes prior to peptide administration. Levels of PP-Ala0, increased significantly after 45mins (Fig 3.36) compared to basal levels of PP-Ala0 cross reactivity and subsequently significantly decreased after 90mins compared to plasma levels at 45mins. Plasma levels of PP-Ala0 at 90mins were not significantly raised compared to basal levels (PP-Ala0 at 90mins: 1809 ± 319pmol/L vs basal: 414 ± 52pmol/L).

At both time points, plasma levels of PP-Ala0 were significantly higher when administered with phosphoramidon compared to PP-Ala0 given alone (45m: PP-Ala0 4916 ± 480pmol/L vs PP-Ala0 + phosphoramidon 11034 ± 703pmol/L; 90m: PP-Ala0 1809 ± 319pmol/L vs PP-Ala0 + phosphoramidon 6484 ± 722pmol/L). Plasma levels of PP-Ala0 were greater than basal levels at 45 minutes only when PP-Ala0 was given alone, but was significantly greater than baseline at both 45 and 90 minutes when given with phosphoramidon.

163

15000 ** *** ### $$

10000

* # 5000

&&& [PP-Ala0] pmol/L [PP-Ala0]

0 basal 45mins 90mins 45mins 90mins + phosphoramidon

Figure 3.36 Plasma levels of PP-Ala0 at 45 and 90mins following a single SC injection of PP-Ala0 at 150nmol/kg to male C57BL/6 mice and in combination with a pre-peptide IP injection of phosphoramidon at 20mg/kg. Basal plasma samples were collected from naïve mice (n=2/6). Data are shown as mean plasma levels of PP-Ala0 ± SEM as determined by RIA. LLOD: 40pmol/L. Significance analysed using one-way ANOVA with Bonferroni’s post hoc test *p≤0.05, **p≤0.01, or *** p≤ 0.001. (Key to significance symbols: black stars are compared to basal levels; blue hash signs are compared to 45min levels without phosphoramidon; red dollar signs are compared to 45min levels with phosphoramidon; green ampersand are compared to 90min levels without phosphoramidon)

3.2.4.2 Effect of phosphoramidon on plasma levels of PP analogues with global modifications in mice

The effect of phosphoramidon was tested on plasma levels of PP analogues shown in Table 3.19. As above, phosphoramidon was used at a concentration of 20mg/kg and administered IP 15minutes prior to SC injection of peptide. Blood was collected from mice sacrificed at 45 and 90minutes post-injection of peptide.

As a control, native PP at 1000 and 25nmol/kg were given in the presence and absence of phosphoramidon. At the higher dose without inhibitor, plasma levels of PP significantly decreased to 13% of the levels seen at 45m (PP at 90mins: 8978 ± 1553pmol/L vs 45mins: 67761 ± 7473pmol/L). At 1000nmol/kg (Fig 3.37(A)), PP co-administered with

164

phosphoramidon, plasma levels of PP at 45minutes were 2-fold higher (141877 ± 13827pmolL) compared to plasma levels of PP in the absence of inhibitor at the same time point (p=0.05). However, by 90mins, even in the presence of inhibitor, plasma levels of PP had significantly decreased to 21% of the levels seen at 45m with phosphoramidon (29091 ± 2976pmolL). Although not statistically significant, the plasma levels of PP at 90mins with inhibitor are 3-times higher compared to the plasma levels of PP at 90mins with no inhibitor.

At 40-times lower dose of PP (25nmol/kg), PP plasma levels are 5-times lower on average, compared to the 1000nmol/kg dose (Fig 3.37(B)). Without phosphoramidon, plasma levels of PP at 90mins were only 50% decreased compared to levels at 45mins. This pattern is repeated in the presence of phosphoramidon except plasma levels at both time points are at least 2-fold higher compared to the respective time points in the absence of NEP inhibitor.

in vivo IC50 (analogue) % NEP bioefficacy

Position: 0 6 19 20 21 22 23 30 IC50 (PP) degradation index LTX (970-977) Lys Tyr Phe Val Gly 0 Ex4 (20-24) Arg Leu Phe Ile Glu 0 PYY Glu Arg Tyr Tyr Ala Ser Leu 0 NPY Asp Arg Tyr Tyr Ser Ala Leu 0 PP Val Gln Tyr Ala Ala Asp Met 1 47 PP-Lys30 Lys 0.7 30 3.0 PP-P0,A23,K30 Pro Ala Lys 1 37 3.0 PP-P0,K19,K30 Pro Lys Lys 0.2 37 4.0 PP-K6,NPY19-23 Lys Arg Tyr Ser Ala 0.2 77 0.1 PP-D6,NPY19-23 Asp Arg Tyr Ser Ala 0.2 90 1.0

Table 3.19 The effect of global modifications on Y4r affinity, susceptibility to degradation by NEP and in vivo bioefficacy.

165

** $$ 200000 35000 A B * $$$ 30000 150000 25000

20000 100000 46% 15000

* pmol/L pmol/L 10000 50000 21% 50% 5000 13% 0 0 45m 90m 45m 90m 45m 90m 45m 90m + phosphoramidon + phosphoramidon

Figure 3.37 Plasma levels of PP following a single subcutaneous injection of PP at (A) 1000nmol/kg and (B) 25nmol/kg to male C57BL/6 mice in combination with a single IP injection of phosphoramidon at 20mg/kg. Blood samples were collected at 45 and 90 minutes post-injection (n=3). Data are shown as mean plasma levels of PP ± SEM as determined by RIA. LLOD: 70pmol/L. Significance analysed using one-way ANOVA with Bonferroni’s post hoc test *p≤0.05, **p≤0.01, or *** p≤ 0.001. (Key to significance symbols: black stars are compared to 45min levels without phosphoramidon; red dollar signs are compared to 45min levels with phosphoramidon)

PP-Lys30 modestly decreased susceptibility to degradation while maintaining affinity to the Y4r. However, anorectic effects in vivo were shown to be 3-fold higher than that of native PP. Plasma levels of PP at 90 mins without phosphoramidon decreased to 26% of plasma levels at 45mins. This pattern is repeated in the presence of phosphoramidon where peptide levels decrease to 36%. Unlike native PP, plasma levels of PP-Lys30 in the presence of phosphoramidon were not significantly higher compared to peptide levels at the same time point without the NEP inhibitor.

A further two PP analogues (PP-P0,A23,K30 and PP-P0,K19,K30) which had modest decreases in susceptibility to NEP degradation in vitro were also measured for circulating levels in the presence and absence of phosphoramidon. Like PP-Lys30, they were 3-4 times more effective at reducing food intake in mice compared to PP. Either in the presence of absence of phosphoramidon, plasma levels of these two analogues did not significantly decrease at 90mins compared to peptide levels at 45mins. Peptide levels of the two

166 analogues in the presence of phosphoramidon were also not significantly higher compared to peptide levels at the same time point without the NEP inhibitor.

Lastly, the effect of phosphoramidon on circulating levels of another two analogues of PP that had unfavourable substitutions with respect to in vitro NEP digests was investigated. The in vivo effects of these PP analogues were either equal to that of native PP (PP- D6,NPY19-23) or 10-times worse (PP-K6,NPY19-23). Plasma levels of PP-D6,NPY19-23 alone at 90minutes post-injection were 58% decreased compared to peptide levels at 45minutes. In the presence of phosphoramidon, the percentage remaining at 90minutes was lower (27%) as the peak at 45mins was significantly higher with NEP inhibitor (>2-fold) compared to plasma levels at 45mins without NEP inhibitor. There was no difference in plasma levels at 90mins post-injection in the either the presence or absence of phosphoramidon.

Plasma levels of PP-K6,NPY19-23 alone at 90minutes post-injection were significantly reduced to 22% of the peptide levels at 45minutes. In the presence of phosphoramidon, the percentage remaining at 90minutes was 15% of the peak at 45mins. There was significantly higher peptide levels at 45mins in the presence of NEP inhibitor (>3-fold, p=0.01) compared to plasma levels at 45mins without NEP inhibitor. There was no difference in plasma levels at 90mins post-injection with or without phosphoramidon.

167

250000 7000 25000 A $$ B 92% C 6000 200000 20000 ** 5000 72% 150000 4000 69% 15000 67% 3000 pmol/L pmol/L 100000 36% pmol/L 10000 26% 2000 50000 5000 1000 0 0 0 45m 90m +45m +90m 45m 90m 45m 90m 45m 90m 45m 90m + phosphoramidon + phosphoramidon + phosphoramidon

4000 ** $$ D 1000 ** $$$ E 800 3000 600 2000 pmol/L pmol/L 400 1000 58% 27% 200 16% 22% 0 0 45m 90m 45m 90m 45m 90m 45m 90m + phosphoramidon + phosphoramidon

Figure 3.38 Plasma levels of peptide at 45mins and 90mins following a single subcutaneous injection of: (A) PP-Lys30 at 1000nmol/kg (green bars), (B) PP-P0,A23,K30 at 25nmol/kg (grey bars), (C) PP- P0,K19,K30 at 1000nmol/kg (purple bars), (D) PP-K6,NPY19-23 at 1000nmol/kg (red bars), and (E) PP-D6,NPY19-23 at 25nmol/kg (blue bars) to male C57BL/6 mice in combination with a single IP injection of phosphoramidon at 20mg/kg (hatched bars). Blood samples were collected at 45 and 90 minutes post-injection (n=3-4). Data are shown as mean plasma levels of PP ± SEM as determined by RIA. LLOD: 70pmol/L. Significance analysed using one-way ANOVA with Bonferroni’s post hoc test *p≤0.05, **p≤0.01, or *** p≤ 0.001. (Key to significance symbols: black stars are compared to 45min levels without phosphoramidon; red dollar signs are compared to 45min levels with phosphoramidon)

168

3.2.4.3 Investigation of plasma levels of PP analogues with different susceptibilities to NEP and contrasting effects in vivo

PP-25 is a DPPIV- and NEP-resistant analogue of PP. It has binding affinity which is over 5- times greater than native PP at the mY2r (PP IC50 0.23 ± 0.033nM vs. 446 IC50 0.04 ± 0.002nM) (Fig 3.39). PP-26 is a similar analogue with mY4 receptor affinity over 3-times greater than native PP (IC50 0.065 ± 0.004nM). When incubated with NEP, HPLC analysis demonstrated that PP-25 underwent 37% degradation while PP-26 only had 8% degradation by NEP (Table 3.20)

mY4r

100

80 PP-25 PP-26 60

20 % Specific Binding % Specific

0 -15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5

Peptide Concentration Log (M)

Figure 3.39 Binding affinities of analogues PP-25 and PP-26 to mouse Y4r expressed as IC50 values calculated from the mean ± SEM of 3-5 separate experiments

in vivo hY4r binding % NEP bioefficacy Position: 0 6 17 19 23 30 index degradation index PP-25 Gly Arg Ala Lys 0.2 37 1.4 PP-26 Gly Thr Leu Arg Ala Lys 0.3 8 6 Table 3.20 The effect of global modifications on PP analogues with similar Y4r affinity but different susceptibilities to degradation by NEP, and differences in in vivo bioefficacy.

169

The effects of PP-25 and PP-26 on acute feeding studies in mice were disparate. PP-26 was equipotent to PP administered at a 8-fold higher dose (Fig 3.40(A)). At the time intervals of 0-1h, 1-2h, 2-4h, and 4-8h post-injection, PP-26 administered at 25nmol/kg significantly reduced food intake compared to the saline control group (p≤0.05). PP administered at 200nmol/kg significantly reduced food intake at 0-1h, 1-2h, and 2-4h compared to the control group (p≤0.05). At the 4-8h time interval, there was no significant difference between the control group and PP. There was also no significant difference between the PP and PP-26 groups at this time point.

PP-25 given at equimolar dose to PP (25nmol/kg) only significantly inhibited food intake in the 0-1h time interval (Fig 3.40(B)). There were no significant changes in food intake at any other time points.

1.0 1.2 A saline B saline PP (200nmol/kg) PP (25nmol/kg) PP-26 (25nmol/kg) 1.0 PP-25 (25nmol/kg) 0.8

0.8 0.6 * ** * ** ** 0.6 0.4 0.4 Food intake (g) intake Food Food intake (g) intake Food *** 0.2 0.2 * *** 0.0 0.0

0-1 4-8 0-1 1-2 2-4 Time (hours) Time (hours)

Figure 3.40 The effect of SC administration of (A) PP (200nmol/kg) and PP-26 (25nmol/kg) and (B) PP (25nmol/kg) and PP-25 (25nmol/kg) on food intake in C57BL/6 mice fasted overnight. PP, PP-25 and PP-26 werecompared to saline (*) using oneway ANOVA with Dunnett’s post hoc test *p≤0.05, **p≤0.01,or *** p≤ 0.001 (n=9-10).

170

C57BL/6 mice were given a subcutaneous injection of either PP, PP-25 or PP-26 at a concentration of 100nmol/kg. Animals were then culled at either 30 minutes or 2 hours

post-injection via CO2 inhalation and then blood collected by cardiac puncture. Each peptide was measured using its own standard curve. Peptide levels for PP at 30m post-injection reached 8737 ± 744pmol/L and significantly decreased at 2h to 2112 ± 224pmol/L (Fig 3.41(A)). At 30m post-injection, peptide levels for PP-25 reached 10426 ± 428pmol/L and again, significantly decreased at 2h to 2844 ± 206pmol/L (Fig 3.41(B)). Peptide levels for PP- 26 at 30m post-injection reached 26862 ± 1026pmol/L and significantly decreased at 2h to 5810 ± 215pmol/L (Fig 3.41(C)). All peptide levels had decreased to 22-27% at 2h post- injection compared to peptide levels at 30m.

15000 15000 30000 C A B

10000 10000 20000

5000 5000 27% 10000 22%

[PP] pmol/L [PP] 24% [PP-25] pmol/L [PP-25]

*** pmol/L [PP-26] ** ***

0 0 0 30m 120m 30 120 30 120

Figure 3.41 Plasma peptide levels at 30mins and 2hrs following a SC injection of (A) PP (white bars), (B) PP-25 (grey bars), and (C) PP-26 (black bars) all administered at 1000nmol/kg. Peptide concentrations were measured by RIA. Significance was measured using student’s t-test. *p≤0.05, **p≤0.01, or *** p≤ 0.001 (n=4)

171

Comparison of peptide levels at 30m (Fig 3.42(A)) demonstrates no difference in peptide levels between PP and PP-25; however levels of PP-26 are significantly higher compared to PP and PP-25 (p≤0.001). This difference is mirrored at the 120m time point where there is no significant difference in peptide levels between PP and PP-25 but levels PP-26 are significantly raised compared to either PP or PP-25 (p<0.001-0.005).

*** *** 30000 $$$ $$ A 6000 B 5000

20000 4000

3000 pmol/L pmol/L 10000 2000 1000

0 0 PP PP-25 PP-26 PP PP-25 PP-26

Figure 3.42 Comparison of plasma peptide levels of PP (white bars), PP-25 (grey bars), and PP-26 (black bars) at (A) 30mins post-injection and (B) 120mins post-injection. All peptides were administered at 1000nmol/kg. Peptide concentrations were measured by RIA. Significance was measured using using one-way ANOVA with Bonferroni’s post hoc test. *p≤0.05, **p≤0.01, or *** p≤ 0.001 (n=4). (Key to significance symbols: black stars are compared to levels of PP; red dollar signs are compared to levels of PP-25)

172

3.2.4.4 Investigation of plasma levels of PP analogues with similar in vivo bioefficacy and different affinities to the Y4 receptor

PP-27 and PP-28 are DPPIV- and NEP-resistant analogues of PP. While both analogues are NEP-resistant compared to native PP, when incubated with purified NEP, HPLC analysis demonstrated that PP-27 underwent 19% degradation while PP-28 was completely resistant to NEP degradation (Table 3.21). The binding affinity of PP-27 is 2-fold greater compared to native PP at the mY4r (PP IC50 0.23 ± 0.033nM vs. PP-27 IC50 0.17 ± 0.028nM). However, PP-

26 binds to the mY4 receptor at 5-times greater affinity than native PP (IC50 0.049 ± 0.007nM) (Fig 3.43).

mY4r 100

75 PP-27 PP-28 50

25 % Specific Binding % Specific

0 -14 -13 -12 -11 -10 -9 -8 -7 -6

Peptide Log [M]

Figure 3.43 Binding affinities of analogues PP-27 and PP-28 to mouse Y4r expressed as IC50 values calculated from the mean ± SEM of 3-5 separate experiments

in vivo hY4r binding % NEP bioefficacy Position: 0 6 11 15 17 19 23 30 index degradation index PP-27 Glu Arg Glu Lys 0.5 19 4 PP-28 Gly Glu Asp Gln Lys Arg Glu Lys 0.2 0 10

Table 3.21 The effect of global modifications on analogues that are both relatively NEP-resistant but differ at their affinity to the Y4r and with modest differences in in vivo bioefficacy.

173

The effects of PP-27 and PP-28 on acute feeding studies in mice were within the same order of magnitude. PP-27 was as 1.5-times more potent than PP given at 5-fold higher dose (Fig 3.44). At the time intervals of 1-2h, and 2-4h post-injection, PP-27 administered at 10nmol/kg significantly reduced food intake compared to the saline control group (p≤0.01). PP administered at 50nmol/kg significantly reduced food intake at 0-1h compared to the control group (p=0.01). At all other time intervals, there was no significant difference between the control group and either PP or PP-27. PP significantly reduced food intake in the first time interval compared to PP-27 (p≤0.05) however, PP-27 significantly reduced food intake in the 2-4h time interval compared to PP (p≤0.001).

. 1.4 saline ** # PP (50nmol/kg) 1.2 PP-27 (10nmol/kg)

1.0

0.8

0.6

(g) intake Food 0.4

0.2 ### ** *** 0.0

0-1 1-2 2-4

Time (hours)

Figure 3.44 The effect of SC administration of PP (50nmol/kg) and PP-27 (10nmol/kg) on food intake in C57BL/6 mice fasted overnight. All groups were compared using one-way ANOVA with Tukey’s post hoc test *p≤0.05, **p≤0.01,or *** p≤ 0.001 (n=9-10).

174

PP-28 given at 5-fold lower dose to PP (50nmol/kg) significantly inhibited food intake in the 0-1, 1-2, and 4-8h time intervals (Fig 3.45). There were no significant changes in food intake compared to vehicle control at any other time point. PP-28 was therefore, on average, 10- fold more potent than PP at reducing food intake. In addition, PP-28 significantly reduced food intake in the 0-1h and 4-8h time intervals compared to PP (p=0.001)

1.2 saline ## PP (50nmol/kg) PP-28 (10nmol/kg) 1.0

0.8 *** ## 0.6

0.4

(g) intake Food * 0.2 **

0.0 0-1 1-2 2-4 4-8

Time (hours)

Figure 3.45 The effect of SC administration of PP (50nmol/kg) and PP-28 (10nmol/kg) on food intake in C57BL/6 mice fasted overnight. All groups were compared using one-way ANOVA with Tukey’s post hoc test *p≤0.05, **p≤0.01,or *** p≤ 0.001 (n=9-10).

To measure circulating levels of the peptides, C57BL/6 mice were given a subcutaneous injection of PP, PP-27 or PP-28 at a concentration of 1000nmol/kg. Animals were then sacrificed at either 15, 45 minutes, or 2 hours post-injection via CO2 inhalation and then blood collected by cardiac puncture. Each peptide was measured using its own standard curve. Peptide levels for PP at 15m post-injection peaked at 34798 ± 8418pmol/L, decreased to 66% at 45m (22825 ± 638pmol/L), and significantly decreased by 120m to 2236 ± 177pmol/L compared to plasma levels of PP at 15m. At 15m post-injection, peptide levels

175 for PP-27 reached 58655 ± 5430pmol/L and did not significantly decrease by the 45m time point (55371 ± 1485pmol/L). However, plasma levels of PP-27 at 120m post-injection significantly decreased to 15599 ± 1678pmol/L compared to peptide levels at both 15 and 45m. Peptide levels for PP-28 at 15m post-injection reached 46257 ± 4860pmol/L and decreased to 77% of the initial levels at 45m to 32636 ± 4429pmol/L. At 120m post- injection, plasma levels of PP-28 had significantly fallen to 10226 ± 2626pmol/L compared to initial plasma levels at 15m.

75000 PP PP-27 A ***$$$ B C 60000 PP-28 94% * * $ 45000 77%

pmol/L 30000 66% 30% 15000 15% 7% 0 15 45 120 15 45 120 15 45 120

Time (min)

Figure 3.46 Plasma peptide levels at 15, 45 and 120mins following a SC injection of (A) PP (white bars), (B) PP-27 (grey bars), and (C) PP-28 (black bars) all administered at 1000nmol/kg. Peptide concentrations were measured by RIA. Significance was measured using using one-way ANOVA with Bonferroni’s post hoc test. *p≤0.05, **p≤0.01, or *** p≤ 0.001 (n=3-4)

176

Comparison of peptide levels at 15m (Fig 3.47) demonstrates no difference in peptide levels between PP, PP-27, and PP-28. At 45m, levels of PP-27 are significantly higher compared to both PP and PP-28 (p<0.01-0.05). At the final time point, plasma levels of PP-27 are still significantly raised compared to PP (p<0.05) but there is no significant difference between PP-27 and PP-28.

75000 15m 45m 120m 60000 ** $

45000

pmol/L 30000

** 15000 7% 0 *

PP PP PP PP-27 PP-28 PP-27 PP-28 PP-27 PP-28

Figure 3.47 Comparison of plasma peptide levels of PP (white bars), PP-27 (grey bars), and PP-28 (black bars) at (A) 15mins post-injection, (B) 45mins post-injection, and (C) 120mins post-injection. All peptides were administered at 1000nmol/kg. Peptide concentrations were measured by RIA. Significance was measured using using one-way ANOVA with Bonferroni’s post hoc test. *p≤0.05, **p≤0.01, or *** p≤ 0.001 (n=3-4)

177

3.3 DISCUSSION

The observation that an intravenous infusion of PP decreases appetite and food intake in normal BMI volunteers without the undesirable side-effects of nausea (Batterham et al., 2003b) highlighted the potential target of this peptide hormone for development as an agent to treat obesity. However, as with many peptide-based drug targets, several properties of the molecule such as its short circulatory half-life would need to be addressed before it could even be considered viable as a therapeutic agent.

For PP, the clearance rate is a particularly limiting factor of the peptide and it was modification of this property which I focused my research on in this chapter. Peptides are cleared from the body by two main processes, proteolytic degradation and renal clearance. The major sites of proteolytic degradation of peptides are the kidney and liver where proteolytic enzymes are found at high concentrations. Using cell membrane extracts from the kidney (renal brush border – RBB) and liver (rat liver microsomes – RLM), I completed a series of in vitro degradation studies to identify critical residues in the molecule with regard to the molecules breakdown. This was followed up with further in vitro degradation studies using purified enzymes and in vivo studies to confirm a possible mechanism. The chapter also continued mechanistic studies by investigating if rational substitutions of residues identified as prone to proteolytic cleavage would result in an improved circulatory half-life and therefore pharmacological action.

Initial in vitro digests utilising tissue membrane extracts showed that PP suffered only 8% degradation after 15minutes incubation with RLM, increasing to 35% after 60minutes incubation. Despite the majority of PP still eluting at the same retention time as the parent compound at 15minutes, MS analysis confirmed degradation had already occurred, as fragments were identifiable.

Analysis by MALDI-TOF of PP incubated with RLM for 10minutes demonstrated the production of fragments: PP1-35 (with and without the oxidised methionine residues), 6-29, 15-35, 2-31 and 14-30. These fragments point to RLM degradation occurring mainly at distal sites to the PP molecule (at either N- or C-terminal) with particular susceptibility between position 2-3, 5-6, 14-15, 29-30 and 30-31.

178

At 60 minutes incubation with RLM, the overall number of fragments decreased as expected; once fragmentation occurs they are more prone to further degradation. There were some common fragments to the earlier time points such as PP1-35 and 14-30. The unique fragments to this later time point were 3-36 and 8-26. This suggests that fragments PP6-29, 15-35 and 2-31 from the 10minute digest of PP were initial breakdown products that eventually get metabolised into other species. However, the susceptible degradation positions still existed between positions 2-3, 14-15 and 29-30. As with shorter incubations of PP with RLM, the fragments produced consisted of core positions of the molecule with the distal parts being more vulnerable to degradation by RLM. Fragments containing cuts at the two most C-terminal residues of PP were also present at both short (10minutes) and longer (60minutes) incubations with RLM.

Initial digests using preparations of RBB showed that PP suffered little (<10%) degradation after 10mins and up to >50% degradation after 60minutes incubation as analysed by HPLC. As well as the decrease in size of the parent peak, earlier novel peaks were also generated.

MS analysis of PP incubated with RBB demonstrated that full length PP is still present after 10 or 60mins incubation however, this peak is at a higher intensity at the early time point suggesting it was present at higher concentrations. The 2 fragments generated after just 10mins were PP3-36 and PP6-33/PP10-36 (which both have the same molecular weight). These fragments point to RBB degradation occurring mainly at distal sites to the PP molecule (at either N- or C-terminal) with particular susceptibility between position 2-3, 5-6, 10-11, and 33-34.

Fragment PP3-36 is a potential product of the enzymatic action of DPPIV and was observed in digests with both RLM and RBB, which is in agreement with known sites of DPPIV expression (Tiruppathi et al., 1990, Tarantola et al., 2012). PYY, a closely related peptide to PP with a high degree of structural similarity is degraded by DPPIV, further suggesting DPPIV may be an important enzyme to investigate for metabolism of PP.

DPPIV cleaves the first two amino acids of the N-terminal of peptides up to around 80 amino acids in length. The preferred substrate of DPPIV is X-Pro-X (where X is any of the naturally occurring mammalian amino acid except Pro). Therefore, I predicted that the N-terminal sequence of PP: Ala1-Pro2-Leu3 would be degraded by DPPIV. To confirm if PP could be

179 degraded by DPPIV, PP was incubated with purified DPPIV and analysed by HPLC. However, initial HPLC studies suggested no change in peak area of the parent compound, no change in retention time and an absence of additional degradative peaks. This result was contradictory to results from tissue preperation studies analysed by MS. Therefore I attempted to optimise the solvent gradient in the event of the lack of resolution was masking the degradation. However, this was not successful and MS analysis was thus used on whole digests. MALDI-TOF MS did confirm that PP was degraded by DPPIV to PP3-36. As expected PP5-36 was not observed as Pro5 at the P3’ cleavage subsite should not be degraded by DPPIV.

Whereas cleavage of PYY1-36 to PYY3-36 alters the peptide affinities to Y receptors, making PYY a Y2r-preferring agonist, PP3-36 had modestly reduced affinity to Y4r and no change in affinity to Y1r or Y2r (data not shown). The bioefficacy of the PP3-36, was tested by comparing its ability to inhibit food intake in mice compared to the parent molecule. Surprisingly, PP3-36 was able to reduce food intake, although its anorectic effects were evident for a shorter time period. While there was no statistical difference between groups treated with PP and PP3-36, compared to the control group, PP3-36 was significantly weaker than PP, particularly at later time points.

The fragment PP6-33 was identified in both RLM and RBB digests of PP, and I hypothesised that cleavage at Arg33 was due to proteolytic degradation by trypsin. Trypsin was considered an important enzyme to investigate as the peptide would be delivered subcutaneously in the clinic. Subcutaneous (SC) administration of peptide therapies introduces prolonged contact of the peptide with the SC environment providing an opportunity for trypsin to degrade the peptide. Trypsin is a specific class of proteases which cleaves the C-terminal to Lys and Arg residues of a peptide. The PP sequence contains 4 Arg residues at positions 25, 26, 33 and 35.

HPLC analysis of PP incubated with trypsin showed the disappearance of the parent molecule and the appearance of two major peaks: RT=14m (PPF1) and RT=18m (PPF2). MS analysis of PPF1 demonstrated that this fraction contained fragments of m/z values 1642 (PP25-26) and 1165 (PP27-35/26-34/28-36/16-25). All 4 fragments of PP with molecular weights of 1165 can all contribute to the intensity of the signal. MS analysis of PPF2

180 contained several fragments of which the main species present was PP1-26. All of the fragments from PPF1 and PPF2 were consistent with the cleavage pattern of trypsin.

Not all fragments identified by membrane digests were successfully attributed to specific enzymes. However, to confirm the roles of a few potential enzymes responsible for sites of defined degradation in the molecule, purified recombinant enzymes which were predicted to cleave at these sites based on amino acid substrate requirements, were used in vitro to investigate degradation with PP and PP analogues.

Many of the cleavage sites identified by the RLM and RBB digests could be sites for cleavage by NEP, these included Val6, Ala21, Leu24, Met30 and Tyr36. NEP is a likely candidate as it is present in RBB (Stewart and Kenny, 1984, Kenny, 1986) and liver (Borscheri et al., 2001, Shousha et al., 2004) at high concs. NEP is a cell-surface metallopeptidase that targets hydrophobic residues such as these.

Incubation of PP with purified NEP 22.11, a relatively highly expressed member of the enzyme family, demonstrated a decrease in the peak area of native PP as analysed by HPLC and the generation of novel peaks with earlier retention times. MS analysis of whole digests of PP with NEP at both short (30m) and long (120m) time points showed only 1 major fragment produced: PP3-21. The amino acid residues at the P1’ subsites (Leu3 and Ala22) are considered small and hydrophobic and are therefore fit with NEP degradation. Unexpectedly, the fragments of NEP digest indentified by MS analysis after the digest had been resolved by HPLC prior to MALDI-MS analysis did not match that found in the whole digest. The new fragments identified were PP5-10, PP1-12, and PP12-26/7-22. The residues at some of these P1’ (Val6, Ala12) and P1 (Ala12) subsites are characteristic NEP target residues however the P1 (Asp10, Arg26) and P1’ (Thr13, Asp23, Tyr27) subsites are not typical NEP target residues.

To assess the role of NEP in the ability of PP to reduce food intake (bioefficacy), PP was administered with and without the potent NEP inhibitor, phosphoramidon (Kenny et al., 1981, Medeiros and Turner, 1994). At the dose used, phosphoramidon alone did not have any effect on food intake compared to saline-treated controls. PP given without phosphoramidon significantly reduced food intake up to 8hrs compared to saline group. Over the entire 24h period post-injection, neither PP alone or PP co-administered with

181 phosphoramidon significantly reduce food intake compared to the control group, however, mice treated with PP and the NEP inhibitor had a >20% decrease in food intake compared to mice treated with PP alone. A limitation of the study is that phosphoramidon could also influence the degradation of endogenous hormones which also influence appetite. This may have contributed to the non-significant difference between treated groups.

An alternative approach from measuring food intake to assess the physiological importance of NEP was to assess plasma levels of PP when administered with and without phosphoramidon to rats. Plasma levels of PP when administered with phosphoramidon remained elevated for longer than PP administered alone. Together this data suggested NEP was partly responsible for the clearance of PP from circulation.

Having identified DPPIV, trypsin and NEP as physiologically relevant enzymes in the clearance of PP, I designed analogues of PP with amino acid substitutions to protect against cleavage by these enzymes. The structure of PP contains a PP fold, a structure known to be important for its biological activity. Therefore changes to the sequence had to be limited to a number of areas of the molecule to avoid disrupting its secondary and tertiary structure. Targeted areas included the N-terminus, a relatively non-conserved area of the PP fold family peptides, and the mid-section α-helix. Changes in the mid-section avoided key, highly conserved sites including Tyr20, Leu24 and Tyr27, and were made with the intention of increasing the helicity. This was achieved by adding residues from the well characterised helical domain of α-latrotoxin found in spider venom, and residues from Ex-4, another well characterised helical domain (discussed in more detail in section 4.1.1).

I designed two strategies to achieve resistance to DPPIV. The first was to extend the N- terminal to create PP-Ala0 and the second was to truncate the N-terminus to produce PP2- 36. Both of these analogues eliminated the DPPIV degradation by having Pro at either position 1 or 3, which was confirmed by MALDI-TOF analysis.

It was important to consider if any benefit from generating DPPIV-resistant molecules would be lost due to reduced receptor affinity and in vivo efficacy. No difference in receptor affinity was noted in the analogues to PP allowing for a direct comparison of bioefficacy in vivo and the assessment of the importance of resistance to DPPIV degradation. Biological activity of both DPPIV-resistant PP analogues were tested in acute feeding studies in mice.

182

In the study comparing matched doses of PP and PP2-36, PP2-36 produced a significantly greater inhibition of food intake than PP over 24hr, confirming DPPIV resistance was beneficial. When PP was compared against PP-Ala0 in an acute feeding study, both peptides significantly reduced food intake up to 4h post injection. However only PP-Ala0 significantly reduced food intake over the whole 0-24h period (p < 0.01 compared with saline) while PP did not.

To confirm this increased efficacy was due to reduced degradation, peptide plasma levels of native PP measured at 90mins post-injection was almost 80% less than that observed at 45 minutes post-injection. However, PP-Ala0 levels remained higher for longer, only 60% of the peptide was cleared between 45 and 90 minutes post-injection. Combining the tissue preparation data, the in vitro degradation with purified enzyme studies, the in vivo efficacy studies and the plasma clearance study, I concluded that DPPIV was an important enzyme in the physiological clearance of PP. The data also suggested designing analogues with resistance to DPPIV degradation would be important in producing longer lasting analogues of PP.

When designing trypsin-resistant analogues, amino acid substitutions were made at position 25, 26, 33 and 35, individually and in various combinations. HPLC analysis demonstrated that individual substitution of position 25 and 26 did not protect PP against trypsin degradation. However, combination substitutions at both 25 and 26 did inhibit PP degradation. Changes at positions 25 and 26 with similarly charged residues had minimal impact on Y4r affinity while changes to neutral amino acids caused a greater loss of receptor affinity. Despite keeping to conservative changes, double substitutions at position 25-26 (PP-Gln25,26 or PP-His25,26) resulted in a 5-fold reduction in receptor affinity.

To assess if PP analogues resistant to trypsin improved their in vivo activity, they were administered to mice and food intake was measured. Due to the disparity of the Y4r affinity values, when assessing the efficacy of analogues, they were tested at twice the dose of PP to allow for a more equal comparison. Analogues with changes at position 25 had a shorter duration of action than PP and analogues with changes at position 26 were approximately as bioefficaious as PP. Acute feeding studies in mice using PP-Gln25,26 and PP-His25,26

183 appeared marginally stronger in the first hour post injection, but overall did not demonstrate any advantage in bioefficacy over native PP.

C-terminal trypsin-resistant modifications at positions 33 and 35 were also initially screened for their affinity to the Y4r. Conservative changes at either position 33 or 35 with similarly charged residues (PP-Lys33, PP-Lys35, PP-His35) had modestly increased IC50 values in the order of 2-3-times worse compared to native PP. However, substitution with Ala residues (PP-Ala33 and PP-Ala35) had a detrimental effect on Y4r binding affinity. PP-Ala35 had an IC50 value 10-fold higher than that of native PP while PP-Ala33 was almost 40-times worse at Y4r binding compared to PP suggesting that position 33 is extremely important in maintaining affinity to the Y4r.

Although Y4r binding affinity is severely affected by Ala substitution at position 33, the bioefficacy of PP-Ala33 in an acute feeding study is not as impaired as its receptor affinity might suggest. While both analogues PP-Ala33 and PP-Lys33 are not better at reducing food intake compared to native PP, PP-Ala33 is of comparative strength to PP. This discrepancy may be the result of greater resistance to trypsin degradation negating any loss of efficacy caused by reduced receptor affinity. However, it could also be due to species variation in Y4 receptors (affinity was measured against the human Y4r while the feeding studies are conducted in mice) where human Y4r may be sensitive to changes at position 33 while mouse Y4r may be more tolerant to similar changes at this position (Berglund et al., 2001).

Interestingly, while both PP-Ala26 and PP-His35 contain modifications which are individually well tolerated when it comes to Y4 receptor affinity, both substitutions combined (PP- Ala26,His35) appear to synergistically lower the binding affinity to the Y4r (almost 30-fold worse compared to native PP). This was not expected as changes to the C-terminus were not expected to be affected mid-section modifications, as the C-terminus projects away from the hairpin-like loop of the PP-fold at a 90° angle (Grundemar et al., 1993).

Although in vitro analysis demonstrated trypsin resistance could be achieved by amino acid substitutions, it was not possible to improve the efficacy of the PP with these changes. This appeared largely to be a result of the reduced receptor affinity which cancelled out any improvement seen in resistance to trypsin in in vivo studies. Modifications of position 33 and 35 completely abolishes the ability of PP to reduce food intake over a 24h period even

184 at higher doses to compensate for the decrease in Y4 receptor affinity. Indeed, the two Arg residues at position 33 and 35 are highly conserved amongst Y receptor ligands and represent important contact sites for all Y-receptors (Merten et al., 2007). Therefore substitution of these Arg residues may disrupt downstream signalling at the Y4r and further receptor activation.

Designing analogues of PP to protect against NEP was challenging as there were numerous factors to consider. For example, Ex-4 has a number of NEP target sites yet remains NEP- resistant while GLP-1 which is rapidly cleaved by NEP and has just as many target sites (Hupe-Sodmann et al., 1995). It has been suggested that, as Ex-4 has a more compact α- helix structure, some target sites are protected by peptidase degradation due to steric hindrance. Larger size peptides may therefore be partly NEP-resistant because they have a stronger tendency to form compact tertiary structures resulting, in the enzyme’s preference for smaller peptides (NEP is indeed often referred to as an oligopeptidase) (Dale, 2006). This observation makes predicting susceptibility to NEP degradation difficult. I therefore conducted a study into what drives the relationship between susceptibility to degradation by NEP, receptor affinity, and in vivo bioefficacy.

Using 2 analogues of PP which had similar affinities to the Y4r, in vitro digests with NEP demonstrated that 1 analogue was more susceptible to NEP degradation than the other. In acute feeding studies with dose titrations, the NEP-resistant analogue and PP had equal potency at reducing food intake when PP is administered at a dose which is 8-times higher than the analogue. The NEP-sensitive analogue administered at an equimolar dose to PP was only able to significantly inhibit food intake in the 0-1h time interval.

When peptide levels were measured in plasma at 30 and 120minutes after subcutaneous administration, all peptides demonstrated significant reductions in plasma levels at 120minutes compared to their respective peptide levels at 30minutes. However, when plasma levels are analysed comparing PP and the 2 analogues at each time point, it is clear that plasma levels of the PP-resistant analogue were significantly increased compared to plasma levels of either PP or the NEP-sensitive PP analogue.

It was concluded from this direct comparison that, although there were no major differences in longevity in circulation between PP and the 2 analogues, the NEP-resistant PP

185 analogue exhibits greater plasma concentrations at both 30 and 120minutes compared to native PP and the NEP-sensitive PP analogue. However, it does not remain in circulation for any longer compared to the other 2 peptides as the same proportion of the NEP-resistant peptide has degraded by 120minutes post-injection (as represented by reduced plasma levels measured by RIA). Despite the same percentage of peptide loss by 120minutes, conceivably the increased plasma levels achieved by the NEP-resistant PP analogue may be above the threshold which will activate the anorectic effects of Y4r agonism (Lamers et al., 1982).

In a second study investigating the importance of NEP degradation to PP, l studied 2 analogues of PP which were both resistant to NEP digests in vitro and both had increased affinity to Y4r compared to PP , however PP-28 analogue had 5-times increased receptor affinity compared to PP-27 which had only a modest 2-fold increase in receptor affinity compared to PP. Acute feeding studies show that the analogue with greater Y4r affinity (PP- 28), had an in vivo bioeffiacy 10-times greater than that of native PP whereas the analogue with lower affinity (PP-27) had a 4-fold greater in vivo anorectic bioefficacy compared to native PP. As with the previous study, plasma levels of all peptides were measured at 45 and 120minutes after a single subcutaneous injection. An earlier measurement at 15minutes was also taken. After 120minutes, plasma levels of all peptides were significantly reduced compared to plasma levels at 15minutes. There was no difference between any of the plasma peptide levels at 15minutes. At 45 and 120minutes, plasma levels of PP-28 were not significantly higher than PP. Plasma levels of PP-27 were significantly increased compared to native PP at both 45 and 120minutes, and significantly raised compared to PP-28 at 45minutes.

As PP-28 performed better in acute feeding studies compared to PP-27, it was surprising to see plasma levels of PP-27 significantly higher than PP-28. Plasma collected from animals administered with PP-27 was therefore chomatographed on a size-exclusion column to fractionate potential breakdown products that were also immunoreactive. However, it was demonstrated that the PP-27 being detected eluted in 1 peak which corresponded to the whole peptide (data not shown). This study suggests that, although increased plasma levels of a PP analogue are significantly increased and sustained as with PP-27, it does not necessarily translate to prolonged anorectic effect in vivo. In fact, PP-28 continued to

186 significantly reduce food intake at longer time points despite plasma levels of PP-28 not being significantly increased compared to that of PP at any of the time points measured. This limitation may have been overcome by the greater affinity of PP-28 at the Y4r and thus the potential for stronger agonism at the receptor.

Amalgamating results from digests with tissues preps and individual enzymes, it was possible to create analogues of PP which combined protection of degradation from the enzymes tested. The changes focused on the N-terminal to protect against DPPIV and the mid-section to protect against NEP, although exact sites were unknown. No changes were made to counter trypsin, as changes at those residues were found to be detrimental to activity.

With every class of changes made to the PP molecule, several properties had to be assessed in order to determine if the change was beneficial or not and why. The properties examined were affinity to Y4r, percentage degradation by NEP in vitro, and bioefficacy as measured by relative effectiveness to native PP in acute feeding studies in mice.

Changes made to the N-terminus of PP were generally well-tolerated at Y4r affinity with the exception of the PP-Cys0(dimer) which had over 2-fold lower affinity at the Y4r. This was largely anticipated as it is known the C-terminal is the part of the peptide which interacts with the receptor active site. NEP-resistance remained fairly unchanged with the exception of Pro0, Thr0, His0 and Cys0(dimer) which were >2-fold more resistant to degradation by NEP. PP-Cys0(dimer) in particular was completely NEP-resistant and, along with PP-Pro0, was more than 2-times more potent at acutely reducing food intake in mice. Based on the assumption that stable secondary and tertiary peptide structure are more resistant to NEP degradation, one can conclude that changes to the N-terminal at sites non-specific to NEP degradation offer greater NEP resistance because of this. Plasma levels of the DPPIV- resistant analogue, PP-Ala0, were elevated by its coadministration with phosphoramidon at both 45 and 90minutes post-injection compared to plasma levels at the same time points without the NEP inhibitor. This data suggests it would be important to inhibit several breakdown mechanisms rather than one alone, to maximise improvement in PK.

Single C-terminal substitutions of Met30 in native PP were also well-tolerated at Y4r affinity and also remained mostly unchanged with susceptibility to degradation by NEP. PP-Glu30

187 was >2-fold more resistant to degradation by NEP compared to native PP and PP-Ile30 was completely NEP-resistant and was also the most efficacious at acute reduction of food intake in mice. PP-Asn30 and PP-Lys30 were also at least 3-times more potent than PP at acute food intake inhibition.

A selection of other single mid-section modifications of PP did not notably improve the parameters measured compared to native PP. Affinity to Y4r could be improved with single substitutions to positions 6 and 19 but these did not translate to an additional increase in potency in vivo.

Combined substitutions at positions 6, 19, 23 and 30 improved affinity at the Y4r. Degradation of these analogues by NEP decreased by >2-fold and the analogue PP-6, in particular, was >3-times more potent in vivo compared to native PP. Analogues with changes at positions 6, 19, 23 and 30 were then investigated in combination with changes that protect against DPPIV. Again, all analogues (PP-9 to PP-17) had improved Y4r affinity and increased resistance against NEP degradation. However, in vivo bioefficacies differed ranging from equal potency to native PP (analogues PP-9 and PP-10) through to 7-times increased bioefficacy compared to native PP (analogue PP-17)

Finally, modifications at position 16 and 17 were explored in combination with all the previous global changes (i.e. substitutions at -1, 0, 6, 19, 23, 30). Single changes at positions 16 and 17 alone had a reduction in the susceptibility to NEP degradation however, there was no improvement in receptor affinity or potency at reducing food intake in acute feeding studies in mice. Combining all of these changes at positions -1, 0, 6, 19, 23, and 30, (analogues PP-19 to PP-24), affinity at Y4r increased 2-5 fold and all analogues tested were completely resistant to degradation by NEP. In vivo bioefficacy also increased from 4 to 7 times compared to native PP.

The same was also true of other analogues of PP with global modifications which had increased susceptibility to in vitro NEP digests (PP-D6,NPY19-23 and PP-K6,NPY19-23). The presence of a section of the NPY α-helix in these analogues considerably increased affinity to Y4r by 5-fold. However, they had 2-3 times more degradation to NEP in vitro compared to native PP. Like PP-Ala0, co-administration of these analogues with phosphoramidon showed significantly higher peptide levels at 45mins post-injection compared to plasma levels at the

188

45mins without the NEP inhibitor. However, by 90mins there was no difference in plasma levels with or without phosphoramidon. This suggests that protection from NEP of these PP analogues is only useful short-term, after which they may become targets for other enzymes that can override the effects of NEP inhibition.

Conversely, PP analogues with global modifications that modestly improved their susceptibility to in vitro NEP digests (PP-P0,A23,K30 and PP-P0,K19,K30) demonstrated insignificant differences in plasma levels in the presence of phosphoramidon at either 45 or 90mins post-injection. The presence of the P0 N-terminal extension makes these 2 analogues DPPIV-resistant and the lack of effect of NEP inhibitor on plasma levels demonstrates that they are also somewhat NEP-resistant in vivo.

The effect of phosphoramidon on circulating plasma levels of the analogue PP-Lys30 appeared halfway between the NEP-resistant and the NEP-susceptible analogues. To this extent, plasma levels of PP-Lys30 decreased significantly from 45 to 90mins post- administration of peptide alone or in combination with phosphoramidon. However, in the presence of phosphoramidon, plasma levels of PP-Lys30 were not much higher than levels without the inhibitor at either time point. Therefore, although phosphoramidon does not appear to prevent degradation in this setting, it is possible that it has been made more prone to degradation by other mechanisms.

In summary, the results from this group of PP analogues suggests that initial resistance to DPPIV is required before there is any beneficial effect of resistance to NEP. Thus, there may be an order of enzyme degradation where DPPIV precedes NEP. It is possible that DPPIV provides the initial breakdown step which destabilises the PP-fold structure and exposes other areas of the molecule to be degraded by other enzymes. In fact, Pro2 found in native PP, forms part of the hydrophobic “zipper” (Nygaard et al., 2006) in the core of the PP-fold structure and is the main interaction partner for Tyr27. Loss of Pro2 following DPPIV degradation causes destabilisation of the PP-fold structure and could make the molecule more unstable in general and, for example, more susceptible to proteolytic degradation.

189

3.4 CONCLUSIONS

This chapter has confirmed that vulnerabilities in a peptide drug candidate can be interpreted from digests with liver and kidney extracts; the routes through which most drugs will take to undergo clearance from the body. From these tissue digests, patterns of cleavage from trypsin, NEP and DPPIV were hypothesised and then tested to confirm the cleavage sites. Once identified, residues around these sites were substituted and not all modifications were found to be beneficial to the affinity of the PP analogue to the Y4r or to their anorectic effect in acute feeding studies in mice. The undesirable substitutions were at Arg33 and Arg35 which, although generated trypsin-resistant analogues, reduced the bioefficacy of the analogue. Utilising only the favourable substitutions (i.e. N-terminal extensions and substitutions at positions 6, 16, 17, 19, 23, and 30), enzyme-resistant analogues were created that may prove more effective pharmacotherapies.

190

CHAPTER 4

INVESTIGATION OF THE IMMUNOGENICITY OF A LONG-ACTING PYY3-36 ANALOGUE IN THE DEVELOPMENT OF A POTENTIAL ANTI-OBESITY AGENT

191

4.1 INTRODUCTION

4.1.1 PYY AS AN ANTI-OBESITY AGENT

The results of PYY administration in humans have made PYY3-36 and its receptor potential targets for development of therapeutic agents for the treatment of obesity (Small and Bloom, 2005, Boggiano et al., 2005, Ueno et al., 2008, Vincent and le Roux, 2008). There were at least two analogues of PYY being developed as treatments for obesity (amylin 2nd gen Y-family mimetics (following from AC162352), 2009; 7TM pharma Obinepitide Y2 + Y4 agonist discontinued in phase II, 2007). However, PYY, like many gut hormones, has a relatively short half-life (Lluis et al., 1989) that limits its use as an anti-obesity agent. By modifying the primary structure of PYY to generate analogues with extended half-life that are still capable of activating the Y2 receptor, and/or to develop a modified-release formula, these could be developed into obesity therapies.

Alanine scans of PYY3-36 and NPY have been used as a method to help identify amino acids important in peptide function and receptor affinity. Substitution of alanine in several positions of PYY3-36 is detrimental to the anorectic effects of PYY3-36 in mice. These included amino acid positions 7, 8, 20, 25, 27 and 29, many of which are required for the hydrophobic interactions to form the PP-fold (Fig 4.1). In addition, substitution of alanine into any of the

5 amino acids at the C-terminal end of PYY3-36 abolishes the anorectic effects of PYY3-36 (Slentz et al., 2004). An NPY alanine scan revealed that the C-terminal hexapeptide is essential for NPY binding to the Y2r (Sjostrom, 2013). These results were consistent with a study investigating the binding of fragments of the NPY molecules, starting from the C- terminal hexapeptide to progressively longer fragments. The C-terminal hexapeptide alone did not show any binding affinity to the Y2r; however long C-terminal fragments encompassing most of the α-helix bound relatively well to the Y2R (Feurle et al., 1980). These results suggest that the C-terminal domain of PYY and NPY is crucial for the Y2r binding (the C-terminal pentapeptide is highly conserved in the PP-fold peptides with identical sequences in PYY and NPY (Tatemoto et al., 1982, Larhammar, 1996a), and the α- helical regions of these 2 peptides, which have a very high homology, are important for higher affinity binding. The importance of the α-helical region of NPY in receptor binding is

192 also demonstrated by large decreases in the affinity to the Y1r, Y2r and Y5r’s when the 19- 23 amino acid segment of the NPY α-helix is exchanged for the PP19-23 segment (Lerch et al., 2005).

NH2

Figure 4.1 Primary amino acid sequence of PYY and schematic representation of stabilising bonds which exist between opposing amino acids of the polyproline- and α-helix.

The native amphipathic α-helix of the PP-fold peptides has two functions in the membrane– compartment model (Sargent and Schwyzer, 1986, Schwyzer, 1995):

1) To direct the peptides to the membrane–water interface 2) To guide the C-terminal receptor binding segment into the correct conformation for activity

The interaction with the membrane stabilises both the α-helix and especially the C terminus, which has been shown to be relatively flexible in solution (Zerbe et al., 2006, Lerch et al., 2004). Recently, it has been suggested that the N-terminal segments bind with low affinity to extracellular Y receptor sections, thereby acting as anchors in the transfer from the

193 membrane-bound state into the genuine binding pocket of the receptor for the peptide hormones (Xu et al., 2003).

We hypothesised that the amphipathic nature of the additional α-helix, and its potentially increased affinity towards the cell membrane, could lead to an accumulation of peptide near the Y receptors. Bringing the peptide from solution to the surface of the cell membrane should increase the probability of the peptide finding the receptor, as well as its ability to activate the receptor multiple times (Pedersen et al., 2010).

The α-helix is the most common motif in protein secondary structures and is involved in the folding and stability of peptide/protein structures and thereby the correct functioning. For example, the α-helical C-terminal region of GLP-1 binds and activates the GLP-1R (Elias et al., 1998, Woloshin and Schwartz, 2014). Ex4, a GLP-1r agonist that shares 53% amino acid identity with GLP-1, binds GLP-1r with a higher affinity and activates the receptor with a higher potency than GLP-1 (Goke et al., 1993). The central region of Ex4 adopts an α-helical conformation and is important for binding with the GLP-1r (Sacks et al., 2009, Franz et al., 2007). Structural analysis of GLP-1 and Ex4 in solution (Runge et al., 2007) in the receptor- bound crystal form suggest that the higher affinity of Ex4 to the GLP-1r is due to superior hydrophilic and hydrophobic interactions in the homologous α-helical regions and a higher propensity of Ex4 to adopt the α-helical conformation in solution (Runge et al., 2008).

Interestingly, the receptor binding α-helical regions of Ex4, and to a lesser extent GLP-1, share a similar homology to an α-helical region in the α-latrotoxin (αLTX) protein (Holz and Habener, 1998) (Table 4.1). αLTX is a toxin isolated from the venom of the black widow spider Latrodectus tredecimguttatus and causes a potent spontaneous discharge of neurotransmitters from nerve endings of motor and sensory neurones (Makris and Foster, 2011). Residues 970-981 of the αLTX protein are highly homologous to the Ex4 α-helical residues 17-28 and are located within the postulated receptor binding domain (Holz and Habener, 1998).

It was therefore investigated whether substituting part of the αLTX (970-975) and Ex4 epitope into the α-helix domain of PYY3-36 would have any effect on its potency and efficacy. The sequence homologies are shown below:

194

Table 4.1 Primary amino acid sequences of the homologous α-helical regions of αLTX, Ex4 and PYY- αLTX. Amino acids positions identical to αLTX are highlighted in grey, Ex4 in blue and those conserved between αLTX, Ex4 and PYY, in yellow.

In previous work, we at the Department of Investigative Medicine, Imperial College London, developed analogues of PYY3-36 utilising several strategies to modify the peptide’s characteristics (Fig 4.2). These include:

1) Modifying the primary sequence of the peptide by substituting amino acids which increase the tendency to form an α-helix sequence while keeping the essential C-terminal pentapeptide intact in a bid to improve receptor binding

2) Introduction of a proline at the N-terminus of PYY3-36, a residue in PYY1-36, PP and NPY which forms hydrophobic interactions with the conserved Tyr27 residue (Glover et al., 1984) to strengthen the PP-fold structure. Reintroducing the proline at the N-terminal end of PYY3-

36 (to make PYY2-36) may help stabilise the peptide by lowering peptide dynamics and increasing resistance to proteolyic degradation (Nygaard et al., 2006, Schwartz et al., 1990).

3) Modifying the peptide charge and therefore isoelectric point (pI). By adjusting the pI to near neutral, this would tend to decrease the solubility of the peptide at physiological pH values, increase the tendency of the peptide to precipitate subcutaneously to create a slow release depot.

4) Introduction of Histidine residues. Histidines interact with metal ions. In combination with a metal salt solution, this strategy of introducing histidine residues is intended to favour peptide aggregate formation

195

While not the primary focus of this work, the importance of the pharmacokinetics for an obesity therapy based on PYY3-36 is critical. With this in mind, and the knowledge that PYY3-36 has a short half-life, two points were considered:

 extended half-life in circulation  slow release (SR) from a subcutaneous (SC) depot

The half-life issue was addressed by assessing the resistance of the peptides to degradation of known peptidases and the increased resistance to this degradation by specific amino acid substitutions. For the SR strategy, the approach taken was based on insulin SR preparations such as insulin glargine (Lantus, Sanofi-Aventis). With these preparations, Zinc is used to causes the peptide to aggregate and form a SC depot.

196

Insertion of αLTX Addition of Proline at N-terminus

Combination of N-terminal extension and αLTX insert

 Affinity to hY2r  Acute effect on food intake  Pharmacokinetics Introduction of 4 Histidine  Effect of chronic administration residues

Removal of αLTX insert

Figure 4.2 Design of PYY analogue. The PYY3-36 analogues designed in the investigation of a longer acting and/or more potent analogue of

PYY3-36 at the human Y2 receptor (hY2r)

197

Following these strategies, analogue PYY2-36-αLTX was created which is an analogue of PYY with an alpha-latrotoxin (αLTX) motif and Pro2 N-terminal extension. This created a highly active Y2R agonist but half-life was insufficiently extended to make it suitable for further development. Substitution of Histidines in PYY2-36-αLTX to create the analogue PYY2-36-αLTX- 4H maintained its activity but also resulted in a slow release from subcutaneous depots when formulated in a zinc chloride solution in the same manner as long acting preparations of insulin which uses zinc as a stabilising agent to promote hexamer formation of the insulin molecule (Farooqi et al., 1999).

4.1.2 IMMUNOGENICITY

Immunogenicity is defined as the ability of a substance (the antigen/drug) to induce an immune response by producing antibodies. Protein and peptide therapeutics are especially prone to provoke an immunological response. The consequences of immunogenicity can range widely from no noticeable effects to rare cases of severe reactions (Kessler, Goldsmith et al. 2006). Therefore immunogenicity cannot safely be ignored.

There are 2 types of immune response: innate and adaptive.

Innate responses are usually nonspecific to any kind of foreign material (pathogen/antigen). Components of this immune system include physical barriers/mechanisms such as skin, cilia in mucous membranes, tears, nasal secretions and saliva. Additionally, many pathogens and infected cells produce chemokines that increase permeability of blood vessels and allows migration of white blood cells (phagocytic cells such as granulocytes (neutrophils), macrophages, and dendritic cells) towards the site of infection. Macrophages release substances that attract other white blood cells (e.g. basophils) and mast cells to the site of infection releasing histamine and heparin that increases blood flow to damaged tissues causing redness and inflammation. They also release chemotaxic substances that attract neutrophils and eosinophils to the area which ingest bacteria and other foreign cells, help immobilise and kill parasites, and secrete enzymes that digest and kill cells (Wren et al., 2000).

198

The adaptive immune response is a specific response against one type of antigen. It is mediated by lymphocytes, which include T cells and B cells. The responses of these cells displays specificity and memory. All lymphocytes are derived from bone marrow stem cells but T cells develop in the thymus while B cells develop in the bone marrow.

B cells are responsible for the production of antibodies that act against extracellular pathogens. T cells are mainly involved with cellular immune responses to intracellular pathogens such as viruses. They also regulate the responses of B cells and the overall immune response.

B cells express specific cell-surface antigen receptors (immunoglobulins) and, when mature, can secret soluble immunoglobulins (antibodies) into the extracellular fluid. Each B cell expresses an immunoglobulin which is specific for a particular antigen. If a B cells binds to its specific antigen, it will multiply and differentiate into plasma cells, which produce large amounts of the secreted antibody. This can be found in blood and tissue fluids and, because they are a soluble version of the original immunoglobulin, they bind to the same antigen that initially activated the B cell (Fig 4.3). Each antibody binds to a restricted part of the antigen called an epitope. A particular antigen can have several epitopes but antibodies are specific for one epitope rather than the whole antigen.

There are several different types of T cells. Helper T cells are required for B cells to divide, differentiate and make antibody.

199

Figure 4.3 Production of antibodies by B cells. The entire population of B cells has the ability to specifically bind to millions of different antigens. When an antigen binds to the few B cells that can recognise it (clonal selection), the cell is induced to undergo proliferation and differentiation to become memory cells and plasma cells. Memory cells ensure that subsequent detection of that same antigen receives a more rapid response, while plasma cells secrete large amounts of antigen- specific antibody. Therefore a primary humoral immune response results from the activation of previously unstimulated naïve B cells, whereas secondary responses are due to stimulation of memory B cells.

200

Normally, antibody production (immunogenicity) is stimulated in response to proteins from outside the body (e.g. from non-human sources, or altered version of human proteins). This immune response causes rapid production of neutralising antibodies in a process mediated by T cell activation of B cells. During development, the immune system is exposed to endogenous proteins, and develops tolerance to them. However, this tolerance can break down (as in the case of autoimmune diseases) and, in this instance, binding antibodies can be produced. This process is normally reversed when the product is withdrawn (Kessler et al., 2006).

When drugs are administered, a number of features may elicit an immune antibody response:

1) Product-related factors:  Structural properties: the protein sequence, the presence or absence of epitopes (sequence recognised as an antigen), the degree of glycosylation influencing the protein degradation, the exposure of antigen sites and solubility.  Formulation and storage which influences the levels of contaminants and impurities. 2) Host-related factors:  Genetic predisposition to immune response (linked to the inheritance of major histocompatibility complex alleles).  With certain conditions, the complete absence of a molecule from the host means that immune cells recognising this molecule are not deleted during maturation in the thymus. When these are introduced to the host, the immune system will recognise these as non-self-antigens. An example is that of haemophilia A, due to the deletion of the Factor VIII gene. When recombinant Factor VIII is introduced to such patients, this is recognised as a non-self-antigen and this leads to the production of neutralising antibodies.  Concomitant illness or auto immune diseases.

201

3) Dose or route of administration:  A higher dose and increased duration of treatment is likely to be more immunogenic as there is a higher concentration or increased presentation of a potential epitope that can be recognised by T cells to elicit antigenicity (Otto et al., 2001, Jarkovska et al., 2004).  Subcutaneous administration is more immunogenic, whereas intravenous and local/topical administration appear to be less immunogenic. The increased rates of injection site reactions following subcutaneous administration may reflect stronger activation of immunocompetent cells, leading to highly efficient antigen presentation (Herzog, 2014). This is probably due to deposition of antigen in the tissues which creates a large contact volume between antigen and immune cells (antigen presenting cells and lymphocytes) distributed in subcutaneous tissue (Fisch et al., 1996).

A summary of immunogenic effects include:

1) No effect. Antibody development can be noticed in a large proportion of patients (8- 60%) given the GLP-1 analogues exenatide and liraglutide but this does not impact on the efficacy or safety of the drug (Buse et al., 2011). 2) Loss of efficacy of the drug due to the antibodies which can neutralise drug activity. Increasing the dose may overcome these antibodies; however the raised dose may lead to other side effects, 3) Alteration of drug clearance. Non-neutralising antibodies may increase the clearance of the drug via the reticuloendothelial system. An example of this are non- neutralising antibodies to interferon alpha which develop during treatment for Hepatitis C (Schellekens, 2002). 4) The antibodies may cross-react with an essential naturally-occurring molecule, e.g. antibodies against megakaryocyte-derived growth factor cross-react with thrombopoietin and cause thrombocytopenia (Neumann and Foote, 2000). 5) Antibody production can induce general effects such as anaphylaxis or allergic reactions. This potentially lethal side effect is nowadays less frequent with the increasing availability of highly purified products.

202

Considering the number of biopharmaceuticals used and the percentage of observed effects among human population, immunogenicity remains a rare side effect.

Measuring immunogenicity is challenging, but it becomes increasingly important as more agents based on endogenous molecules are produced. Two steps are required to measure immunogenicity. The first involves measuring the ability of serum antibodies to bind exogenous peptides. If serum has a high affinity for exogenous peptides, this suggests a high level of antibodies, which may subsequently elicit an immunogenic reaction. Several types of assays can be used, including a Radioimmunoprecipitation Assay (RIPA) or Enzyme- Linked Immunosorbent Assay (ELISA). These measure both neutralising and non-neutralising antibodies. These assays are rarely standardised, so can be difficult to compare. The second step required to detect a neutralising antibody is a bioassay that measures the ability of the serum to neutralise the biological effects of the exogenous peptide (Jahn and Schneider, 2009). It should be noted, however, that any system examining immunogenicity where peptides from one species are tested in a second species can cause false positives (i.e. a drug based on the human sequence of a peptide administered to a rodent may cause an immune reaction which may not be applicable in humans).

Molecule size determines, in part, the immunogenicity of a drug. Polypeptides and proteins with molecular weights >10000 Da have a relatively high incidence of immunogenicity, whereas compounds with molecular weights <1000 Da have relatively low immunogenicity. Immunogenicity of compounds between this size range is unpredictable (Considine et al., 1996). Low molecular weight compounds can be made more immunogenic by covalent bonding to proteins to form hapten-protein complexes. This sometimes exploited deliberately, as when raising antibodies using carbodiimide coupling (Deen et al., 1990).

203

A number of approaches to prevent or reduce immunogenicity have been proposed: (Schellekens, 2002):

 PEGylation, the covalent attachment of polyethylene glycol (PEG) to lysine molecules on a protein’s surface reduces immunogenicity, possibly due to a physical “shielding” of the epitope by PEG.  Altering protein structure so that the immune-eliciting epitope is eliminated, either through replacing specific amino acids in the protein, or through exon shuffling within the protein-coding regions of the gene.  Humanisation of monoclonal antibodies, by replacing nonhuman regions with corresponding human sequences, may eliminate antigenic epitopes.

204

4.1.3 HYPOTHESES AND AIMS

4.1.3.1 Hypotheses

 PYY2-36-αLTX-4H is capable of reducing food intake in animal models of obesity

 PYY2-36-αLTX-4H is immunogenic  The αLTX motif is the most immunogenic epitope in the peptide

4.1.3.2 Aims

1) To investigate the immunogenicity of PYY2-36(αLTX-4H) and the potential active region of the peptide responsible for this reaction. 2) To design PYY analogues by amino acid substitutions, PEGylation, and/or changes in formulation, specifically aiming to reduce immunogenicity. 3) To incorporate changes into analogues that maintains or improves efficacy and affinity at the Y2 receptor. To confirm receptor specificity by testing affinity to other members of the Y-receptor family. 4) To investigate the acute effects of analogues on food intake. 5) To characterise the chronic effects of analogues on food intake, body weight, and immunogenicity in rodent models of obesity 6) To investigate the pharmacokinetics of PYY analogues

205

4.2 RESULTS

4.2.1 CHARACTERISATION OF PYY2-36(αLTX-4H)

4.2.1.1 Y1r and Y2r Receptor affinity

PYY3-36 bound to the hY2r with an IC50 of 0.25 ± 0.01nM while PYY2-36(αLTX-4H) had an IC50 which was approximately 3-fold worse (PYY2-36(αLTX-4H) IC50: 0.80 ± 0.07nM) than that of the endogenous ligand (Fig 4.4(A)).

PYY1-36 and PYY2-36 bound with similar affinities to the hY1r (PYY1-36 IC50: 0.25 ± 0.07nM vs.

PYY2-36 IC50: 0.32 ± 0.02 nM) while PYY3-36 had an IC50 which was approximately 4.5-fold worse (PYY3-36 IC50: 1.12 ± 0.25nM) than that of the endogenous hY1r ligand, PYY1-36 (Fig

4.4(B)). Specific Y2r agonist (Y2A), NPY-Leu28,31, had an IC50 which was approximately 180- fold worse (Y2A IC50: 46.88 ± 2.17nM) compared to that of PYY1-36.

PYY2-36(αLTX-4H) had a hY1r binding affinity that was 4 times worse that of PYY1-36 (Fig 4.4(D))

Finally, as efficacy of appetite reduction of these PYY analogues was assessed in mice, binding to the mouse Y2 receptor (mY2r) was completed for comparison (Fig 4.4(C)). PYY2-

36(αLTX-4H) bound to the mY2r with the same affinity as to the hY2r (PYY3-36 IC50: 0.24 ±

0.07nM, PYY2-36 (αLTX-4H) IC50: 0.78 ± 0.07nM)

206

PYY IC : 0.25 ± 0.01nM PYY1-36 IC50: 0.25  0.07nM 100 3-36 50 100 PYY2-36 IC50: 0.32  0.02nM PYY2-36(LTX-4H) IC50: 0.80 ± 0.07nM PYY3-36 IC50: 1.12  0.25nM 80 80 Y2A IC50: 46.88  2.17nM

60 60

40 40 % Specific Binding Specific % % Specific Binding Specific % 20 20 A B 0 0 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 log [Peptide] M log [Peptide] M

PYY IC : 0.24  0.07nM PYY IC : 0.25  0.07nM 100 50 100 1-36 50 PYY2-36(LTX-4H) IC50: 0.78  0.07nM PYY2-36(LTX-4H) IC50: 1.08nM

80 80

60 60

40 40

% Specific Binding Specific % Binding Specific % 20 20 C D 0 0 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 log [Peptide] M log [Peptide] M

Figure 4.4 Binding affinities of PYY and analogues to human and mouse Y2r and human Y1r. (A)

Binding affinity of PYY3-36 and PYY2-36(αLTX-4H) to the hY2r; (B) Binding affinity of PYY1-36, PYY2-36,

PYY3-36, and Y2A to the hY1r; (C) Binding affinities of PYY3-36 and PYY2-36 (αLTX-4H) to the mY2r; (D)

Binding affinities of PYY1-36 and PYY2-36 (αLTX-4H) to the hY1r. In all cases, binding affinities are

expressed as IC50 values calculated from the mean ± SEM of 3-5 separate experiments

207

4.2.1.2 Comparison of PYY2-36(αLTX-4H) against PYY3-36 on food intake in mice

PYY3-36 and PYY2-36(αLTX-4H) at 100nmol/kg significantly reduced food intake in mice at 0-1h post injection (p < 0.01 vs. saline for PYY3-36 and p < 0.001 for PYY2-36- αLTX-4H). Whilst a single s.c. injection of PYY3-36 (100nmol/kg) resulted in a significant reduction of food intake in the first time period only, PYY2-36(αLTX-4H) at the same dose, significantly reduced food intake during the additional time points of 2-4 and 4-8h post injection when compared with saline control (2-4h: saline 0.49 ± 0.04g vs. PYY2-36(αLTX-4H) 0.06 ± 0.04g, p < 0.05; 4-8h: saline 0.99 ± 0.09g vs. PYY2-36(αLTX-4H) 0.67 ± 0.05g, p < 0.05).

PYY2-36(αLTX-4H) significantly reduces food intake compared to PYY3-36 at the time intervals of 2-4 and 4-8h (2-4h: PYY2-36(αLTX-4H) 0.06 ± 0.04g vs. PYY3-36 0.36 ± 0.05g, p < 0.05; 4-

8h:PYY2-36(αLTX-4H) 0.67 ± 0.05g vs PYY3-36 0.89 ± 0.04g, p < 0.05). There were no significant changes in food intake at the earlier time points between PYY3-36 and PYY2-36(αLTX-4H) (Fig 4.5).

208

5 saline 4 PYY3-36 (100nmol/kg) 1.2 PYY2-36(LTX-4H) (100nmol/kg) 1.0 *

0.8 * ** 0.6 *** * Food intake (g) intake Food 0.4

0.2 * 0.0

0-1 1-2 2-4 4-8 8-24

Time (hours)

Figure 4.5 Effect of subcutaneous administration of 100nmol/kg PYY3-36 and PYY2-36(αLTX-4H) on food intake in fasted C57BL/6 mice (n=4-5). Peptides or vehicle (saline) administered in the early light phase. Data are shown as mean ± SEM food intake for 0-1, 1-2, 2-4, 4-8 and 8-24 hour intervals. One-way ANOVA with a Dunnett’s post-hoc used to test for significance vs. saline control (black stars) and a Tukey’s post-hoc to test for significance between the treatment groups at each time point (red stars) (*=p<0.05; **=p<0.01; ***=p<0.001)

4.2.1.3 Acute effect of a dose response of PYY2-36(αLTX-4H) on food intake in mice

At the lowest dose of 100nmol/kg (Fig 4.6), a single s.c. injection of PYY2-36(αLTX-4H) significantly reduced food intake in mice during the time periods 0-1, and 1-2h post injection (0-1h: p < 0.01 vs. saline and 1-2h: p < 0.001 vs. saline). At higher doses of 300 and

500nmol/kg, PYY2-36(αLTX-4H) caused significant decrease in food intake at 0-1h when compared to the saline control group (saline: 0.98 ± 0.03g vs. 300nmol/kg PYY2-36(αLTX-4H):

0.68 ± 0.02g, p < 0.01; 500nmol/kg PYY2-36(αLTX-4H): 0.66 ± 0.04g, p < 0.01) but not during the time period of 1-2h. All doses of PYY2-36(αLTX-4H) caused significant decrease in food intake at 2-4 and 4-8h when compared to saline (p < 0.001 vs. saline for all groups at both time periods). At 8-24h post injection, the lowest dose of PYY2-36(αLTX-4H) did not

209 significantly reduce food intake, however, there was a significant dose-dependent reduction of food intake with the 300 and 500nmol/kg doses (saline: 3.57 ± 0.11g vs. 300nmol/kg PYY2-

36(αLTX-4H): 2.46 ± 0.34g p < 0.05; 500nmol/kg PYY2-36(αLTX-4H): 1.46 ± 0.16g p < 0.001).

PYY2-36(αLTX-4H) at all the doses tested, significantly reduced food intake over the whole 0- 24h period (all p < 0.001 compared with saline) (Fig 4.7).

6 saline

4 PYY2-36(LTX-4H) (100nmol/kg) * PYY2-36(LTX-4H) (300nmol/kg) 2 *** PYY2-36(LTX-4H) (500nmol/kg)

1.0 0.8 **

0.6 Food intake (g) intake Food 0.4

0.2 *** *** *** 0.0

0-1 1-2 2-4 4-8 8-24 Time (hours)

Figure 4.6 Effect of subcutaneous dose response of 100, 300, and 500nmol/kg PYY2-36(αLTX-4H) on food intake in fasted C57BL/6 mice (n=7-8). Peptides or vehicle (saline) administered in the early light phase. Data are shown as mean ± SEM food intake for 0-1, 1-2, 2-4, 4-8 and 8-24 hour intervals. One-way ANOVA with a Dunnett’s post-hoc test used to test for significance vs. saline control (*=p<0.05; **=p<0.01; ***=p<0.001)

210

0-24 hour 6

5 ***

(g) intake Food

saline

100nmol/kg300nmol/kg500nmol/kg

Figure 4.7 Cumulative 24hour food intake after administration of 100, 300, and 500nmol/kg PYY2-

36(αLTX-4H) (n=7-8). Data are shown as mean ± SEM food intake. One-way ANOVA with a Dunnett’s post-hoc test used to test for significance vs. saline control (*=p<0.05; **=p<0.01; ***=p<0.001)

211

4.2.1.4 The importance of Zn in the pharmacokinetic of a single SC injection of PYY2-36(αLTX-4H) in rat

Following a single SC injection of PYY3-36 at 50mg/ml formulated in zinc chloride solution to achieve a 1:1 Zn to peptide molar ratio, a maximum plasma concentration (Cmax) of 55,603 ± 2,807pmol/L was observed at 30mins post injection (Fig 4.8(A)). At 3h post injection, plasma concentrations of PYY3-36 had decreased to significantly lower levels of 895 ± 209pmol/L.

The levels of PYY3-36 in plasma remained low at 24h post injection (1312 ± 217pmol/L) which was not significantly different from PYY3-36 plasma levels seen at 3h.

The Cmax reached following a single s.c. injection of 50mg/ml PYY2-36(αLTX-4H) in 1:1 zn was at 48h post injection at about 3,400 ± 813pmol/L (Fig 4.8(B)). Prior to Tmax (time taken to achieve maximum plasma concentrations), plasma levels rose slowly from 30mins (333 ± 56pmol/L) to 3h (808 ± 93pmol/L) and then approximately doubled by 24h (1947 ± 593pmol/L) post injection.

*** * A 80000 *** * 60000 4000 40000 B 2000 ns 3000 1500 (pmol/L) 2000 LTX-4H) (pmol/L) LTX-4H) 3-36 1000  ( 1000 PYY

500 2-36

of concentration Plasma Plasma concentration of concentration Plasma 0 PYY 0 30mins 3h 24h 30mins 3h 24h 48h

Figure 4.8 Pharmacokinetics after a single s.c. injection of (A) 1mg/animal PYY3-36 and (B)

0.5mg/animal PYY2-36(αLTX-4H), both at 50mg/ml (n=3 at all time points). Data are shown as mean ± SEM plasma concentrations. One-way ANOVA with a Bonferroni’s post-hoc test used to test for significance between time points (*=p<0.05; **=p<0.01; ***=p<0.001)

212

Following a single SC injection of PYY2-36(αLTX-4H) in rats, at a concentration of 20mg/ml, formulated in zinc chloride solution to achieve a 1:1 molar ratio of Zn to peptide, a Cmax of 714 ± 54pmol/L was observed at 24h post injection (Fig 4.9). Conversely the same dose of

PYY2-36(αLTX-4H) formulated in normal saline (0.9% w/v) had a Cmax of 683 ± 58pmol/L at 60mins post injection. The plasma levels at 15mins post injection are significantly higher in the injection of PYY2-36(αLTX-4H) not formulated with Zn (549 ± 44pmol/L without Zn; 285 ± 19pmol/L 1:1 Zn).

Plasma concentration of PYY2-36(αLTX-4H) formulated without Zn remains approximately 2- fold higher than the formulation containing Zn until its Cmax at 60mins. At 24h post injection, no significant difference in plasma concentrations were observed, although a trend for the Zn-formulated peptide to have increased levels was noted (546 ± 46pmol/L without Zn; 714 ± 54pmol/L 1:1 Zn p = 0.786).

800 *** PYY2-36(LTX-4H) without Zn

** 600

400

(pmol/L) LTX-4H)  ( PYY2-36(LTX-4H) with 1:1 Zn

2-36 200 Plasma concentrationPlasma of PYY

0 15m 30m 60m 24h

Figure 4.9 Pharmacokinetics after SC administration of 20mg/ml PYY2-36(αLTX-4H) in saline not containing Zn (solid circles/line) and in the presence of 1:1 Zn (open circles, dotted line) (n=4 at all time points). Data are shown as mean ± SEM plasma concentrations as measured by RIA. Unpaired student’s t-test was used to test for significance at each time point (*=p<0.05; **=p<0.01; ***=p<0.001) 213

Longer studies were completed where rats received a single SC injection of PYY2-36(αLTX-4H)

(50mg/ml) formulated in 1:1 molar Zn ratio. Again, Cmax was reached at 48h post injection

(2733 ± 147pmol/L) (Fig 4.10). The AUC from 0h to day7 (AUC7d) for PYY2-36(αLTX-4H) 1:1 Zn 6 was 0.35 × 10 pmol.h/L. The profile of PYY2-36(αLTX-4H) showed a steady rise to Cmax with no initial burst, followed by a slow decrease in plasma levels with only a 30% reduction from

Cmax at day 7.

3000

2000

LTX-4H) (pmol/L) LTX-4H)



( 1000

2-36 Plasma concentration of concentration Plasma PYY 0

0 1h 3h 6h day 1 day 2 day 4 day 7

Figure 4.10 Pharmacokinetic profile of a single subcutaneous injection PYY2-36(αLTX-4H) at

50mg/ml. A basal plasma sample was collected immediately prior to administration of PYY2-36(αLTX- 4H) (1mg/rat) and further plasma samples collected at 1, 3 and 6h, 1, 2, 4 and 7 days later (n=2-5). Data are shown as mean ± SEM plasma concentrations as measured by RIA.

214

4.2.1.5 Effect of chronic administration of PYY2-36(αLTX-4H) on food intake and body weight in DIO mice

Chronic administration of PYY2-36(αLTX-4H) attenuated weight gain in DIO mice (Fig 4.11(B)). At the start of the study, groups were stratified by body weight and there was no significant difference between their mean body weights (body weight on day 0: vehicle: 31.7 ± 0.8g vs.

3000nmol/kg PYY2-36(αLTX-4H): 31.0 ± 0.6g, p > 0.05).

Daily SC injection of 3000nmol/kg PYY2-36(αLTX-4H) in a slow release formulation (1:1 molar ratio of peptide to zinc) significantly decreased food intake on day 1 of the study and a cumulative reduction in food intake was maintained throughout the study (cumulative food intake on day 29: vehicle: 73.1 ± 2.0g vs. 3000nmol/kg; PYY2-36(αLTX-4H): 61.9 ± 1.2g, p <

0.001) (Fig 4.11(A)). The mean body weight of PYY2-36(αLTX-4H) treated animals was significantly lower than that of the vehicle group 24h after the first administration, and continuing throughout the study (body weight on day 1: vehicle: 32.3 ± 0.8g vs.

3000nmol/kg PYY2-36(αLTX-4H): 30.0 ± 0.6g, p < 0.001; body weight on day 29: vehicle: 38.0

± 1.4g vs. 3000nmol/kg PYY2-36(αLTX-4H): 32.5 ± 0.5g, p < 0.01) (Fig 4.11(B)).

80 *** 70 A Vehicle

30 PYY2-36(LTX-4H) (3000nmol/kg)

(g) intake food Cumulative 20

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Day 215

Day 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

-2 Vehicle

-4

-6 PYY2-36(LTX-4H) (3000 nmol/kg)

-8

-10 % body weight loss (corrected% toloss weight body mean body weight of vehicle group) vehicle of weight body mean B -12 ***

Figure 4.11 Effect of subcutaneous administration of PYY2-36(αLTX-4H) (3000nmol/kg) on (A) cumulative food intake (data shown as mean ± SEM) and (B) body weight (data shown as mean percentage weight loss corrected to mean vehicle body weight ± SEM), in C57BL/6 DIO mice (n=13). Peptide and vehicle (saline) administered daily. GEE and Mann-Whitney statistical tests used to test significance (***=p<0.001 vs. vehicle).

216

4.2.1.6 Effects of chronic administration of PYY2-36(αLTX-4H) on food intake and body weight in male Wistar rats

There was no significant difference between the average body weights of the vehicle control group, and the PYY2-36(αLTX-4H) group at the start of the study.

Daily SC injection of PYY2-36(αLTX-4H) 1:1 Zn at 500nmol/kg did not cause a significant reduction in food intake or body weight change. Daily SC injection of PYY2-36(αLTX-4H) 1:1 Zn at 3000nmol/kg significantly decreased food intake on day 6 of the study and continued to cause a decrease in food intake until day 13 (cumulative food intake on day 13: vehicle:

406.7 ± 7.5g vs. 3000nmol/kg PYY2-36(αLTX-4H): 368.3 ± 5.2g, p < 0.05) (Fig 4.12(A)).

PYY2-36(αLTX-4H) (3000nmol/kg/day) significantly reduced body weight from day 6 onwards

(body weight day 13: vehicle: 477 ± 7g vs. 3000nmol/kg PYY2-36(αLTX-4H): 458 ± 8g, p < 0.05) (Fig 4.12(B)).

400 A 350 vehicle

300

250

200 PYY2-36(LTX-4H) (500nmol/kg)

PYY2-36(LTX-4H) (3000nmol/kg) 150 * cumulative food intake (g) intake food cumulative 100

0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Day

217

Day 0 1 2 3 4 5 6 7 8 9 10 11 12 13

1 vehicle

PYY2-36(LTX-4H) (500 nmol/kg)

-1

-2

% body weight loss (corrected to% loss weight body

mean body weight of vehicle group) vehicle of weight body mean -3 PYY2-36(LTX-4H) (3000 nmol/kg) B -4 *

Figure 4.12 Effect of subcutaneous administration of PYY2-36(αLTX-4H) (500 and 3000nmol/kg) on (A) cumulative food intake (data are shown as mean ± SEM) and (B) body weight (data are shown as mean percentage weight loss corrected to mean vehicle body weight ± SEM) in male Wistar rats (n=6-14). Peptide and vehicle (saline) administered daily. GEE and Mann-Whitney statistical tests used to test significance (*=p<0.05, PYY2-36(αLTX-4H) 3000nmol/kg vs. saline).

218

4.2.2 IMMUNOGENICITY

4.2.2.1 Immunogenicity of chronic administration of PYY2-36(αLTX-4H)

Analogue PYY2-36(αLTX-4H) underwent further investigation and its in vivo properties investigated in the canine species (outsourced to Huntingdon Life Sciences, Cambridgeshire, UK). As part of the study, plasma samples were collected from dogs following 10 injections of PYY2-36(αLTX-4H) (5, 25 and 100mg/kg) each 3 days apart. Using the radioimmunoprecipitation assay (RIPA), plasma samples were tested for the presence of antibodies against the therapeutic agent (antidrug antibodies – ADAs) and against the native peptide from which the analogue was structured on. Results showed the presence of antibodies against PYY2-36(αLTX-4H) in all tested dogs who received the compound (Table 4.2).

Dilution Neat 1:200 1:700 1:2000 Average binding (%) 84.8 45 35.5 29.5

Table 4.2 Immunogenicity of plasma from dog pharmacokinetic study 7 days after last injection – testing the presence of antibodies against PYY2-36(αLTX-4H) and the antibody titre.

In light of these results, terminal plasma samples collected from the chronic rat study

(section 4.2.1.6) were tested for the presence of antibodies against PYY2-36(αLTX-4H). Positive samples were identified as those with increased percentage binding of radiolabel (≥4-fold) compared to control groups. Results showed that plasma from 13 of the 14 rats injected with 500nmol/kg PYY2-36(αLTX-4H) and plasma from all the rats injected with

3000nmol/kg PYY2-36(αLTX-4H) contained antibodies against PYY2-36(αLTX-4H) (table 3.2). Further tests of the plasma demonstrated from 2 of the 14 rats injected with 500nmol/kg

PYY2-36(αLTX-4H) and plasma from 3 of the 6 rats injected with 3000nmol/kg PYY2-36(αLTX- 4H) contained antibodies against native PYY (Table 4.3).

219

Day 14 - Terminal plasma

PYY3-36 PYY2-36(αLTX-4H) immunogenicity immunogenicity Dose Positive % binding of whole Positive % binding of whole Treatment group (nmol/kg) rats group (20µl sample) rats group (20µl sample) Saline - 5.1 - 12.4

PYY2-36(αLTX-4H) 500 2/14 9.3 13/14 56.5

PYY2-36(αLTX-4H) 3000 3/6 22.9 6/6 68.3

Table 4.3 Immunogenicity of terminal plasma from the rat chronic study – testing the presence of

antibodies against PYY3-36 and PYY2-36(αLTX-4H).

4.2.2.2 Effect of Zn precipitation on immunogenicity

The amount of Zn in the formulation was investigated as a potential contributor to the

immunogenicity of PYY2-36(αLTX-4H). PYY2-36(αLTX-4H) was administered subcutaneously to rats at a concentration of 20mg/ml (0.5mg/rat) formulated with zinc at a molar ratio of either 0, 0.125, 0.25, 0.5 and 1:1 every 3 days and samples collected by tail venesection on

day 15 and 30. As a control PYY3-36 was tested using the same protocol and formulated with zinc at a molar ratio of either 0 or 1:1. Results showed that there was no change in the

percentage binding of iodinated-PYY3-36 between day 15 and day 30 (Table 4.4). An

approximate 2-3 fold increase in percentage binding of iodinated PYY2-36(αLTX-4H) was

observed with all molar ratios of zinc to PYY2-36(αLTX-4H) between day 15 and 30 (Table 4.4) which reflects an increase in levels of ADAs.

220

15 days after immunisation 30 days after immunisation # positive % binding of whole # positive % binding of whole Peptide/condition rats group (30ul sample) rats group (30ul sample) PYY3-36 (0 Zn) 0/5 9.5 0/5 9.1 PYY3-36 (1 Zn:1) 0/5 10.1 0/5 9.2 PYY2-36(αLTX-4H) (0 Zn) 0/5 10 4/5 33.2 PYY2-36(αLTX-4H) (0.125 Zn:1) 0/6 9.7 3/6 23.7 PYY2-36(αLTX-4H) (0.25 Zn:1) 1/4 19.9 3/5 39.1 PYY2-36(αLTX-4H) (0.5 Zn:1) 1/5 14.4 3/5 43.1 PYY2-36(αLTX-4H) (1 Zn:1) 0/5 12.4 3/5 36.5

Table 4.4 Effect of zinc chloride on immunogenicity. Plasma samples were collected from rats which

received SC injections of PYY3-36 or PYY2-36(αLTX-4H) and were assessed for ability to bind

radiolabelled PYY or PYY2-36(αLTX-4H).

4.2.2.3 Immunogenicity of PYY2-36(αLTX-4H) fragments

Fragments of PYY2-36(αLTX-4H) consisting of residues 2-28, 2-14, 7-36, 13-36 and 27-36 were 125 tested for their ability to compete with I-PYY2-36(αLTX-4H) for binding to ADAs (Fig 4.13). The source of antibodies used for these tests were pooled samples from dogs, rats, and 125 sheep which bound significant levels of I-PYY2-36(αLTX-4H).

Results are summarised in Table 4.5. In plasma from rat, dog and sheep, the fragment 7-36

had the closest cross-reactivity to full length PYY2-36(αLTX-4H), across all cold peptide doses tested (approximately 33% cross-reactive at 0.1pmol, 50% at 1pmol, and almost 80% at 10pmol). In dog and rat plasma, both the N and C terminal truncated fragments, 13-36 and 2-28, cross reacted with the ADAs with a trend of fragment 13-36 cross reacting with higher affinity. The shorter fragments, 2-14 and 27-36, also cross reacted with ADAs in dog and rat plasma.

Tests with sheep plasma showed fragments 2-14 had low affinity to the ADAs, and fragment 2-28 had approximately 40% cross-reactivity at all doses of the cold peptide tested. In sheep plasma fragment 13-36 has poor cross-reactivity at 0.1pmol of cold peptide and good cross-

221 reactivity at 10pmol of cold peptide. Conversely, fragment 2-28 had high cross-reactivity at 0.1pmol of cold peptide compared to low cross reactivity at 10pmol of cold peptide.

Figure 4.13 Fragments of PYY2-36(αLTX-4H) tested for cross reactivity with plasma containing ADAs. P = proline at position 2.

222

Concentration Dog Rat Sheep Peptide of peptide antibody antibody antibody (pmol)

0.1 1 1 1 Full 1 1 1 1 length 10 1 1 1

0.1 0.34 0.54 0.69 1 0.53 0.40 0.97 7-36 10 0.77 0.42 1

0.1 0.13 0.52 0.30 1 0.17 0.23 0.50 13-36 10 0.33 0.22 0.74

0.1 0.08 0.01 0.31 1 0.01 0.03 0.03 2-14 10 0.01 0 0.08

0.1 0.12 0.15 0.42 1 0.16 0.10 0.42 2-28 10 0.24 0.22 0.47

0.1 0.03 0 0.63 1 0.04 0 0.31 27-36 10 0.07 0 0.32

Table 4.5 Summary of the displacement ratio of PYY2-36(αLTX-4H) fragments with dog, rat and sheep plasma. Results are expressed as the ratio of the percentage binding of the fragments at various concentrations, in comparison with full length PYY2-36(αLTX-4H).

223

125 To assess specificity of the ADAs, the ability to displace I-PYY2-36(αLTX-4H) at low concentrations (0.1pmol) of competing non-radiolabelled peptide or fragments was used. The average cross reactivity from the three species for each fragment is expressed as a ratio of the cross reactivity of PYY2-36(αLTX-4H) and the results are summarised in Table 4.6.

At low concentrations of peptide, fragment 7-36 shares the most similar binding to full length PYY2-36(αLTX-4H) (50% cross-reactivity), followed by 13-36 at 30%. Fragments 2-28 and 27-36 have similar cross-reactivity at 0.1pmol of cold peptide (20%) while fragment 2-14 had low levels of cross-reactivity to the anti-PYY2-36(αLTX-4H) antibodies across all the species tested.

The order of cross-reactivity is much the same when ratios from all concentrations tested were averaged, suggesting the fragments specificity correlates with their avidity.

Average ratio of % binding Average ratio at all doses

of 0.1pmol peptide of peptide (compared to

Peptide (compared to full length full length PYY2-36(αLTX-

PYY2-36(αLTX-4H)) 4H))

Full length 1 1 7-36 0.52 0.63

13-36 0.32 0.35

2-14 0.13 0.06

2-28 0.23 0.26

27-36 0.22 0.16

Table 4.6 Average of the displacement ratios of PYY2-36(αLTX-4H) fragments against dog, rat and sheep antibodies. Results are expressed as the average ratio of the percentage binding of the fragments at low (0.1pmol) concentrations and across all concentrations, in comparison with full length PYY2-36(αLTX-4H).

224

4.2.2.4 Immunogenicity of PYY2-36(αLTX-4H) analogues

A series of analogues, all variations of PYY2-36(αLTX-4H), were tested for their ability to 125 compete with I-PYY2-36(αLTX-4H) for binding to PYY2-36(αLTX-4H) antibodies (Fig 4.14). This test was completed as part of a strategy to identify the specific amino acid(s) or sequence of amino acids which bound to the antibodies raised when 125I-PYY2-36(αLTX-4H) was injected into dog, rat and sheep.

Groups of analogues based on two variations of PYY2-36(αLTX-4H) were designed: those that contain the αLTX sequence (peptides 2-5) and those without the αLTX sequence (peptides 6- 11). In each group, the removal of the Pro2 extension was investigated as was the progressive decrease in the number of His residues.

Figure 4.14 Analogues of PYY2-36(αLTX-4H) tested for cross reactivity to PYY2-36(αLTX-4H) antibodies. P = proline at position 2.

225

A screen of the PYY2-36(αLTX-4H) analogues at 0.1, 1 and 10 pmol to bind to the plasma from rat, dog and sheep containing ADAs was completed and is summarised in Table 4.7.

With ADAs from dog and rat, peptides 2, 3, and 4 bound with almost full cross reactivity, as 125 assessed by their ability to displace I-PYY2-36(αLTX-4H). Peptides 5 and 6 had reduced cross-reactivity (approximately 60%) to the dog and rat plasma. Peptides 7, 8, and 10 125 competed with similar cross-reactivity, displacing approximately 45% of I-PYY2-36(αLTX- 4H). Peptides 9 and 11 bound with the least affinity to ADAs in dog and rat (approximately 30% binding).

ADAs from sheep were found to be less discriminating with analogues of PYY2-36(αLTX-4H) tested at high concentrations. All peptides tested bound at ≥ 60% compared to PYY2-36(αLTX- 4H) itself. However, at low concentrations of competing unlabelled peptide, only peptides 2, 3, 4, and 5 bound with 100% cross-reactivity. Peptides 6-11 had low cross-reactivity, 125 displacing only a small percentage of I-PYY2-36(αLTX-4H).

226

Concentration of Dog Rat Sheep Key Peptide peptide (pmol) antibody antibody antibody

0.1 1 1 1 1 1 1 1 1 PYY (αLTX-4H) 2-36 10 1 1 1

0.1 0.9 0.8 1 1 0.8 1 1 2 PYY (αLTX-4H) 3-36 10 0.9 1 1

0.1 0.9 0.6 0.9 1 0.7 0.7 1 3 PYY (αLTX-3H) 2-36 10 0.8 0.9 1

0.1 0.8 0.7 0.9 1 0.8 0.7 0.9 4 PYY (αLTX-3H) 2-36 10 0.9 0.7 1

0.1 0.7 0.3 0.9 1 0.6 0.6 1 5 PYY (αLTX-2H) 2-36 10 0.6 0.7 1

0.1 0.7 0.3 0.5 1 0.7 0.6 0.7 6 PYY (4H) 2-36 10 0.8 0.7 0.8

0.1 0.5 0.1 0.5 1 0.6 0.3 0.8 7 PYY (2H) 2-36 10 0.7 0.7 1

0.1 0.5 0.1 0.4 1 0.4 0.3 0.7 8 PYY (2H) 2-36 10 0.6 0.5 0.9

0.1 0.4 0.1 0.5 1 0.3 0.2 0.6 9 PYY (1H) 2-36 10 0.4 0.3 0.6

227

0.1 0.3 0.1 0.5 1 0.5 0.4 0.9 10 PYY (1H) 3-36 10 0.7 0.8 1

0.1 0.4 0 0.4

11 PYY2-36 1 0.3 0.1 0.5 10 0.3 0.2 0.8

Table 4.7 Summary of the displacement ratio of PYY2-36(αLTX-4H) analogues with dog, rat and sheep plasma. Results are expressed as the ratio of the percentage binding of the fragments at various concentrations, in comparison with full length PYY2-36(αLTX-4H).

To assess specificity of the ADAs, the ability of low concentrations (0.1pmol) of 125 non-radiolabelled PYY2-36(αLTX-4H) analogues to displace I-PYY2-36(αLTX-4H) was tested. Results show analogues containing the αLTX sequence (peptides 2-5) binding with a high degree of cross reactivity, greater than 60%; and those without the αLTX motif (peptides 6- 11) binding less than 50% (Table 4.8).

Amongst all peptides, (either in the presence or absence of the αLTX sequence), the ability of the analogue to bind ADAs decreases with decreasing His residues.

228

Average ratio of % binding of Average ratio at all doses Key Peptide 0.1pmol peptide (compared of peptide (compared to to PYY2-36-αLTX-4H) PYY2-36-αLTX-4H)

1 PYY2-36(αLTX-4H) 1 1

2 PYY3-36(αLTX-4H) 0.90 0.93

3 PYY2-36(αLTX-3H) 0.80 0.83

4 PYY2-36(αLTX-3H) 0.80 0.82

5 PYY2-36(αLTX-2H) 0.63 0.71

6 PYY2-36(4H) 0.50 0.64

7 PYY2-36(2H) 0.37 0.58

8 PYY2-36(2H) 0.33 0.49

9 PYY2-36(1H) 0.33 0.38

10 PYY3-36(1H) 0.30 0.58

11 PYY2-36 0.27 0.33

Table 4.8 Average ratios of PYY2-36(αLTX-4H) fragments against dog, rat and sheep ADAs. Results are expressed as the average ratio of the percentage binding of the fragments at low (0.1pmol) concentrations and across all concentrations, in comparison with full length PYY2-36(αLTX-4H).

229

4.2.3 ASSESSMENT OF ALTERNATIVES TO PYY2-36(αLTX- 4H)

Two alternative strategies were explored in an attempt to produce an analogue of PYY which maintained the anorexigenic and pharmacokinetic properties of PYY2-36(αLTX-4H) without the immunogenic side effect. To the above described studies a PEGylated version,

PYY2-36(αLTX-4H)-PEG, was produced and tested. In parallel to the investigation of PYY2-

36(αLTX-4H)-PEG, analogue PYY2-36(4H) was also selected for further investigation as a result of the immunogenicity screens using peptide fragments and analogues (section 4.2.2.3 and 4.2.2.4).

4.2.3.1 PEGylation of PYY2-36(αLTX-4H) – PYY2-36(αLTX-4H)-PEG

4.2.3.1.1 Y1r and Y2r RBA

PYY3-36 bound to the hY2r with an IC50 of 0.19 ± 0.01nM while PYY2-36(αLTX-4H) had an IC50 which was approximately 3-fold worse (PYY2-36(αLTX-4H) IC50: 0.57 ± 0.11nM) than that of the endogenous ligand (Fig 4.15(A)).

PYY1-36 and PYY2-36 bound with similar affinities to the hY1r (PYY1-36 IC50: 0.25 ± 0.07nM vs.

PYY2-36 IC50: 0.32 ± 0.02 nM) while PYY3-36 had an IC50 which was approximately 4.5-fold worse (PYY3-36 IC50: 1.12 ± 0.25nM) than that of the endogenous hY1r ligand, PYY1-36 (Fig

4.15(B)). Specific Y2r agonist (Y2A), [Leu28, Leu31] NPY, had an IC50 which was approximately 180-fold worse (Y2A IC50: 46.88 ± 2.17nM) compared to that of PYY1-36.

PYY2-36(αLTX-4H) had a hY1r binding affinity that was 3.5 times worse that of PYY1-36. The binding affinity of PYY2-36(αLTX-4H)-PEG compared to the unPEGylated peptide at the hY1r was 2.5-fold worse (IC50: 2.21 ± 0.30nM) and thus almost 9 times worse than that of PYY1-36 (Fig 4.15(D))

Finally, as efficacy of appetite reduction of these PYY analogues was assessed in mice, binding to the mouse Y2 receptor (mY2r) was completed for comparison (Fig 4.15(C)). PYY2-

36(αLTX-4H) bound to the mY2r with the same affinity as to the hY2r (PYY3-36 IC50: 0.24 ±

230

0.07nM, PYY2-36 (αLTX-4H) IC50: 0.54 ± 0.11nM) however, PYY2-36(αLTX-4H)-PEG was almost

5-fold worse compared to the unPEGylated peptide at the mY2r (IC50: 2.61 ± 0.42nM) and

twice as bad as PYY2-36(αLTX-4H)-PEG at the hY2r (IC50: 1.32 ± 0.17nM).

PYY IC : 0.19 ± 0.01nM PYY IC : 0.25  0.07nM 100 3-36 50 100 1-36 50 PYY2-36 IC50: 0.32  0.02nM PYY2-36(LTX-4H) IC50: 0.57 ± 0.11nM

PYY3-36 IC50: 1.12  0.25nM PYY2-36(LTX-4H)-PEG IC50: 1.32 ± 0.17nM 80 80 Y2A IC50: 46.88  2.17nM

60 60

40 40 % Specific Binding Specific % % Specific Binding Specific % 20 20 A B

0 0 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 log [Peptide] M log [Peptide] M

PYY IC : 0.24  0.07nM PYY IC : 0.25  0.07nM 100 50 100 1-36 50

PYY2-36(LTX-4H) IC50: 0.54  0.11nM PYY2-36(LTX-4H) IC50: 0.87  0.09nM

PYY2-36(LTX-4H)-PEG IC50: 2.61  0.42nM PYY2-36(LTX-4H)-PEG IC50: 2.21  0.30nM 80 80

60 60

40 40

% Specific Binding Specific % Binding Specific % 20 20 C D 0 0 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 log [Peptide] M log [Peptide] M

Figure 4.15 Binding affinities of PYY and PYY analogues to human and mouse Y2r and human Y1r. (A)

Binding affinity of PYY3-36 and PYY2-36(αLTX-4H) to the hY2r; (B) Binding affinity of PYY1-36, PYY2-36,

PYY3-36, and Y2A to the hY1r; (C) Binding affinity of PYY3-36 and PYY2-36(αLTX-4H) to the mY2r; (D)

Binding affinities of PYY1-36 and PYY2-36(αLTX-4H) to the hY1r. In all cases, binding affinities are

expressed as IC50 values calculated from the mean ± SEM of 3-5 separate experiments

231

4.2.3.1.2 Effect of PYY2-36(αLTX-4H)-PEG on food intake in mice

At the lowest dose of 200nmol/kg, a single SC injection of PYY2-36(αLTX-4H)-PEG significantly reduced food intake in mice in time 0-1, and 1-2h interval post injection (Fig 4.16; p < 0.01 vs. saline at both time intervals). At higher doses of 1000 and 4000nmol/kg, PYY2-36(αLTX- 4H)-PEG caused significant decrease in food intake at 0-1, 1-2, 2-4, and 4-8h when compared to the saline control group (4-8h saline: 1.11 ± 0.05g vs. 1000nmol/kg PYY2-

36(αLTX-4H)-PEG: 0.50 ± 0.10g, p<0.01; 4000nmol/kg PYY2-36(αLTX-4H)-PEG: 0.20 ± 0.03g, p<0.001). At 8-24h post injection, none of the doses of PYY2-36-αLTX-4H-PEG tested significantly reduce food intake. The lowest dose of PYY2-36(αLTX-4H)-PEG did not significantly reduce food intake over the whole 0-24h period, however the two higher doses did (1000nmol/kg p<0.05; 4000nmol/kg p < 0.001, both compared to saline) (Fig 4.17).

7 saline 5 PYY2-36(LTX-4H)-PEG (200nmol/kg)

PYY2-36(LTX-4H)-PEG (1000nmol/kg) 3 PYY (LTX-4H)-PEG (4000nmol/kg) 1.4 2-36 1.2 1.0

0.8 ** *** ** Food intake (g) intake Food 0.6 * 0.4 ** *** 0.2 ** *** 0.0

0-1 1-2 2-4 4-8 8-24

Time (hours)

Figure 4.16 Effect of subcutaneous administration of 200, 1000 and 4000nmol/kg PYY2-36(αLTX-4H)- PEG on food intake in fasted mice C57BL/6 mice (n=5). Peptides or vehicle (saline) administered in the early light phase. Data are shown as mean ± SEM food intake food intake for 0-1, 1-2, 2-4, 4-8 and 8-24 hour intervals. One-way ANOVA with a Dunnett’s post-hoc test used to test for significance vs. vehicle control (*=p<0.05; **=p<0.01; ***=p<0.001).

232

0-24 hour 7.0 6.5

6.0 * 5.5

5.0 4.5 ***

(g) intake Food 4.0 3.5

3.0

saline 200nmol/kg 1000nmol/kg4000nmol/kg

Figure 4.17 Cumulative 24hour food intake after administration of 200, 1000, and 4000nmol/kg PYY2-

36(αLTX-4H)-PEG (n=5). Data are shown as mean ± SEM food intake. One-way ANOVA with a Dunnett’s post-hoc test used to test for significance vs. saline control (*=p<0.05; **=p<0.01; ***=p<0.001)

233

4.2.3.1.3 Pharmacokinetics of PYY2-36(αLTX-4H)-PEG with Zn in rat

The Cmax observed following a single SC injection of 50mg/ml PYY2-36(αLTX-4H)-PEG administered in a 1:1 molar Zn ratio resulted in a Cmax of 43649 ± 6851pmol/L at 24h post injection (Fig 4.18) compared to Cmax at 48h for the unPEGylated version (Fig 4.10). Plasma levels of PYY2-36(αLTX-4H)-PEG decreased by more than half Cmax by day 2 (14832 ±

3405pmol/L; 66% reduction from Cmax) and were further reduced by day 4 (4666 ±

1860pmol/L; 89% reduction from Cmax). The AUC7d for PYY2-36(αLTX-4H)-PEG 1:1 zn was 2.30 6 × 10 pmol.h/L which is approximately 6.5-fold higher than the non-PEGylated PYY2-36(αLTX- 4H).

60000

40000

(pmol/L) LT-4H)  ( 20000 2-36

Plasma concentration Plasma

PYY of 0

0h 4h day 1 day 2 day 4 day 7

Figure 4.18 Pharmacokinetic profile of a single subcutaneous injection of PYY2-36(αLTX-4H)-PEG to male Wistar rats. A basal plasma sample was collected immediately prior to administration of PYY2-

36(αLTX-4H)-PEG (50mg/ml (1mg/rat)) and further plasma samples collected at 4h and 1, 2, 4 and 7 days later (n=4), data are shown as mean plasma levels of PYY2-36(αLTX-4H)-PEG ± SEM as determined by RIA.

234

4.2.3.1.4 Immunogenicity of PYY2-36(αLTX-4H)-PEG

PYY2-36(αLTX-4H)-PEG formulated in saline administered once every 3 days over a 30 day period to rats. As a control PYY2-36(αLTX-4H) was inoculated without zinc using the same protocol. The number of rats testing positive for the presence of antibodies against the test compound was lower in those treated with the PEGylated form (Table 4.9)

15 days after immunisation 30 days after immunisation

% binding of % binding of # positive whole group # positive whole group Peptide/condition rats (30ul sample) rats (30ul sample)

PYY2-36(αLTX-4H) (0 Zn) 0/5 10 4/5 33.2

PYY2-36(αLTX-4H)-PEG (0 Zn) 0/5 8.9 1/5 12.1

Table 4.9 Effect of PEGylation on immunogenicity. Plasma samples were collected from rats which received SC injections of PYY2-36(αLTX-4H) or PYY2-36(αLTX-4H)-PEG and were assessed for ability to bind radiolabelled PYY2-36(αLTX-4H) or PYY2-36(αLTX-4H)-PEG.

235

4.2.3.2 Removal of αLTX – PYY2-36(4H)

Analogue PYY2-36(4H) was shown to be less immunogenic than PYY2-36(αLTX-4H) (Table 4.8), and was therefore tested for its bioefficacy and pharmacokinetic profile.

4.2.3.2.1 Y1r and Y2r RBA

PYY3-36 and PYY2-36(4H) bound with similar affinities to the hY2r (PYY3-36 IC50: 0.25nM ±

0.01nM vs. PYY2-36(4H) IC50: 0.27 ± 0.02 nM) (Fig 4.19(A)).

Control peptides for hY1r (PYY1-36, PYY2-36, PYY3-36, and Y2A) were as previously seen in section 3.1.1.2. At the hY1r, PYY2-36(4H) had a binding affinity that was 4-6 times worse than that of PYY1-36 (Fig 4.19(D))

Finally, as we would be testing the efficacy of appetite reduction of these PYY analogues in mice, we tested binding against the mouse Y2 receptor (mY2r) for comparison (Fig 4.19(C)).

Both peptides bound to the mY2r with the same affinity as to the hY2r (PYY3-36 IC50: 0.24nM

± 0.07nM, PYY2-36(4H) IC50: 0.22 ± 0.03nM)

236

PYY1-36 IC50: 0.25  0.07nM 100 PYY IC50: 0.25  0.01nM 100 PYY2-36 IC50: 0.32  0.02nM PYY2-36(4H) IC50: 0.27  0.02nM PYY3-36 IC50: 1.12  0.25nM 80 80 Y2A IC50: 46.88  2.17nM

60 60

40 40 % Specific Binding Specific % Binding Specific % 20 20 A B

0 0 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 log [Peptide] M log [Peptide] M

100 PYY1-36 IC50: 0.25  0.07nM

100 PYY IC50: 0.24  0.07nM PYY2-36(4H) IC50: 1.52nM

PYY2-36(4H) IC50: 0.22  0.03nM 80 80

60 60

40 40 % Specific Binding Specific % % Specific Binding Specific % 20 20 C D 0 0 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 log [Peptide] M log [Peptide] M

Figure 4.19 Binding affinities of PYY and PYY analogues to human and mouse Y2r and human Y1r. (A)

Binding affinity of PYY3-36 and PYY2-36(4H) to the hY2r. (B) Binding affinity of PYY1-36, PYY2-36, PYY3-36,

and Y2A to the hY1r. (C) Binding affinity of PYY3-36 and PYY2-36(4H) to the mY2r. (D) Binding affinity of

PYY1-36 and PYY2-36(4H) to the hY1r. In all cases, binding affinities are expressed as IC50 values calculated from the mean ± SEM of 3-5 separate experiments.

237

4.2.3.2.2 Comparison of PYY2-36(4H) against PYY3-36 on food intake in mice

A single s.c. injection of PYY3-36 at 100nmol/kg significantly reduced food intake in mice during the time periods 0-1 and 1-2h post injection (p < 0.01 vs. saline at 0-1h and p < 0.05 at 1-2h). However, at the same dose, a single s.c. injection of PYY2-36(4H) did not significantly reduced food intake during the first time point of 0-1h but significantly reduced food intake during the following time periods of 1-2 and 2-4h when compared with saline control

(saline: 0.42 ± 0.04g vs. PYY2-36(4H): 0.04 ± 0.01g, at 1-2h p < 0.001; saline: 0.62 ± 0.04g vs.

PYY2-36(4H): 0.27 ± 0.08g, at 2-4h p < 0.05). At 8-24h post injection, only PYY3-36 caused a significant increase in food intake compared to the saline group (saline: 3.61 ± 0.09g vs.

PYY3-36: 4.08 ± 0.13g p < 0.05), however there was also a trend (although not significant) for

PYY2-36(4H) to cause an increase in food intake at this time point. There were no other significant changes in food intake at any other time points (Fig 4.20).

7 saline 5 PYY3-36 (100nmol/kg) *

PYY (4H) (100nmol/kg) 3 2-36 1.2 1.0

0.8 0.6

Food intake (g) intake Food 0.4 ** * * 0.2 ** 0.0

0-1 1-2 2-4 4-8 8-24

Time (hours) Figure 4.20 Effect of subcutaneous administration of 100nmol/kg PYY3-36 and PYY2-36(4H) on food intake in fasted C57BL/6 mice (n=5). Peptides or vehicle (saline) administered in the early light phase. Data are shown as mean ± SEM food intake for 0-1, 1-2, 2-4, 4-8 and 8-24 hour intervals. One- way ANOVA with a Dunnett’s post-hoc test used to test for significance vs. saline control (*=p<0.05; **=p<0.01; ***=p<0.001)

238

4.2.3.2.3 Effect of acute administration of PYY2-36(4H) on food intake in mice (dose response study without Zn formulation)

A single s.c. injection of PYY2-36(4H) at 100, 300 and 500nmol/kg significantly reduced food intake in mice during the time periods 0-1, 1-2, and 2-4h post injection (Fig 4.21; p < 0.001 vs. saline for all groups at 0-1 and at least p < 0.05 at 1-2 and 2-4h). At 4-8h post injection,

only 300 and 500nmol/kg doses of PYY2-36(4H) caused significant decrease in food intake

when compared to the saline control group (saline: 0.83 ± 0.08g vs. 300nmol/kg PYY2-36(4H):

0.42 ± 0.02g, p < 0.01 and 500nmol/kg PYY2-36(4H): 0.21 ± 0.05g, p < 0.001). All doses of

PYY2-36(4H) caused a significant increase in food intake in the 8-24h time interval compared

to the saline group (saline: 3.71 ± 0.05g vs. 100nmol/kg PYY2-36(4H): 4.21 ± 0.08g;

300nmol/kg PYY2-36(4H): 4.33 ± 0.10g; 500nmol/kg PYY2-36(4H): 4.34 ± 0.14g; all p < 0.01).

Only the 300 and 500nmol/kg doses of PYY2-36(4H) significantly reduced food intake over the whole 24h period (both p < 0.001 compared with saline) (Fig 4.22).

6 saline ***

5 PYY2-36(4H) (100nmol/kg) **

4 PYY2-36(4H) (300nmol/kg) 3 PYY (4H) (500nmol/kg) 1.2 2-36

1.0

0.8

0.6 *** Food intake (g) intake Food * ** 0.4 *** 0.2 * *** *** ** 0.0

0-1 1-2 2-4 4-8 8-24 0-24

Time (hours)

Figure 4.21 Effect of subcutaneous dose response of 100, 300, and 500nmol/kg PYY2-36(4H) on food intake in fasted mice C57BL/6 mice (n=5-7). Peptides or vehicle (saline) administered in the early light phase. Data are shown as mean ± SEM food intake for 0-1, 1-2, 2-4, 4-8 and 8-24 hour intervals. One-way ANOVA with a Dunnett’s post-hoc test used to test for significance vs. saline control (*=p<0.05; **=p<0.01; ***=p<0.001)

239

6.5 0-24 hour

6.0

*** 5.5

5.0

(g) intake Food 4.5

4.0

saline 100nmol/kg300nmol/kg500nmol/kg

Figure 4.22 Cumulative 24hour food intake after administration of 100, 300, and 500nmol/kg PYY2-

36(4H) (n=5-7). Data are shown as mean ± SEM food intake. One-way ANOVA with a Dunnett’s post- hoc test used to test for significance vs. saline control (*=p<0.05; **=p<0.01; ***=p<0.001)

240

4.2.3.2.4 Effect of Zinc on acute administration of PYY2-36(4H) on food intake in mice

As previously found, a single s.c. injection of PYY2-36(4H) at 500nmol/kg (without Zn) can significantly reduce food intake in mice at 0-1, 1-2, 2-4 and 4-8h post injection (Fig 4.21; p <

0.001 vs. saline at 0-1, 1-2, 4-8h; p < 0.05 vs. saline at 2-4h). By the 8-24h time period, PYY2-

36(4H) without Zn did not continue to inhibit food intake, however overall during the 0-24h period, it did significantly reduce food intake when compared to the saline control group

(saline: 5.98 ± 0.12g vs. PYY2-36(4H) no Zn: 4.82 ± 0.14g p < 0.01). In the presence of 1:1

molar ratio of Zn, PYY2-36(4H) at 500nmol/kg significantly inhibited food intake at all time periods measured post injection (p < 0.001 vs. saline at 0-1, 1-2, 2-4, 4-8, 8-24, and 0-24h) (Fig 4.23 and Fig 4.24).

7 *** saline 5 *** ** PYY (4H) no Zn (500nmol/kg) 2-36 3 *** PYY2-36(4H) 1:1 Zn (500nmol/kg) *** 1 1.0

0.8

0.6 * Food intake (g) intake Food *** 0.4 *** *

0.2

*** *** 0.0

0-1 1-2 2-4 4-8 8-24 0-24

Time (hours)

Figure 4.23 Effect of Zn on subcutaneous administration of 500nmol/kg PYY2-36(4H) on food intake in fasted C57BL/6 mice (n=8). Peptides or vehicle (saline) administered in the early light phase. Data are shown as mean ± SEM food intake for 0-1, 1-2, 2-4, 4-8 and 8-24 hour intervals. One-way ANOVA with a Dunnett’s post-hoc test used to test for significance vs. saline control (*=p<0.05; **=p<0.01; ***=p<0.001)

241

0-24 hour

6 ***

5 **

*** (g) intake Food 2

no Zn saline 1:1 Zn

Figure 4.24 Cumulative 24hour food intake after administration of 500nmol/kg PYY2-36(4H) with and without 1:1 Zn (n=8). Data are shown as mean ± SEM food intake. One-way ANOVA with a Dunnett’s post-hoc test used to test for significance vs. saline control (*=p<0.05; **=p<0.01; ***=p<0.001)

242

4.2.3.2.5 Pharmacokinetics of PYY2-36(4H) investigating dose response of Zn formulation

The Cmax observed following s.c. injection of 50mg/ml PYY2-36(4H) (no Zn) was at 30mins post injection at about 2,728 ± 306pmol/L. The same dose of PYY2-36-4H administered in a 0.5:1 molar Zn ratio resulted in a Cmax of about 4,466 ± 663pmol/L at 24h post injection. The time taken to achieve maximum plasma concentrations of PYY2-36(4H) (Tmax) was further delayed when the same dose of PYY2-36(4H) was administered in a 1:1 molar Zn ratio resulting in Cmax of about 6,141 ± 772pmol/L at 48h post injection (Fig 4.25).

The area under the curve over the 48h period (AUC48h) for PYY2-36(4H) without Zn was

26,366pmol.h/L. AUC48h for PYY2-36(4H) in 0.5:1 and 1:1 Zn was 171,306 and

157,708pmol.h/L respectively. The relative bioavailability of PYY2-36(4H) 0.5:1 Zn was 650% of PYY2-36(4H) administered without zn, and 600% relative bioavailabilty for PYY2-36(4H) 1:1 zn compared to that of PYY2-36(4H) without Zn.

7000 1:1 Zn 6000

5000

4000

(4H) (pmol/L) 0.5:1 Zn 3000 2-36

PYY 2000 Plasma concentration of concentration Plasma

1000 0 Zn 0

0 30m 3h 24h 48h

Figure 4.25 Pharmacokinetic profile in rat of PYY2-36(4H) when administered SC at 50mg/ml dose with various amounts of Zn in the formulation (n=3 at all time points). A basal plasma sample was collected immediately prior to administration of PYY2-36(4H) and further plasma samples collected at

30m, 3, 6, 24, and 48h later. Data are shown as mean plasma levels of PYY2-36(4H) ± SEM as determined by RIA. 243

Longer time course studies of the pharmacokinetic profile in rat of a single SC injection of

PYY2-36(4H) (50mg/ml) formulated in 1:1 molar Zn ratio demonstrated Cmax was reached at

24h post injection (10,860 ± 2,102pmol/L) (Fig 4.26). Plasma levels of PYY2-36(4H) remain elevated at 2 and 4 days post-injection (day 2: 6,990pmol/L ± 1,045pmol/L; day 4: 7,799 ±

1,282pmol/L) both approximately a 35% decrease from Cmax level.

14000

12000

10000

8000

(4H) (4H) (pmol/L) 6000

2-36 4000

Plasma concentration Plasma of PYY of 2000

0 1h 3h 6h day 1 day 2 day 4 day 7

Figure 4.26 Pharmacokinetic profile of a single subcutaneous injection PYY2-36(4H). A basal plasma sample was collected immediately prior to administration of PYY2-36(4H) (50mg/ml - 1mg/rat) and further plasma samples collected at 1, 3 and 6h, 1, 2, 4 and 7 days later. At all time points, n≥3. Data are shown as mean plasma levels of PYY2-36-4H ± SEM as determined by RIA.

6 The AUC7d for PYY2-36(4H) 1:1 Zn was 1 × 10 pmol.h/L compared with the AUC7d for PYY2- 6 36(αLTX-4H) 1:1 Zn, which was 0.35 × 10 pmol.h/L (section 4.2.1.4).

244

4.2.4 FURTHER CHARACTERISATION OF PYY2-36(4H)

4.2.4.1 Comparison of acute effect of subcutaneous administration of PYY2-36(4H) and PYY2-36(αLTX-4H) on food intake in mice

PYY2-36(4H) and PYY2-36(αLTX-4H) at 100nmol/kg significantly reduced food intake in mice at 0-1 and 1-2h post injection (p < 0.001 vs. saline for both peptides). Both peptides additionally reduced food intake to a significant degree during the 2-4h time period (saline:

0.53 ± 0.05g vs. PYY2-36(4H): 0.32 ± 0.04g p < 0.01; vs. PYY2-36(αLTX-4H): 0.08 ± 0.02g p <

0.001). At 8-24h post injection, both peptides caused a significant increase in food intake compared to the saline group (saline: 3.73 ± 0.06g vs. PYY2-36(4H): 4.09 ± 0.08g p < 0.05; vs.

PYY2-36(αLTX-4H): 4.34 ± 0.12g p < 0.001). PYY2-36(αLTX-4H) significantly reduced food intake over the whole 0-24h period (p < 0.001 compared with saline) (Fig 4.27).

PYY2-36(αLTX-4H) significantly reduces food intake compared to PYY2-36(4H) at the time intervals of 1-2, 2-4 and 4-8h (1-2h p < 0.05; 2-4h p < 0.001; 4-8h p < 0.001). It also significantly reduced food intake over the whole 0-24h period compared to PYY2-36(4H) (Fig 4.28; p < 0.05).

245

7 saline

5 PYY2-36(4H) (100nmol/kg) *** * PYY2-36(LTX-4H) (100nmol/kg) 3 *** 1.2 1.0

0.8 *** 0.6 *** Food intake (g) intake Food *** 0.4 * **

0.2 *** *** 0.0

0-1 1-2 2-4 4-8 8-24

Time (hours)

Figure 4.27 Effect of subcutaneous administration of 100nmol/kg PYY2-36(4H) and PYY2-36(αLTX-4H) on food intake in fasted C57BL/6 mice (n=11-12). Peptides or vehicle (saline) administered in the early light phase. Data are shown as mean ± SEM food intake for 0-1, 1-2, 2-4, 4-8 and 8-24 hour intervals. One-way ANOVA with a Dunnett’s post-hoc test used to test for significance vs. saline control (*=p<0.05; **=p<0.01; ***=p<0.001)

6.5 * saline

PYY2-36-4H 6.0 PYY2-36-LTX-4H *** 5.5

5.0

Food intake (g) intake Food 4.5

4.0

0-24 hour

Figure 4.28 Cumulative 24hour food intake after administration of 100nmol/kg PYY2-36(4H) and PYY2-

36(αLTX-4H) (n=11-12). Data are shown as mean ± SEM food intake. One-way ANOVA with a Dunnett’s post-hoc test used to test for significance vs. saline control (*=p<0.05; **=p<0.01; ***=p<0.001)

246

4.2.4.2 Pharmacokinetics of PYY2-36(4H) at high concentrations – Zn dose range finding

The Cmax observed following s.c. injection of 200mg/ml PYY2-36(4H) (0.25:1 molar Zn ratio – lowest ratio tested) was at 24h post injection at about 120,000 ± 6790pmol/L. The same dose of PYY2-36(4H) administered in a 0.5:1 molar Zn ratio resulted in a Cmax of about 63,500

± 7840pmol/L at 24h post injection also. The Tmax of PYY2-36(4H) is delayed when the same dose of PYY2-36(4H) is administered in a 1:1 molar Zn ratio resulting in Cmax of about 27,000 ± 2914pmol/L at 7 days post injection (Fig 4.29).

6 The AUC12d for PYY2-36(4H) (0.25:1 Zn) was 8.0 × 10 pmol.h/L. AUC12d for PYY2-36(4H) in 0.5:1 and 1:1 Zn was 6.8 × 106and 5.9 × 106pmol.h/L respectively. The relative bioavailability of

PYY2-36(4H) in 0.25:1 and 0.5:1 Zn was fairly similar within the error margins of the RIA. PYY2-

36(4H) administered with 1:1 Zn had 75% of the relative bioavailability of PYY2-36(4H) administered with the lowest amount of Zn.

The PK profile of PYY2-36(4H) 1:1 Zn up to 7days is in stark contrast to PYY2-36(4H) 1:1 Zn at 50mg/ml (4 times lower concentration than tested here at 200mg/ml) (fig 3.22; section

3.3.2.5). Tmax has increased from 24h to 7days, AUC7d has increased 6-fold, however Cmax has only increased by 3-fold.

247

0.25:1 Zn 100000 50000 0.5:1 Zn 30000

1:1 Zn

(4H) (4H) (pmol/L) 20000

2-36

10000 PYY Plasma concentration of concentration Plasma 0

0h 3h day 1 day 4 day 7 day 12

Figure 4.29 Pharmacokinetics after administration of PYY2-36(4H) at 200mg/ml dose containing various amounts of Zn in the formulation (n=3-4 at all time points). A basal plasma sample was collected immediately prior to administration of PYY2-36-4H and further plasma samples collected at

3h, 1, 4, 7, and 12 days later. Data are shown as mean plasma levels of PYY2-36-4H ± SEM as determined by RIA.

4.2.4.3 Comparison of pharmacokinetics of PYY2-36(4H) and PYY2-36(αLTX- 4H) at high concentrations with 1:1 Zn

The Cmax observed following a single s.c. injection of 200mg/ml PYY2-36(4H) containing 1:1 Zn was at 4 days post injection at about 5,457 ± 476pmol/L. The same dose of PYY2-36(αLTX-4H), also administered in a 1:1 molar Zn ratio resulted in a Cmax of 3,194 ± 569pmol/L at 48h post injection (Fig 4.30).

The AUC8d for PYY2-36(4H) 33,086pmol.h/L compared with the AUC8d for PYY2-36(αLTX-4H) 1:1

Zn, which was 19,414pmol.h/L. The relative bioavailability of PYY2-36(αLTX-4H) 1:1 Zn was only 60% that of PYY2-36(4H) also with 1:1 Zn.

248

4000 7000

6000 PYY (LTX-4H) Plasmaconcentration of

2-36 PYY 3000 5000 2-36

4000 (pmol/L)(4H) 2000

LTX-4H) (pmol/L) LTX-4H) 3000  ( PYY2-36(4H)

2-36 2000 1000 Plasma concentration of concentration Plasma PYY 1000

0 0

0 1 2 4 5 8 Days after SC injection

Figure 4.30 Pharmacokinetic profile after administration of PYY2-36(4H) and PYY2-36(αLTX-4H) both at 200mg/ml dose containing 1:1 Zn in the formulation (n=3-4 at all time points). A basal plasma sample was collected immediately prior to administration of peptides and further plasma samples collected at 1, 2, 4, 5, and 8 days later. Data are shown as mean ± SEM plasma concentrations as determined by RIA.

4.2.4.4 Effect of chronic administration of PYY2-36(4H) on food intake and body weight in DIO mice

There was no significant difference between the average body weights of the vehicle control

group and the PYY2-36(4H) group at the start of the study.

Daily s.c. injection of PYY2-36(4H) 1:1 Zn at 300nmol/kg from day 0-9, followed by daily s.c.

injection of PYY2-36(4H) 1:1 Zn at 1200nmol/kg from day 10-20, followed by daily s.c.

injection of PYY2-36(4H) 1:1 Zn at 2400nmol/kg from day 21-28, significantly decreased food intake on day 1 of the study and continued to cause a decrease in food intake until day 28

249

(cumulative food intake by day 28: vehicle: 80.6 ± 1.3g vs. 2400nmol/kg PYY2-36(4H): 67.7 ± 1.2g, p < 0.001) (Fig 4.31(A)).

The PYY2-36(4H) group had a lower average body weight than the vehicle group and the difference in body weight was significant from day 1 until day 28 (body weight on day 1: vehicle: 43.5 ± 1.2g vs. 300nmol/kg PYY2-36(4H): 41.7 ± 0.7g, p < 0.01; body weight on day

28: vehicle: 47.8 ± 1.4g vs. 2400nmol/kg PYY2-36(4H): 42.8 ± 1.1g, p < 0.001) (Fig 4.31(B)).

*** 80 A Vehicle 70

PYY2-36(4H) 40

Food intake (g) intake Food 30

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Day

250

Day 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 2

Vehicle -2

-4

-6 PYY2-36(4H)

vehicle corrected vehicle -8 B -10 % body weight change from day 0, day from % change weight body -12 ** ***

Figure 4.31 Effect of subcutaneous administration of increasing doses PYY2-36(4H) on (A) food intake and (B) body weight, in C57BL/6 DIO mice (n=7/14). Peptide and vehicle (saline) administered daily. Data are shown as mean ± SEM. Shaded grey area is the dose at 300nmol/kg, followed by 1200nmol/kg in the white area, followed by 2400nmol/kg in the days of the shaded red area. GEE and Mann-Whitney statistical tests used to test significance (**=p<0.01; ***=p<0.001 vs. vehicle).

251

4.2.4.5 Effect of chronic administration of PYY2-36(4H) on food intake and body weight in rats

There was no significant difference between the average body weights of the vehicle control

group and the PYY2-36(4H) group at the start of the study.

Daily s.c. injection of PYY2-36(4H) 1:1 Zn at 300nmol/kg from day 0-16, followed by daily s.c.

injection of PYY2-36(4H) 1:1 Zn at 600nmol/kg from day 17-28, significantly decreased food intake on day 1 of the study and continued to cause a decrease in food intake (FI) until day

28 (cumulative FI by day 28: vehicle: 823.2 ± 18.7g vs. 600nmol/kg PYY2-36(4H): 749.9 ± 9.2g,

p < 0.05) (Fig 4.32(A)). The PYY2-36(4H) group had a lower average body weight than the vehicle group and the difference in body weight was significant from day 3 until day 28

(body weight on day 3: vehicle: 491 ± 12g vs. 300nmol/kg PYY2-36(4H): 483 ± 10g, p < 0.05;

body weight on day 28: vehicle: 544 ± 15g vs. 600nmol/kg PYY2-36(4H): 512 ± 9g, p < 0.001) (Fig 4.32 (B)).

*** ** * 800 A Vehicle 700

600

500

PYY2-36(4H) 400

Food intake (g) intake Food 300

200

100

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Day

252

Day 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 1

Vehicle -1

-2

-3

-4 PYY2-36(4H)

-5

-6 % Body weight change, vehicle corrected from day 0 day corrected from vehicle % change, weight Body ***

Figure 4.32 Effect of subcutaneous administration of increasing doses PYY2-36(4H) on (A) food intake and (B) body weight, in male Wistar rats (n=9). During the days of the unshaded white area, the dose was 300nmol/kg, followed by 600nmol/kg for the duration of the shaded grey area. Peptide and vehicle (saline) administered daily. Data are shown as mean ± SEM. GEE and Mann-Whitney statistical tests used to test significance (*=p<0.05; **=p<0.01; ***=p<0.001 vs. vehicle).

253

4.2.4.6 Investigation of immunogenicity of PYY2-36(4H)

At the end of the chronic studies described above, blood was collected from the animals and plasma screened for immunogenicity, by testing for the presence of antibodies against PYY

and antibodies against PYY2-36(4H). Plasma samples from studies with daily administration of 125 PYY2-36(4H) did not show any increase in binding of I-PYY2-36(4H) in rats, after 28 days daily 125 injections. Nor was there any increase in binding of I-PYY3-36 from both rat and mouse 125 chronic studies. Plasma from five out of fifteen mice which received PYY2-36(4H) bound I-

PYY2-36(4H) by the end of the study (average PYY2-36(4H) group binding is 12.6%) (Table 4.10)

% PYY (4H) Dose Dosing % PYY immunogenicity 2-36 Reduction immunogencity Species (nmol/kg) regimen weight cumulative (day) loss # positive Avg % # positive Avg % FI rats binding* rats binding* 300 0-16 Rat 5.3 8.9 0 4.5 0 5.5 600 17-28 300 0-9 Mouse 1200 10-20 9.2 16.1 0 6.2 5/15 12.6 2400 21-28

*Average binding of the group is corrected against control groups

Table 4.10 Summary of the chronic data studies with PYY2-36(4H) and immunogenicity of terminal

plasma samples – testing the presence of antibodies against PYY3-36 and PYY2-36(4H).

254

4.3 DISCUSSION

The observation that intravenous PYY3-36 administration to both lean and obese humans resulted in a decrease in food intake (Batterham et al., 2002, Batterham et al., 2003a) has raised interest in PYY3-36 and its receptor as potential targets for development of therapeutic agents for the treatment of obesity. However, there are a number of considerations with developing peptide therapeutics. The first is avoiding unwanted side effects, which have limited development of other obesity therapies. Next is prolonging the duration of action of the peptide, as the short half-life of native PYY limits its clinical use. The first part of this work details studies investigating a PYY analogue as a potential therapeutic agent (section 4.2.1). However during these studies it became apparent that the designed analogue provoked an immunogenic response (section 4.2.2.1) when administered on multiple occasions to rats and dogs. Therefore the second part of this chapter presents a summary of the investigations I completed to identify the potential causes of immunogenicity and the approaches used to reduce this non-desirable side effect.

When designing potential analogues of PYY3-36, it was important to consider the analogues’ affinity to the NPY receptors. PYY3-36 binds selectively to the Y2r but also has affinity (albeit low) to the Y1r, Y5r and Y4r. Maintaining the selectivity at the Y2r is a critical requirement to prevent off-target effects. In particular, Y1r activation results in orexigenic effects (as with NPY binding to hY1r in the arc (Jung and Kim, 2013) in contrast to the inhibition of food intake that is exhibited with Y2r activation. Species specificity also had to be considered as the vast majority of the efficacy work for a potential human drug was completed in the mouse, however the hY2r and mY2r share 98% amino acid sequence identity (Lo et al.,

2005). From the receptor ligand studies I completed, PYY2-36(αLTX-4H) was shown to have selectivity for the Y2r over the Y1r. Compared with PYY3-36 (fig. 3.1), PYY2-36(αLTX-4H) was shown to be about 3-fold worse at both human and mouse Y2r receptors. This 3-fold decrease in affinity at the Y2r would impact on doses chosen for in vivo studies (sections

3.1.3 and 3.1.5). At the hY1r, PYY2-36(αLTX-4H) bound approximately 16-times worse compared with PYY1-36 which is in line with having similar receptor selectivity as PYY3-36 for the Y2r.

255

I then looked at the pharmacokinetic properties of the PYY analogues. There are two aspects of pharmacokinetics that are important to consider. The first is that of a ‘burst effect’: PYY is known to cause nausea in human when plasma levels rise rapidly (le Roux et al., 2008b). The second is the rate of release, elimination and degradation of the peptides. Peptide therapeutics have to be given as subcutaneous injections, due to low bioavailability when given orally. A low frequency of injections will improve patient compliance with the therapy, and so slow release and elimination are ideal. Indeed, any analogue of PYY3-36 to be developed as a potential therapeutic agent had to provide at least 24hr coverage following a single administration, whilst not having any bursts of release causing spikes in plasma levels.

Reducing the burst effect was facilitated by the addition of 4 His residues and using a zinc chloride solution. The addition of 4 His residues in the analogue PYY2-36(αLTX-4H) prevented a rapid rise in hormone levels, with a steady rise to Cmax at 60mins. Although zinc chloride had no effect on the initial burst in levels of native PYY3-36, when the analogue PYY2-

36(αLTX-4H) was administered with zinc chloride, this further diminished the rate of increase in plasma levels, with a Tmax at 48h post injection. The zinc is thought to prolong peptide release by forming stable hexamers that allow slow release from a subcutaneous depot (Brange and Langkjoer, 1993, Brange and Langkjaer, 1997). This slow rise will likely limit the onset of nausea.

These features also affected the length of time the peptide remained present in the blood. Native PYY has a half-life of 8 minutes, measured after IV administration reached a steady state (Lluis et al., 1989). By contrast, after a bolus SC administration, the addition of Pro2 and the αLTX-4H sequence in PYY2-36(αLTX-4H) demonstrated elevated peptide levels until 24 hours. This difference in PK profile may be due to the route of administration or the resistance of PYY2-36(αLTX-4H) to hepatic or renal enzymatic degradation or resistance to renal clearance. Formuation of native PYY3-36 with zinc chloride did not demonstrate extended elevation of plasma levels which declined rapidly and had decreased by 98% within 3 hours. However, the complex of PYY2-36(αLTX-4H) with zinc chloride showed elevated levels were maintained for 7 days, after one SC injection.

256

However, a pharmacokinetic profile alone cannot determine the length of efficacy of a drug in vivo. These can be investigated in feeding studies. During the acute feeding studies in overnight fasted mice, both PYY3-36, and PYY2-36(αLTX-4H) (not formulated with Zn) demonstrated similar potency as PYY3-36 at reducing food intake initially, with both peptides significantly inhibiting food intake in the 0-1h interval (figure 3.2). However, PYY3-36 only caused a transient reduction of food intake while PYY2-36(αLTX-4H) continued to significantly reduce food intake compared to both vehicle and PYY3-36 in the 2-4 and 4-8h time intervals post-injection. This extended length of action could be due to a difference in release from the subcutaneous injection site or a longer circulatory half-life in plasma. To further investigate this, a more complex PK investigation would be required where the clearance rate from steady state by IV infusion would need to be determined.

While acute studies can screen compounds rapidly and obtain information on the potency, efficacy, and duration of action of a compound in vivo, effects of a drug over a longer dosing period (e.g. 28 days) are also needed in order to demonstrate a maintained reduction of food intake and body weight without escape from the compound tested. Published work from other groups (Reidelberger et al., 2008, Chelikani et al., 2007, Chelikani et al., 2006) have shown a modest reduction in body weight after chronic administration of PYY3-36 in rats, testing various dosing and administration regimes.

Currently available peptide therapeutics, such as insulin and GLP-1 analogues, are administered as subcutaneous injections as bioavailability of peptides is low after oral administration. Reduced frequency of injections improves patient compliance, and it would be ideal to develop a once-weekly PYY analogue formulation. Although a once-weekly injection would be ideal, I decided to administer the peptides daily because high doses of

PYY2-36(αLTX-4H) would be required to provide weekly coverage as small mammals have very high peptide metabolism rates.

Chronic feeding studies were conducted where PYY2-36(αLTX-4H) (formulated with zinc) was administered daily to rats for 14 days, and mice for 29 days. The analogue caused a sustained inhibition of food intake and a reduction of body weight (figure 3.8 and 3.9). The percentage reduction in body weight caused by 3000nmol/kg PYY2-36(αLTX-4H) was greater in mice than in rats over a 14 day period, likely to be a result of using an obese mouse model

257 and a lean rat model. Stress may have also influenced the studies as rats are more susceptible than mice to stress induced by daily SC injections, which reduces the anorectic effect of PYY3-36 (Abbott et al., 2006). Nevertheless, demonstrating the anorectic effect of chronic administration of PYY analogues in both rats and mice provides greater support that the peptide would be anorectic in humans compared to only testing in a single model organism.

Several studies have shown that continuous administration of PYY3-36 only transiently reduces food intake and body weight in rodents (Pittner et al., 2004). This brings into question if receptor down-regulation or tachyphylaxis (Savini, 1964) occurs when PYY3-36 is repeatedly administered (Chelikani et al., 2007). In the presented chronic studies where peptides were administered once daily by subcutaneous injection, a continuous effect on food intake and body weight was observed providing evidence against receptor down- regulation or tachyphylaxis occurring. Moreover, when doses were increased during the study (“dose ramping”) to prevent nausea during the early phase of the study, animals still responded acutely to the increase in dose, suggesting there was no desensitisation in the system.

In a formal toxicology study of PYY2-36(αLTX-4H) conducted in dog, it was found that plasma from animals which had received multiple doses of the peptide bound iodinated PYY2- 125 36(αLTX-4H) ( I-PYY2-36(αLTX-4H)). When plasma samples from the chronic rat study were 125 tested (section 3.2), a similar pattern was observed with I-PYY2-36(αLTX-4H) binding to the 125 plasma. The likely cause of I-PYY2-36(αLTX-4H) binding to plasma would be the presence of anti-drug antibodies (ADAs) against the compound present in the plasma, caused by an immunogenic reaction. This side effect was not entirely unexpected as PYY2-36(αLTX-4H) is a protein which could contain immunogenic sequences (Kessler et al., 2006) resulting from the helix-forming sequences of the αLTX protein (Holz and Habener, 1998). Also, the subcutaneous route of administered is known to be the more immunogenic than others such as intraperitoneal or intravenous (Schellekens, 2002, Kessler et al., 2006). As mentioned above, immunogenic responses range from no demonstrable effects to loss of drug efficacy. The types of ADAs produced can also range from non-neutralising to those that severely cross-react with endogenous molecules. Although immunogenicity cannot be predicted from one species to another (Schellekens, 2002), and the analogue tested in rat

258 and dog was based on human sequence PYY2-36 (which could cause false positives) it was decided unsafe to continue developing this peptide in the event of the induction of immunogenicity in man. However, understanding why this analogue caused an immunogenic reaction will help develop less immunogenic peptides in the future.

PYY2-36(αLTX-4H) is a relatively small protein with a molecular weight of ~4kDa (Considine et al., 1996), and is a highly purified, synthetically synthesised peptide with >95% purity (section 2.1.2). It also contains only naturally occurring amino acids. These are all factors which are likely to reduce the risk of an immune response. However, there are a number of features that may cause an immunogenic reaction.

The first possibility was the use of zinc to form a subcutaneous depot of PYY2-36(αLTX-4H) was postulated to be a possible cause of immunogenicity. Zinc has been shown to cause immunogenic reactions when used in this way with crystalline zinc-insulin (Kosinski et al.,

2012). However, a study of the immunogenicity of PYY3-36 and PYY2-36(αLTX-4H) formulated with a range of zinc to peptide molar ratios found no correlation between presence of ADAs and zinc (section 3.2.2). Based on these result, the presence of Zn in the formulation of PYY2-

36(αLTX-4H) was determined not to be important in the generation of ADAs.

Subsequently, I investigated the part of the molecule that bound to the anti-PYY2-36(αLTX- 4H) antibodies, and thus which part of the peptide initiates the immune response. The three different animal species which developed ADAs were tested in case of any species variation in antibodies. All three species produces similar types of ADAs. They all bound fragment 7- 36 with 60% cross-reactivity and 13-36 to a lesser extent. Fragments 2-14, 2-28, and 27-36 did not bind well to the ADA which suggested that the middle section was important for ADA binding but only when presented together with the C-terminal end of the peptide (table 3.4). As the substituted αLTX sequence was the most altered part of the analogue, and therefore the most “foreign” to the molecule, this finding was not unexpected (Schellekens, 2002). The importance of the C-terminal for immunogenicity was harder to establish as fragment 27-36 only bound with low affinity by itself. It may possibly serve to stabilise the mid-section of the peptide to make it more recognisable by the ADA.

Results from the fragments cross-reactivity suggested the ADA recognised a tertiary structure and not merely a sequence or particular end of the peptide. Therefore variations

259 of PYY2-36(αLTX-4H) were created (fig.3.11) to elucidate which substitutions/eliminations could be made which would not bind the ADA and therefore potentially not cause their formation. PYY2-36(αLTX-4H) analogues which still contain the αLTX mid-section but have alterations in either the Pro2 residue or any of the four His residues still bind very well to 125 the ADA. However there was a trend in decrease of I-PYY2-36(αLTX-4H) binding with a decrease in the number of His residues (the positions of which do not appear to correlate with high binding). Removal of the αLTX mid-section gave a series of analogues which have much lower cross-reactivity with the ADA but the trend still existed for a decrease in affinity with a decrease in His residues. As His residues are important in forming good subcutaneous depots when formulated with Zn, this observation could indicate that aggregation is partly responsible for increased immunogenicity. While this discovery is not surprising, native PYY2-

36 bound to ADAs 30% as well as PYY2-36-αLTX-4H which indicates a basal cross-reactivity of unaltered PYY (table 3.6).

While completing the above studies, a strategy to reduce the immunogenicity PYY2-36(αLTX- 4H) itself was investigated. The addition of polyethylene glycol (PEG) to molecules is an accepted method of reducing immunoreactivity (Balthasar et al., 2005) of the molecule possibly due to a mechanical “shielding” of the epitope by PEG.

Studies with PYY2-36(αLTX-4H)-PEG, showed that although PEGylation did reduce the immunogenic response to exposure of the molecule by approximately 60% (section 3.3.1.4), the bioefficacy of PYY2-36(αLTX-4H)-PEG was also reduced (section 3.3.1.2). This could conceivably be due to the PEG group disrupting affinity to the Y2 receptor (section 3.3.1.1) by either steric hindrance or its location on the molecule (in the same way that PEG reduces immunogenicity). Indeed Shechter et al. 2005 demonstrated that PEGylation (40kDa) of

PYY3-36 abolishes all bioactivity (does not reduce FI at equivalent doses to normal PYY3-36). But it was concluded that this was not completely due to the steric hindrance presented by the PEG group as acetylation (a very small group – COCH3) of the two most N-terminal amino acids (Nα-Nε-diacetyl-PYY3-36) also abolished bioactivity. However, reversible

PEGylation of PYY3-36 restored some bioactivity, increased the duration of FI reduction, and therefore its “functional half-life”. Conversely, Ortiz et al. (Ortiz et al., 2007) showed that

PEGylated PYY13-36 causes a greater reduction of food intake than its non-PEGylated counterpart (i.e. PYY13-36). The C-terminal section of PYY was used for their analogue

260 because it is more selective for Y2r (DeCarr et al., 2007). Therefore the location of the PEG group affects bioefficacy.

The size of the PEG group must also be considered, as PYY must cross the blood brain barrier to function effectively (Harder et al., 2004). Molecules up to 40kDa can cross fenestrated capillaries of the BBB (Lockie et al., 2012). PYY2-36(αLTX-4H)-PEG (4.7kDa) and PEG-PYY13-36 from Ortiz et al. (23kDa) fall below this level, while PEG-PYY3-36 from Shechter et al is above this level, and may partly explain the loss in bioactivity of this molecule.

As the rationale for the design of PYY2-36(αLTX-4H)-PEG was solely to reduce immunogenicity, the method of PEGylation to produce a long-acting PYY analogue was at odds with the strategy of a slow-release formulation and perhaps may not be jointly utilised with the zinc formulation for this analogue of PYY. PYY2-36(αLTX-4H) was already designed to have extended bioefficacy without Zn (section 3.1.2). So the use of PEG for this drug to prolong its bioefficacy would be redundant and the presence of the PEG moiety was indeed found to decrease affinity for the Y2r compared to the non-PEGylated peptide, decrease bioactivity at acute feeding studies (fig. 3.12) and worsen pharmacokinetics (fig. 3.14). The poor PK profile was likely to be a consequence of the PEG group interfering with the subcutaneous depot formation in a Zn formulation (Lee et al., 2005)

The rationale for designing PYY2-36(αLTX-4H)-PEG was to reduce immunogenicity of the peptide. PEGylation has been used to increase functional half-life of molecules (Hatoum and

Kaplan, 2013). However, in this case, PYY2-36(αLTX-4H)-PEG was actually found to have reduced bioactivity (fig 3.12) and poorer pharmacokinetics (3.14) than the original analogue. This is likely to be a consequence of the PEG group interfering with the formation of the zinc-complexed subcutaneous depot (Lee et al., 2005)

Considering all the data generated from the investigation of immunogenicity, it was decided that a refined analogue of PYY2-36(αLTX-4H) was the best strategy to reduce immunogenicity with minimal consequence on other properties. From the immunogenicity screen of analogues (table 3.5), PYY2-36(4H) was selected for its significantly reduced binding of ADAs

(50% decrease compared to PYY2-36(αLTX-4H)) while still maintaining as much of the original sequence as possible. PYY2-36(4H) was shown to have high affinity to the mouse and human

261

Y2r, while maintaining similar selectivity as the native ligand, PYY3-36 for the Y2r over the Y1r (fig. 3.15).

Taking into consideration all the data generated from the immunogenicity investigations, it was decided that a refined analogue of PYY2-36(αLTX-4H) was the best strategy to reduce immunogenicity with minimal consequences on other properties. From the immunogenicity screen of analogues (table 3.5), PYY2-36(4H) was selected for its similar levels of antibody production as PYY2-36 (50% lower than PYY2-36(αLTX-4H), but double PYY2-36). PYY2-36(4H) has a high affinity to both mouse and human Y2 receptors, and has a similar selectivity for the

Y2r over the Y1r as PYY3-36 (fig 3.15).

The pharmacokinetic properties of PYY2-36(4H) when delivered with differing amounts of zinc were investigated. Without Zn, PYY2-36(4H) levels peak at 30minutes and return to basal levels at 24hrs, like native PYY3-36. With increasing doses of zinc, Tmax also increases, from 30mins with 0 Zn, to 24hrs with 0.5:1zin, to 48hr with 1:1 zinc. It should be noted that the concentration of PYY2-36(4H) when given with 1:1 Zn was higher than the maximum concentration of PYY2-36(4H) at any point when given without zinc; this may be due to the

‘true’ Cmax for the analogue without zinc occurring before the first 30min sampling point, or because the zinc depot allows accumulation of the peptide within the blood that exceeds its degradation.

Zinc also affected the relative bioavailability of the peptide: it increased it by 6.5 times when given with 0.5:1 zinc, and 6-fold higher bioavailability when given with 1:1 zinc. However, this again may be affected by missing higher peaks of peptide level when given without zinc, if they peak before the first 30minute sampling point.

The dose of peptide also affects pharmacokinetics. PYY2-36(4H) was given either at 200mg/ml or at 50mg/ml (fig 3.22; section 3.3.2.5), in complex with 1:1 zinc. At both concentrations, 1:1 zinc gave a desirable PK profile, with no initial burst, and a smooth, slow-release curve for one-week coverage (fig 3.25). At 50mg/ml, Tmax was at 24hrs; at 200mg/ml, it is prolonged to 7 days. The Cmax is increased 3-fold with the 200mg/ml dose compared to 50mg/ml, and the AUC is increased 6 fold. The 200mg/ml dose would equate to 1mg/kg in man to give one week coverage, as metabolism in humans is relatively slow compared to rodents.

262

PYY2-36(4H) was also compared to PYY2-36(αLTX-4H). At 50mg/ml with 1:1 zinc, the exposure level of PYY2-36(4H) is approximately 3 times that of PYY2-36(αLTX-4H). PYY2-36(4H) shows a lower percentage of Cmax level of peptide on day 7. This suggests that the αLTX region was important in either the subcutaneous depot formation, or in maintaining stability in the plasma. At the higher dose of 200mg/ml, there is less difference in exposure levels between

PYY2-36(4H) and PYY2-36(αLTX-4H) when given with 1:1 zinc. The levels of PYY2-36(4H) are approximately double that of PYY2-36(αLTX-4H), and the levels of both peptides remain elevated above basal through the time points sample, so that at 8 days post injection, PYY2-

36(4H) is 9x above basal levels, and PYY2-36(αLTX-4H) is 10x above basal levels. A reduction of only 30% from Cmax was observed for both peptides at 8 days post injection.

The effect of PYY2-36(4H) on food intake was also examined. Acute administration of PYY2-

36(4H) produced a dose-dependent inhibition of food intake in mice (fig 3.17 and 3.18), and reduced food intake to a similar extent as PYY3-36 in the 0-2hr interval. However, even when administered without zinc, PYY2-36(4H) continued to significantly reduce food intake at the 2-

4hr time period when compared with both vehicle and PYY3-36. When administered with zinc (fig 3.19), food intake was significantly reduce at 8-24hours, similar to PYY2-36(αLTX-4H) without zinc. When directly compared, PYY2-36(4H) reduces food intake with a similar potency to PYY2-36(αLTX-4H), but with a reduced duration of action. As with PYY2-36(αLTX-

4H), PYY2-36(4H) was administered daily to rats and mice for 28 days. Chronic administration of PYY2-36(4H) caused a sustained inhibition off food intake and a reduction in body weight in both rats and mice (fig 3.27 and 3.28). Immunogenicity was measured at the end of these studies and rats did not develope ADAs towards native PYY3-36 or PYY2-36(4H). Plasma from mice were shown not to possess ADAs towards native PYY3-36 however a third tested positive for ADAs towards the drug. Of those that did, the percentage binding of the animals were just above the cut-off threshold for positive identification of ADAs suggesting only a weak immunogenic reaction and an overall low average binding to ADAs for the whole group.

263

4.4 CONCLUSIONS

PYY2-36(αLTX-4H) displayed many properties which suggested a potentially useful agent for development as a treatment of obesity. However, the level of antibody production provoked by chronic administration of the agent in preclinical studies was considered too high to continue the peptides development. Investigation of the cause of immunogenicity suggested that no particular sequence was responsible, and the tertiary shape was important. However, the αLTX domain was important for the binding of ADAs. The removal of this region increased the clearance rate of the peptide following a single subcutaneous injection. However, the activity of the peptide remained favourable and the peptide’s ability to cause an immunogenic response was similar to that of native peptide.

PYY analogue PYY2-36(4H) has been selected to undergo a formal assessment of safety for administration to humans. Whilst the majority of this work is outsourced to facilities regulated by good laboratory practice (GLP), samples generated from these studies will continue to be routinely tested for immunogenicity.

264

CHAPTER 5

GENERAL DISCUSSION

265

5.1 INTRODUCTION

The work in this thesis consisted of 2 sections. The first examined the development of an enzyme-resistant, longer-acting analogue of the satiety molecule PP. For the purpose of rational analogue design, I investigated how the native peptide is degraded by tissue preparations and individual enzymes known to degrade hormones. Based on these results, I aimed to investigate the effects of altering different domains of the primary sequence of PP to attempt to create more potent and/or longer-acting analogues. The second part of my work investigated the immunogenic properties of a novel analogue of PYY3-36. I aimed to standardise the method in which immunogenicity was detected, and measured and investigated the contribution of the Zn delivery system and modified domains of the analogue towards its immunogenicity.

Data analysed by the Health and Social Care Information Centre (HSCIC, 2014) between 1993-2014 showed that in England, adults with normal BMI decreased by 9% and the proportion of adults that were obese increased by almost 100%. The underlying causes of the UK’s (and most of the developed world’s) obesity epidemic is due to present-day “obesogenic” environmental factors (such as easy access to cheap, high-energy food sources and the reduced requirement for physical exercise) and evolutionary factors that predispose to weight gain (Weinsier et al., 1998; Bloom et al., 2008). Although lifestyle modification through caloric restriction and enhanced physical activity remain the first line treatment for obesity, the most effective treatment is bariatric surgery. However, it is impractical to use surgery to treat the increasing numbers of obese patients (Christou and Efthimiou, 2009; Garb et al., 2009; Martins et al., 2010) due to the need for specialist personnel and centres, high costs, and the risk of complications and/or mortality.

Recent findings have demonstrated that GLP-1 and PYY are increased after bariatric surgery (Duan et al., 2014), which may contribute to the reduced appetite and loss of body weight seen after surgery. Mimicking the post-bariatric state by peripherally administering gut hormones to the obese may therefore be a logical, less costly therapeutic approach (Korner et al., 2005; Le Roux et al., 2006a). A number of gut hormone therapies are currently in development (Tan and Bloom, 2013b).

266

A further attraction of using gut hormones is that, as their physiological purpose is to regulate food intake, biological therapies based on these hormones will have a high specificity and low toxicity. This contrasts with previously approved anti-obesity treatments such as fenfluoramine, dexfenfluoramine, fen-phen, sibutramine, and rimonabant. These were all synthetic drugs, modelled on small molecules with ubiquitously-expressed receptors. As described in section 1.3.4, these drugs have all been associated with significant side-effects due to their wide-range of effects, and have therefore all been withdrawn from the market.

Nevertheless, gut hormone therapies do present their own challenges. Native peptides have short half-lives in circulation, which limits their efficacy and ease of administration. Therefore logical therapeutic development requires consideration of peptide characteristics, interaction with targets, structural analysis, stability studies, pharmalogical parameters, sequence modifications and high throughput screening. Moreover, all drugs may be subject to side effects, including the development of immunogenicity, and therefore must undergo rigorous screening before they can be fully developed for human use.

267

5.2 DEVELOPMENT OF AN ENZYME-RESISTANT PP ANALOGUE

PP is an attractive target when developing an anti-obesity therapy as it was shown to decrease food intake and appetite without the undesirable side-effect of nausea (Batterham et al., 2003b). The development of PP as an anti-obesity agent has to overcome its short half-life allowing less frequent administration, reduced cost of manufacturing, improved patient tolerance and compliance. To improve on its half-life, this chapter aimed to investigate the weakness points in the molecule through the use of liver and kidney membrane preparations. These organs were chosen for their role in the metabolism and clearance of the majority of pharmacotherapies and it is possible to predict in vivo metabolic clearance using in vitro techniques with good correlation (Houston, 1994, Kenny and Stephenson, 1988).

In both tissues, I found PP was degraded to PP3-36. This breakdown occurred through the action of DPPIV enzyme which is present in preparations of liver and kidney, which was confirmed by incubation of PP with purified enzyme. Unlike the deactivation of GLP-1 by DPPIV, PP3-36 remained bioactive albeit for shorter time periods compared to full length PP. Moving the target sequence of Pro2 through removal of Ala1 (DesAla-PP) or extension by the addition of an extra residue (e.g. PP-Ala0) protected against DPPIV in vitro without altering Y4 receptor affinity. In vivo tests showed that these analogues of PP were effective at reducing food intake for extended periods of time compared to the native molecule. Results suggested than protection from DPPIV was achievable with minimal detrimental effect on the molecule’s other properties resulting in an improved pharmacological action. Other methods of conferring DPPIV-resistance were considered including dimerisation (PP-Cys0 homodimer) however, not only was this analogue more costly and time-consuming to synthesise, it bound >2-times less effectively at the Y4r.

Products which matched those you would expect from trypsin degradation were observed in in in vitro digests of PP with RLM and RBB tissue extracts. As well as the generation of these products of breakdown, trypsin was hypothesised to be a relevant enzyme to investigate due to subcutaneous enzymic activity that is sensitive to trypsin inhibitors (Ogiso et al.,

268

2000). All Arg residues in the PP molecule were targeted by trypsin as confirmed using purified enzyme. Most single substitutions of these residues did not greatly affect receptor affinity; however replacement of Arg33 or Arg35 with an Ala residue decreased receptor affinity significantly. In vitro analysis of these analogues demonstrated that resistance to trypsin is only acquired through double substitutions at Arg25,26. None of the analogues designed to protect against trypsin were more effective at reducing food intake than native PP. In fact, the Ala33 and Ala35 substitution were totally inactive in vivo despite being tested at higher dose levels than required for PP to inhibit food intake.

There were other fragments from digests with the tissue extracts whose target sites were less well-defined. A common feature was the middle location of the target residues and often they were found to be small and hydrophobic amino acids. Amongst the numerous endopeptidases, I decided to investigate NEP as it is found in the tissues being studied as has previously been implicated in the breakdown of other gut hormones such as GLP-1 and PYY. Co-administration of PP with the metallopeptidase inhibitor, phosphoramidon (Kenny et al., 1981, Medeiros and Turner, 1994), indeed demonstrated that the anorectic effects of PP can be sustained. However, this effect may not be exclusively attributed to the preservation of PP by phosphoramidon as other NEP-targeted satiety signals such as GLP-1 (Hupe-Sodmann et al., 1995, Goke et al., 1993) and PYY (Addison et al., 2011) may equally be saved from degradation, although the concentration of these endogenous signals will be minor in comparison to the pharmacological doses of PP used. In addition, as phosphoramidon is not a specific NEP inhibitor, it may be inhibiting the enzymic actions of other metallopeptidases which could potentially contribute to PP degradation. Furthermore, measurement of circulating plasma levels of PP in the presence of phosphoramidon shows 65% higher concentrations compared to PP alone at longer time points suggesting that phosphoramidon (and therefore metallopeptidases) does indeed have a role in the breakdown of PP in vivo.

Based on the fragments identified through tissue digests, key residues in the secondary structure domains of the PP molecule were revealed: position 6 located in the polyproline region, and positions 16, 17, 19, 23, and 30 in the α-helical region. Through alignment of these sections against sequence homologies found in related molecules, cycles of peptide design and selection were driven by in vitro screenings at the Y4 receptor, NEP digests, and

269 anorectic bioactivity in vivo. Analogues of PP were ultimately developed that had increased affinity to the Y4r and improved resistance to NEP digests in vitro. These analogues also exhibited prolonged effects on inhibition of food intake and/or increased potency in vivo (equal anorectic effects compared to native PP but at significantly lower doses). However, it was not always found that analogues with greater receptor affinity were more potent in vivo, nor that resistance to NEP in vitro consistently correlated with extended in vivo bioefficacy.

I proposed a scheme that initial resistance to DPPIV is required before there is any beneficial effect of resistance to NEP suggesting there is an order of enzyme degradation where DPPIV precedes NEP or trypsin. It is possible that DPPIV provides the initial breakdown step which destabilises the PP-fold structure and exposes other areas of the molecule to be degraded by other enzymes. Since Pro2 in native PP is the main interaction partner for Tyr27 (Nygaard et al., 2006), its loss following DPPIV cleavage can cause destabilisation of the PP-fold structure thus making the molecule more unstable and more susceptible to proteolytic degradation. This would also somewhat explain why the fragments of PP degradation using tissue extracts and purified enzymes do not always correspond. It may also explain why protecting against one enzyme (such as trypsin) is not always effective in producing analogues with enhanced biological effect. Thus, despite identifying PP fragments with trypsin target sites after incubation with tissue extracts, it may play a secondary role in the degradation of PP and perhaps, only in the presence of other enzymes.

Another detail that this entire process does not take into account are the enzymes present in blood, plasma or the subcutaneous fat relevant to drugs administered by this route. Although the enzymes that could potentially be investigated are great, this thesis does not aim to study the multitude of potential enzyme candidates and their individual roles in generating an enzyme-resistant peptide analogue. However, I have demonstrated that it is possible to design a peptide drug based on an endogenous satiety molecule that has improved pharmacokinetic and pharmacodynamic qualities to make it a viable pharmacotherapy for obesity.

A limitation of the MALDI-TOF MS method used in this chapter was that it was not quantitative. However, there is usually a correlation between peak signal intensity and

270 concentration of peptide generating the signal. Peak intensity in MALDI has significant analytical variation (for example, depending on peptide ionisation efficiencies, which are influenced by a peptide's composition and the chemical environment). This phenomenon is poorly understood because not only is peak intensity related to the concentration of the individual protein, but also to its primary structure, and to the complexity of the sample. In other words, the sensitivity of a mass spectrometer varies between peptides. Thus, two peptides at equal concentration will not necessarily have equal peak intensities (Timm et al., 2008). When identifying the main fragments produced in digests, it is therefore not entirely desirable to choose the principle degradation products based on intensity signal of the corresponding peak.

It is probably more correct to fractionate digests before analysis by MALDI-TOF. However, as found with the results from this chapter, the total number of identified fragments increased when analysing separate HPLC fractions of digests. This may be due to the reduced noise:signal ratio when examining individual fractions by HPLC compared to the MS analysis of whole digests. This method of analysis would reveal MS peaks which may have been supressed by other ions present in a whole digest. A preferred method may be LC-MS/MS which directly analyses individual peaks as they elute from the HPLC column and thus adjusting sensitivity for each peak on the MS as it arrives.

FUTURE WORK

Apart from Y4r affinity and susceptibility to degradation, there are a number of parameters that can be investigated using peptide analogues. These could include Y4r selectivity compared to Y1r, Y2r and Y5r; receptor activation through the peptide’s ability to decrease cAMP production; bioavailability of the peptide to the receptors expressed in the CNS; mechanisms affecting the ‘on-time’ of a peptide at its receptor (Kd - dissociation constant); any potential receptor signalling bias. Therefore, future work may aim to investigate these parameters in investigating relationships where the altered bioactivity of an analogue is not explained by changes in receptor affinity or susceptibility to proteolytic degradation.

271

5.3 IMMUNOGENICITY OF A LONG-ACTING

ANALOGUE OF PYY3-36

PYY3-36 is potent peripheral satiety signal, released from the GI tract following a meal (Morinigo et al. 2008; Rodieux et al. 2008; Holdstock et al. 2008; Vidal et al. 2009). The reduced appetite and weight loss observed in post-bariatric patients has been suggested to be partly due to increased circulating satiety signals such as PYY and GLP-1 (Degen et al. 2005; Sloth et al. 2007a; Sloth et al. 2007b; Le Roux et al. 2008; Gantz et al. 2007). The peripheral administration PYY3-36 has been shown to decrease in food intake in lean and obese human volunteers (Batterham et al. 2002; Batterham et al. 2003a; Degen et al. 2005; Le Roux et al. 2008) and therefore appears to be a promising anti-obesity agent.

In the second half of my thesis I investigated the immunogenicity of an analogue of PYY

(PYY2-36(αLTX-4H)) which we had designed and initially chosen as the candidate to take through to clinical trials for an obesity therapy. The immunogenicity of a novel drug is animportant factor to consider in the design of a peptide therapy. As the lead candidate,

PYY2-36(αLTX-4H) had passed all the necessary requirements of an enhanced analogue PYY3-

36. Although receptor affinity was 3-fold worse compared to native PYY3-36 across human and mouse variants, it maintained selectivity at the Y2r over Y1r. In vivo, PYY2-36(αLTX-4H) was more potent than native PYY3-36 and had prolonged anorectic effects. Pharmacokinetics of this compound were significantly improved as circulating levels remaining raised for 24h without formulation with zinc and up to 7days in the presence of equimolar zinc. Chronic administration of this drug in both rats and mice caused sustained reductions in food intake and body weight. The drug was then outsourced for toxicokinetic studies and after 10 dosings in dogs, plasma was sent to us for analysis of ADAs. The high level of immunoprecipitation of radiolabelled drug suggested the presence of ADAs, which was also found in the chronic rodent studies. The antibody production provoked by chronic administration of the agent was considered too high to continue with development of this analogue of PYY3-36.

272

The immune assessment for ADAs normally comprises 3 steps: a screening step to detect the presence of binding ADAs, a confirmation step to confirm specificity of the response and finally characterisation of the ADAs. At the screening step, samples are positive if they have values greater than the assay cut point which is defined as values greater than signal:noise ratio. This means that if the noise amongst the control (vehicle-treated) group is high, the cut point is also relatively higher and vice versa. Low cut points can generate false positives and high cut points can potentially produce false negatives. The most suitable cut point provides moderate risk in producing false positives or negatives.

After identifying positive samples, they are analysed in the specificity confirmation assay where the signal of high binding of the radiolabelled drug is displaced in the presence of increasing concentrations of unlabelled drug (competition assay). The samples that displace to a pre-determined extent are reported as positive for binding ADAs and are normally further characterised for neutralising activity in a biological assay. However, I did not do any characterisation of ADAs in this thesis as my aim was to redesign the analogue of PYY to reduce the immunogenicity while still retaining as many of its beneficial pharmacotherapeutic qualities.

Initial studies found that the presence of zinc in the drug formulation did not affect the development of ADAs. The presence of zinc also did not induce the formation of ADAs when formulated with native PYY3-36. ADA-positive samples from rat, dog and sheep were used in competition assays in order to investigate the epitope of the ADA using peptide fragments of the drug. Unsurprisingly, there was greater displacement with the bigger fragments; however, the least displacement was found with the N-terminal fragment which did not contain the αLTX sequence. I concluded that it was impossible to determine exactly the particular sequence responsible for binding to the ADA; however the tertiary shape of the molecule was important.

Finally, I tested the effect of the number of substituted His residues and their location in the competition assay with ADA-postive samples. There was a modest decrease in displacement with decreasing numbers of substituted His residues in the drug, suggesting the epitope of the ADA partly involves these His residues. However there was significant decrease in displacement when the αLTX sequence was removed altogether, which further confirms this

273 region as the main sequence recognised by the ADAs. With the removal of the αLTX sequence, there was little effect of the numbers of His residues present.

It was important to preserve as many His residues as possible as the method of the slow release of this drug depended on the presence of His to increase its isoelectric point to near neutral pH. This would make the drug less soluble at the physiological pH of the SC injection site. His residues are also thought to have a role in further delaying the release of the drug in combination with zinc (Bal et al., 2001).

PEGylation of PYY2-36(αLTX-4H) was also investigated for ability to develop ADAs, as well as all the properties that would make this version of the drug a suitable replacement therapy. While it retained selectivity for the Y2r over the Y1r, it bound with almost 7-times less affinity compared to native PYY3-36, and 2-fold worse compared to the unPEGylated version. It was also less potent in vivo as a 8-fold higher dose was needed to produce the same inhibition of food intake as the unPEGylated version. Remarkably, although an extended effect on bioactivity was expected, this was found not to be the case in acute feeding studies in mice. This can partly be explained by the poor PK profile seen in rats. Time to Cmax was reduced from 48h for the unPEGylated drug, to 24h for the PEGylated drug. Disappearance rate of the PEGylated drug was also increased with more than 50% loss seen at 24h after Tmax. This is in stark contrast to the PK of the original drug which had raised circulating plasma levels up to 7 days post-injection. The only redeemable quality of the PEGylated PYY analogue was its reduced immunogenic response. Although less immunogenic, due to all the other less desirable properties of PYY2-36(αLTX-4H)-PEG, it was abandoned in favour of redesigning the analogue without the αLTX sequence to create PYY2-

36(4H).

PYY2-36(4H) bound with similar affinity to native PYY3-36 at the Y2r, though was more selective for the Y2r over the Y1r, compared to PYY3-36. Acute administration in vivo showed a dose-dependent prolonged anorectic effect compared to native PYY3-36; however it was less potent compared to the original drug. The inhibition of food intake could be further sustained in the presence of zinc formulation. This was confirmed in a zinc dose response in a PK study where higher amounts of zinc delayed the release of peptide the most. While not a 7day PK profile like the original PYY3-36 analogue, PYY2-36(4H) had a modest 4 day PK profile

274 that was still significantly extended compared to PYY2-36(αLTX-4H)-PEG. This PK profile is enhanced at higher concentrations of drug so that at 200mg/ml, PYY2-36(4H) has the same

PK as PYY2-36(αLTX-4H).

Although PYY3-36 has been shown to cause a modest decrease in food intake and body weight in human volunteers, this was only achieved using high doses of IV-infused peptide which also often induced nausea (le Roux et al., 2008b, Sloth et al., 2007, Degen et al.,

2005). The SC administration of PYY3-36 had no effect on feeding even at much higher pharmacological doses (Sloth et al., 2007). A stable analogue of PYY3-36 such as PYY2-36(4H) would have a longer plasma residence time and thus may result in higher efficacy at decreasing appetite, food intake and body weight.

I demonstrated that chronic administration of PYY2-36(4H) to rats and mice, like PYY2-36(αLTX- 4H), produced sustained reductions in food intake and body weight. Most importantly, screening of plasma for ADAs after 28days treatment showed no samples positive for ADAs in rats and a few low signal positive samples in mice. Analogue PYY2-36(4H) was thus selected to undergo formal of safety assessments for administration to humans. While the majority of this work is outsourced to facilities regulated by good laboratory practice (GLP), samples generated from these studies were continued to be routinely tested for immunogenicity.

It is also encouraging that rodents remained acutely sensitive to the effects of the PYY analogue even after 28 days of daily injections suggesting that receptor desensitization and/or tolerance previously hypothesised to be associated with continuous administration of PYY and other gut hormones in osmotic minipumps, may not occur when following this administration regimen (Pittner et al., 2004, Vrang et al., 2006, Chelikani et al., 2007).

275

FUTURE WORK

While the extended bioefficacy of the drug can be partly explained by enhanced PK, receptor potency would be a useful parameter to test, as well as potency of the peptide to the receptors expressed in the CNS. Future work would therefore aim to elucidate the mechanism by which this analogue of PYY is superior to the native peptide. This could include ICV administration of the drug or administration of the analogue in an osmotic minipump which would remove the delayed-release effect of the analogue and overcome the short half-life of native PYY. Another feature worth investigating is the effect of SC depot on integrity of the drug.

Further work is warranted in species such as the dog, where side effects of nausea (vomiting) are apparent, in order to compare the effects of slow-release administration regimes and the effects of long-acting analogues such as PYY2-36(4H) with and without zinc in their utility as potential anti-obesity therapies.

In conclusion, this thesis has developed an analogue of PP and PYY3-36 with improved enzyme resistance and improved affinity for the Y4r or Y2r receptor. In vivo experiments have shown that the analogues inhibit food intake acutely and chronically. During the development of PP, a number of PP analogues were generated which were useful in elucidating the complex structure-activity relationship between PP and the Y4r. Additionally, issues with immunogenicity of a novel peptide drug have been solved without too much compromise on bioactivity. Further development of the PP and PYY3-36 lead candidates may provide a new obesity treatment with greater efficacy than those currently on the market.

276

REFERENCES

277

REFERENCES

ABBOTT, C. R., MONTEIRO, M., SMALL, C. J., SAJEDI, A., SMITH, K. L., PARKINSON, J. R., GHATEI, M. A. & BLOOM, S. R. 2005a. The inhibitory effects of peripheral administration of peptide YY(3- 36) and glucagon-like peptide-1 on food intake are attenuated by ablation of the vagal- brainstem-hypothalamic pathway. Brain Res, 1044, 127-31. ABBOTT, C. R., SMALL, C. J., KENNEDY, A. R., NEARY, N. M., SAJEDI, A., GHATEI, M. A. & BLOOM, S. R. 2005b. Blockade of the neuropeptide Y Y2 receptor with the specific antagonist BIIE0246 attenuates the effect of endogenous and exogenous peptide YY(3-36) on food intake. Brain Res, 1043, 139-44. ABBOTT, C. R., SMALL, C. J., SAJEDI, A., SMITH, K. L., PARKINSON, J. R., BROADHEAD, L. L., GHATEI, M. A. & BLOOM, S. R. 2006. The importance of acclimatisation and habituation to experimental conditions when investigating the anorectic effects of gastrointestinal hormones in the rat. Int J Obes (Lond), 30, 288-92. ABDALLAH, B., GOULA, D., GHORBEL, M., GUISSOUMA, H., BENOIST, C., SEUGNET, I. & DEMENEIX, B. A. 1998. Nonviral gene transfer for studying signaling in comparative developmental biology. Ann N Y Acad Sci, 839, 87-92. ABDALLAH, B., SACHS, L. & DEMENEIX, B. A. 1995. Non-viral gene transfer: applications in developmental biology and gene therapy. Biol Cell, 85, 1-7. ABENHAIM, L., MORIDE, Y., BRENOT, F., RICH, S., BENICHOU, J., KURZ, X., HIGENBOTTAM, T., OAKLEY, C., WOUTERS, E., AUBIER, M., SIMONNEAU, G. & BEGAUD, B. 1996. Appetite- suppressant drugs and the risk of primary pulmonary hypertension. International Primary Pulmonary Hypertension Study Group. N Engl J Med, 335, 609-16. ABU-HAMDAH, R., RABIEE, A., MENEILLY, G. S., SHANNON, R. P., ANDERSEN, D. K. & ELAHI, D. 2009. Clinical review: The extrapancreatic effects of glucagon-like peptide-1 and related peptides. J Clin Endocrinol Metab, 94, 1843-52. ADDISON, M. L., MINNION, J. S., SHILLITO, J. C., SUZUKI, K., TAN, T. M., FIELD, B. C., GERMAIN-ZITO, N., BECKER-PAULY, C., GHATEI, M. A., BLOOM, S. R. & MURPHY, K. G. 2011. A role for metalloendopeptidases in the breakdown of the gut hormone, PYY 3-36. Endocrinology, 152, 4630-40. ADRIAN, T. E. 1978. Pancreatic polypeptide. J Clin Pathol Suppl (Assoc Clin Pathol), 8, 43-50. ADRIAN, T. E., BLOOM, S. R., BRYANT, M. G., POLAK, J. M., HEITZ, P. H. & BARNES, A. J. 1976. Distribution and release of human pancreatic polypeptide. Gut, 17, 940-44. ADRIAN, T. E., FERRI, G. L., BACARESE-HAMILTON, A. J., FUESSL, H. S., POLAK, J. M. & BLOOM, S. R. 1985. Human distribution and release of a putative new gut hormone, peptide YY. Gastroenterology, 89, 1070-7. ADRIAN, T. E., GREENBERG, G. R., BESTERMAN, H. S. & BLOOM, S. R. 1978. Pharmacokinetics of pancreatic polypeptide in man. Gut, 19, 907-9. ADRIAN, T. E., UTTENTHAL, L. O., WILLIAMS, S. J. & BLOOM, S. R. 1986. Secretion of pancreatic polypeptide in patients with pancreatic endocrine tumors. N Engl J Med, 315, 287-91. AHREN, B., JOHNSON, S. L., STEWART, M., CIRKEL, D. T., YANG, F., PERRY, C. & FEINGLOS, M. N. 2014. HARMONY 3: 104-Week Randomized, Double-Blind, Placebo- and Active-Controlled Trial Assessing the Efficacy and Safety of Albiglutide Compared With Placebo, Sitagliptin, and Glimepiride in Patients With Type 2 Diabetes Taking Metformin. Diabetes Care, 37, 2141-8. ALVAREZ, E., RONCERO, I., CHOWEN, J. A., THORENS, B. & BLAZQUEZ, E. 1996. Expression of the glucagon-like peptide-1 receptor gene in rat brain. J Neurochem, 66, 920-7. AMORI, R. E., LAU, J. & PITTAS, A. G. 2007. Efficacy and safety of incretin therapy in type 2 diabetes: systematic review and meta-analysis. Jama, 298, 194-206.

278

ANAND, B. K. & BROBECK, J. R. 1951. Localization of a "feeding center" in the hypothalamus of the rat. Proc Soc Exp Biol Med, 77, 323-4. APOVIAN, C. M., ARONNE, L., RUBINO, D., STILL, C., WYATT, H., BURNS, C., KIM, D., DUNAYEVICH, E. & FOR THE, C. O. R. I. I. S. G. 2013. A randomized, phase 3 trial of naltrexone SR/bupropion SR on weight and obesity-related risk factors (COR-II). Obesity, 21, 935-943. ARAVICH, P. F. & SCLAFANI, A. 1983. Paraventricular hypothalamic lesions and medial hypothalamic knife cuts produce similar hyperphagia syndromes. Behav Neurosci, 97, 970-83. ARITA, Y., KIHARA, S., OUCHI, N., TAKAHASHI, M., MAEDA, K., MIYAGAWA, J., HOTTA, K., SHIMOMURA, I., NAKAMURA, T., MIYAOKA, K., KURIYAMA, H., NISHIDA, M., YAMASHITA, S., OKUBO, K., MATSUBARA, K., MURAGUCHI, M., OHMOTO, Y., FUNAHASHI, T. & MATSUZAWA, Y. 1999. Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res Commun, 257, 79-83. ASAKAWA, A., INUI, A., UENO, N., FUJIMIYA, M., FUJINO, M. A. & KASUGA, M. 1999. Mouse pancreatic polypeptide modulates food intake, while not influencing anxiety in mice. Peptides, 20, 1445-8. ASAKAWA, A., INUI, A., YUZURIHA, H., UENO, N., KATSUURA, G., FUJIMIYA, M., FUJINO, M. A., NIIJIMA, A., MEGUID, M. M. & KASUGA, M. 2003. Characterization of the effects of pancreatic polypeptide in the regulation of energy balance. Gastroenterology, 124, 1325-36. ASTRUP, A., CARRARO, R., FINER, N., HARPER, A., KUNESOVA, M., LEAN, M. E., NISKANEN, L., RASMUSSEN, M. F., RISSANEN, A., ROSSNER, S., SAVOLAINEN, M. J. & VAN GAAL, L. 2012. Safety, tolerability and sustained weight loss over 2 years with the once-daily human GLP-1 analog, liraglutide. Int J Obes (Lond), 36, 843-54. ASTRUP, A., RÖSSNER, S., VAN GAAL, L., RISSANEN, A., NISKANEN, L., AL HAKIM, M., MADSEN, J., RASMUSSEN, M. F. & LEAN, M. E. J. 2009. Effects of liraglutide in the treatment of obesity: a randomised, double-blind, placebo-controlled study. The Lancet, 374, 1606-1616. AVENELL, A., BROOM, J., BROWN, T. J., POOBALAN, A., AUCOTT, L., STEARNS, S. C., SMITH, W. C., JUNG, R. T., CAMPBELL, M. K. & GRANT, A. M. 2004. Systematic review of the long-term effects and economic consequences of treatments for obesity and implications for health improvement. Health Technol Assess, 8, iii-iv, 1-182. BAGGIO, L. L., HUANG, Q., BROWN, T. J. & DRUCKER, D. J. 2004a. Oxyntomodulin and glucagon-like peptide-1 differentially regulate murine food intake and energy expenditure. Gastroenterology, 127, 546-58. BAGGIO, L. L., HUANG, Q., BROWN, T. J. & DRUCKER, D. J. 2004b. A recombinant human glucagon- like peptide (GLP)-1-albumin protein (albugon) mimics peptidergic activation of GLP-1 receptor-dependent pathways coupled with satiety, gastrointestinal motility, and glucose homeostasis. Diabetes, 53, 2492-500. BAGNOL, D., LU, X. Y., KAELIN, C. B., DAY, H. E., OLLMANN, M., GANTZ, I., AKIL, H., BARSH, G. S. & WATSON, S. J. 1999. Anatomy of an endogenous antagonist: relationship between Agouti- related protein and proopiomelanocortin in brain. J Neurosci, 19, RC26. BAL, W., DYBA, M., SZEWCZUK, Z., JEZOWSKA-BOJCZUK, M., LUKSZO, J., RAMAKRISHNA, G. & KASPRZAK, K. S. 2001. Differential zinc and DNA binding by partial peptides of human protamine HP2. Mol Cell Biochem, 222, 97-106. BALLANTYNE, G. H., GOLDENRING, J. R., SAVOCA, P. E., KRANZ, H. K., ADRIAN, T. E., BILCHIK, A. J. & MODLIN, I. M. 1993. Cyclic AMP-mediated release of peptide YY (PYY) from the isolated perfused rabbit distal colon. Regul Pept, 47, 117-26. BALTHASAR, N., DALGAARD, L. T., LEE, C. E., YU, J., FUNAHASHI, H., WILLIAMS, T., FERREIRA, M., TANG, V., MCGOVERN, R. A., KENNY, C. D., CHRISTIANSEN, L. M., EDELSTEIN, E., CHOI, B., BOSS, O., ASCHKENASI, C., ZHANG, C. Y., MOUNTJOY, K., KISHI, T., ELMQUIST, J. K. & LOWELL, B. B. 2005. Divergence of melanocortin pathways in the control of food intake and energy expenditure. Cell, 123, 493-505.

279

BARDEN, J. A., CUTHBERTSON, R. M., POTTER, E. K., SELBIE, L. A. & TSENG, A. 1994. Stabilized structure of the presynaptic (Y2) receptor-specific neuropeptide Y analog N-acetyl[Leu- 28,Leu-31]NPY(24-36). Biochim Biophys Acta, 1206, 191-6. BARSON, J. R., MORGANSTERN, I. & LEIBOWITZ, S. F. 2013. Complementary roles of orexin and melanin-concentrating hormone in feeding behavior. Int J Endocrinol, 2013, 983964. BASTL, C., FINKELSTEIN, F. O., SHERWIN, R., HENDLER, R., FELIG, P. & HAYSLETT, J. P. 1977. Renal extraction of glucagon in rats with normal and reduced renal function. Am J Physiol, 233, F67-71. BATAILLE, D., GESPACH, C., TATEMOTO, K., MARIE, J. C., COUDRAY, A. M., ROSSELIN, G. & MUTT, V. 1981. Bioactive enteroglucagon (oxyntomodulin): present knowledge on its chemical structure and its biological activities. Peptides, 2 Suppl 2, 41-4. BATTERHAM, R. L., COHEN, M. A., ELLIS, S. M., LE ROUX, C. W., WITHERS, D. J., FROST, G. S., GHATEI, M. A. & BLOOM, S. R. 2003a. Inhibition of food intake in obese subjects by peptide YY3-36. N Engl J Med, 349, 941-8. BATTERHAM, R. L., COWLEY, M. A., SMALL, C. J., HERZOG, H., COHEN, M. A., DAKIN, C. L., WREN, A. M., BRYNES, A. E., LOW, M. J., GHATEI, M. A., CONE, R. D. & BLOOM, S. R. 2002. Gut hormone PYY(3-36) physiologically inhibits food intake. Nature, 418, 650-4. BATTERHAM, R. L., FFYTCHE, D. H., ROSENTHAL, J. M., ZELAYA, F. O., BARKER, G. J., WITHERS, D. J. & WILLIAMS, S. C. 2007. PYY modulation of cortical and hypothalamic brain areas predicts feeding behaviour in humans. Nature, 450, 106-9. BATTERHAM, R. L., HEFFRON, H., KAPOOR, S., CHIVERS, J. E., CHANDARANA, K., HERZOG, H., LE ROUX, C. W., THOMAS, E. L., BELL, J. D. & WITHERS, D. J. 2006. Critical role for peptide YY in protein-mediated satiation and body-weight regulation. Cell Metab, 4, 223-33. BATTERHAM, R. L., LE ROUX, C. W., COHEN, M. A., PARK, A. J., ELLIS, S. M., PATTERSON, M., FROST, G. S., GHATEI, M. A. & BLOOM, S. R. 2003b. Pancreatic polypeptide reduces appetite and food intake in humans. J Clin Endocrinol Metab, 88, 3989-92. BAURA, G. D., FOSTER, D. M., PORTE, D., JR., KAHN, S. E., BERGMAN, R. N., COBELLI, C. & SCHWARTZ, M. W. 1993. Saturable transport of insulin from plasma into the central nervous system of dogs in vivo. A mechanism for regulated insulin delivery to the brain. J Clin Invest, 92, 1824- 30. BECKH, K., MONNIKES, H., LOOS, S., ARNOLD, R. & KOOP, H. 1992. Low hepatic clearance of peptide YY (PYY) in the perfused rat liver. Regul Pept, 37, 205-12. BEINBORN, M., WORRALL, C. I., MCBRIDE, E. W. & KOPIN, A. S. 2005. A human glucagon-like peptide- 1 receptor polymorphism results in reduced agonist responsiveness. Regul Pept, 130, 1-6. BELL, G. I., SANCHEZ-PESCADOR, R., LAYBOURN, P. J. & NAJARIAN, R. C. 1983. Exon duplication and divergence in the human preproglucagon gene. Nature, 304, 368-71. BELLINGER, L. L. & BERNARDIS, L. L. 2002. The dorsomedial hypothalamic nucleus and its role in ingestive behavior and body weight regulation: lessons learned from lesioning studies. Physiol Behav, 76, 431-42. BENET, L. Z. 1978. Effect of route of administration and distribution on drug action. J Pharmacokinet Biopharm, 6, 559-85. BENOIT, S. C., AIR, E. L., COOLEN, L. M., STRAUSS, R., JACKMAN, A., CLEGG, D. J., SEELEY, R. J. & WOODS, S. C. 2002. The catabolic action of insulin in the brain is mediated by melanocortins. J Neurosci, 22, 9048-52. BERG, A. H., COMBS, T. P. & SCHERER, P. E. 2002. ACRP30/adiponectin: an adipokine regulating glucose and lipid metabolism. Trends Endocrinol Metab, 13, 84-9. BERGLUND, M. M., HIPSKIND, P. A. & GEHLERT, D. R. 2003. Recent developments in our understanding of the physiological role of PP-fold peptide receptor subtypes. Exp Biol Med (Maywood), 228, 217-44.

280

BERGLUND, M. M., LUNDELL, I., ERIKSSON, H., SOLL, R., BECK-SICKINGER, A. G. & LARHAMMAR, D. 2001. Studies of the human, rat, and guinea pig Y4 receptors using neuropeptide Y analogues and two distinct radioligands. Peptides, 22, 351-6. BERNTSON, G. G., ZIPF, W. B., O'DORISIO, T. M., HOFFMAN, J. A. & CHANCE, R. E. 1993. Pancreatic polypeptide infusions reduce food intake in Prader-Willi syndrome. Peptides, 14, 497-503. BERTENSHAW, G. P., TURK, B. E., HUBBARD, S. J., MATTERS, G. L., BYLANDER, J. E., CRISMAN, J. M., CANTLEY, L. C. & BOND, J. S. 2001. Marked differences between metalloproteases meprin A and B in substrate and peptide bond specificity. J Biol Chem, 276, 13248-55. BERTHOUD, H. R. & POWLEY, T. L. 1991. Morphology and distribution of efferent vagal innervation of rat pancreas as revealed with anterograde transport of Dil. Brain Res, 553, 336-41. BIANCHI, E., CARRINGTON, P. E., INGALLINELLA, P., FINOTTO, M., SANTOPRETE, A., PETROV, A., EIERMANN, G., KOSINSKI, J., MARSH, D. J., POCAI, A., SINHAROY, R. & PESSI, A. 2013. A PEGylated analog of the gut hormone oxyntomodulin with long-lasting antihyperglycemic, insulinotropic and anorexigenic activity. Bioorg Med Chem, 21, 7064-73. BIEDZINSKI, T. M., BATAILLE, D., DEVAUX, M. A. & SARLES, H. 1987. The effect of oxyntomodulin (glucagon-37) and glucagon on exocrine pancreatic secretion in the conscious rat. Peptides, 8, 967-72. BJORBAEK, C. 2009. Central leptin receptor action and resistance in obesity. J Investig Med, 57, 789- 94. BLOMQVIST, A. G., SODERBERG, C., LUNDELL, I., MILNER, R. J. & LARHAMMAR, D. 1992. Strong evolutionary conservation of neuropeptide Y: sequences of chicken, goldfish, and Torpedo marmorata DNA clones. Proc Natl Acad Sci U S A, 89, 2350-4. BODEN, G., MASTER, R. W., OWEN, O. E. & RUDNICK, M. R. 1980. Human pancreatic polypeptide in chronic renal failure and cirrhosis of the liver: role of kidneys and liver in pancreatic polypeptide metabolism. J Clin Endocrinol Metab, 51, 573-8. BOEY, D., LIN, S., KARL, T., BALDOCK, P., LEE, N., ENRIQUEZ, R., COUZENS, M., SLACK, K., DALLMANN, R., SAINSBURY, A. & HERZOG, H. 2006. Peptide YY ablation in mice leads to the development of hyperinsulinaemia and obesity. Diabetologia, 49, 1360-70. BOGGIANO, M. M., CHANDLER, P. C., OSWALD, K. D., RODGERS, R. J., BLUNDELL, J. E., ISHII, Y., BEATTIE, A. H., HOLCH, P., ALLISON, D. B., SCHINDLER, M., ARNDT, K., RUDOLF, K., MARK, M., SCHOELCH, C., JOOST, H. G., KLAUS, S., THONE-REINEKE, C., BENOIT, S. C., SEELEY, R. J., BECK-SICKINGER, A. G., KOGLIN, N., RAUN, K., MADSEN, K., WULFF, B. S., STIDSEN, C. E., BIRRINGER, M., KREUZER, O. J., DENG, X. Y., WHITCOMB, D. C., HALEM, H., TAYLOR, J., DONG, J., DATTA, R., CULLER, M., ORTMANN, S., CASTANEDA, T. R. & TSCHOP, M. 2005. PYY3-36 as an anti-obesity drug target. Obes Rev, 6, 307-22. BONGERS, J., LAMBROS, T., AHMAD, M. & HEIMER, E. P. 1992. Kinetics of dipeptidyl peptidase IV proteolysis of growth hormone-releasing factor and analogs. Biochim Biophys Acta, 1122, 147-53. BONIC, A. & MACKIN, R. B. 2003. Expression, purification, and PC1-mediated processing of human proglucagon, glicentin, and major proglucagon fragment. Protein Expr Purif, 28, 15-24. BOOTH, A. G. & KENNY, A. J. 1974. A rapid method for the preparation of microvilli from rabbit kidney. Biochem J, 142, 575-81. BORSCHERI, N., ROESSNER, A. & ROCKEN, C. 2001. Canalicular immunostaining of neprilysin (CD10) as a diagnostic marker for hepatocellular carcinomas. Am J Surg Pathol, 25, 1297-303. BRANGE, J. & LANGKJAER, L. 1997. Insulin formulation and delivery. Pharm Biotechnol, 10, 343-409. BRANGE, J. & LANGKJOER, L. 1993. Insulin structure and stability. Pharm Biotechnol, 5, 315-50. BRAY, G. A., BLACKBURN, G. L., FERGUSON, J. M., GREENWAY, F. L., JAIN, A. K., MENDEL, C. M., MENDELS, J., RYAN, D. H., SCHWARTZ, S. L., SCHEINBAUM, M. L. & SEATON, T. B. 1999. Sibutramine produces dose-related weight loss. Obes Res, 7, 189-98. BRAY, G. A. & TARTAGLIA, L. A. 2000. Medicinal strategies in the treatment of obesity. Nature, 404, 672-7.

281

BROADWELL, R. D. & BRIGHTMAN, M. W. 1976. Entry of peroxidase into neurons of the central and peripheral nervous systems from extracerebral and cerebral blood. J Comp Neurol, 166, 257- 83. BROBERGER, C., JOHANSEN, J., JOHANSSON, C., SCHALLING, M. & HOKFELT, T. 1998. The neuropeptide Y/agouti gene-related protein (AGRP) brain circuitry in normal, anorectic, and monosodium glutamate-treated mice. Proc Natl Acad Sci U S A, 95, 15043-8. BROBERGER, C., LANDRY, M., WONG, H., WALSH, J. N. & HOKFELT, T. 1997. Subtypes Y1 and Y2 of the neuropeptide Y receptor are respectively expressed in pro-opiomelanocortin- and neuropeptide-Y-containing neurons of the rat hypothalamic arcuate nucleus. Neuroendocrinology, 66, 393-408. BUCHWALD, H., AVIDOR, Y., BRAUNWALD, E., JENSEN, M. D., PORIES, W., FAHRBACH, K. & SCHOELLES, K. 2004. Bariatric surgery: a systematic review and meta-analysis. Jama, 292, 1724-37. BUETER, M., LOWENSTEIN, C., OLBERS, T., WANG, M., CLUNY, N. L., BLOOM, S. R., SHARKEY, K. A., LUTZ, T. A. & LE ROUX, C. W. 2010. Gastric bypass increases energy expenditure in rats. Gastroenterology, 138, 1845-53. BULLOCK, B. P., HELLER, R. S. & HABENER, J. F. 1996. Tissue distribution of messenger ribonucleic acid encoding the rat glucagon-like peptide-1 receptor. Endocrinology, 137, 2968-78. BURDAKOV, D., GERASIMENKO, O. & VERKHRATSKY, A. 2005. Physiological changes in glucose differentially modulate the excitability of hypothalamic melanin-concentrating hormone and orexin neurons in situ. J Neurosci, 25, 2429-33. BURNHAM, N. L. 1994. Polymers for delivering peptides and proteins. Am J Hosp Pharm, 51, 210-8; quiz 228-9. BUSE, J. B., DRUCKER, D. J., TAYLOR, K. L., KIM, T., WALSH, B., HU, H., WILHELM, K., TRAUTMANN, M., SHEN, L. Z. & PORTER, L. E. 2010. DURATION-1: exenatide once weekly produces sustained glycemic control and weight loss over 52 weeks. Diabetes Care, 33, 1255-61. BUSE, J. B., GARBER, A., ROSENSTOCK, J., SCHMIDT, W. E., BRETT, J. H., VIDEBAEK, N., HOLST, J. & NAUCK, M. 2011. Liraglutide treatment is associated with a low frequency and magnitude of antibody formation with no apparent impact on glycemic response or increased frequency of adverse events: results from the Liraglutide Effect and Action in Diabetes (LEAD) trials. J Clin Endocrinol Metab, 96, 1695-702. CABRELE, C. & BECK-SICKINGER, A. G. 2000. Molecular characterization of the ligand-receptor interaction of the neuropeptide Y family. J Pept Sci, 6, 97-122. CALVO, J. C., YUSTA, B., MORA, F. & BLAZQUEZ, E. 1995. Structural characterization by affinity cross- linking of glucagon-like peptide-1(7-36)amide receptor in rat brain. J Neurochem, 64, 299- 306. CARO, J. F., KOLACZYNSKI, J. W., NYCE, M. R., OHANNESIAN, J. P., OPENTANOVA, I., GOLDMAN, W. H., LYNN, R. B., ZHANG, P. L., SINHA, M. K. & CONSIDINE, R. V. 1996. Decreased cerebrospinal-fluid/serum leptin ratio in obesity: a possible mechanism for leptin resistance. Lancet, 348, 159-61. CHAN, E. K., MACKEY, M. A., SNOVER, D. C., SCHNEIDER, P. D., RUCKER, R. D., JR., ALLEN, C. E. & BUCHWALD, H. 1984. Suppression of weight gain by glucagon in obese Zucker rats. Exp Mol Pathol, 40, 320-7. CHANDARANA, K., GELEGEN, C., IRVINE, E. E., CHOUDHURY, A. I., AMOUYAL, C., ANDREELLI, F., WITHERS, D. J. & BATTERHAM, R. L. 2013. Peripheral activation of the Y2-receptor promotes secretion of GLP-1 and improves glucose tolerance. Mol Metab, 2, 142-52. CHANG, A. M., JAKOBSEN, G., STURIS, J., SMITH, M. J., BLOEM, C. J., AN, B., GALECKI, A. & HALTER, J. B. 2003. The GLP-1 derivative NN2211 restores beta-cell sensitivity to glucose in type 2 diabetic patients after a single dose. Diabetes, 52, 1786-91.

282

CHATURVEDI, D. N., KNITTEL, J. J., HRUBY, V. J., CASTRUCCI, A. M. & HADLEY, M. E. 1984. Synthesis and biological actions of highly potent and prolonged acting biotin-labeled melanotropins. J Med Chem, 27, 1406-10. CHATURVEDI, P. R., DECKER, C. J. & ODINECS, A. 2001. Prediction of pharmacokinetic properties using experimental approaches during early drug discovery. Curr Opin Chem Biol, 5, 452-63. CHAUDHRI, O. B., PARKINSON, J. R., KUO, Y. T., DRUCE, M. R., HERLIHY, A. H., BELL, J. D., DHILLO, W. S., STANLEY, S. A., GHATEI, M. A. & BLOOM, S. R. 2006. Differential hypothalamic neuronal activation following peripheral injection of GLP-1 and oxyntomodulin in mice detected by manganese-enhanced magnetic resonance imaging. Biochem Biophys Res Commun, 350, 298-306. CHELIKANI, P. K., HAVER, A. C., REEVE, J. R., JR., KEIRE, D. A. & REIDELBERGER, R. D. 2006. Daily, intermittent intravenous infusion of peptide YY(3-36) reduces daily food intake and adiposity in rats. Am J Physiol Regul Integr Comp Physiol, 290, R298-305. CHELIKANI, P. K., HAVER, A. C. & REIDELBERGER, R. D. 2007. Intermittent intraperitoneal infusion of peptide YY(3-36) reduces daily food intake and adiposity in obese rats. Am J Physiol Regul Integr Comp Physiol, 293, R39-46. CHEN, H. Y., TRUMBAUER, M. E., CHEN, A. S., WEINGARTH, D. T., ADAMS, J. R., FRAZIER, E. G., SHEN, Z., MARSH, D. J., FEIGHNER, S. D., GUAN, X. M., YE, Z., NARGUND, R. P., SMITH, R. G., VAN DER PLOEG, L. H., HOWARD, A. D., MACNEIL, D. J. & QIAN, S. 2004. Orexigenic action of peripheral ghrelin is mediated by neuropeptide Y and agouti-related protein. Endocrinology, 145, 2607-12. CHEN, Y. E. & DRUCKER, D. J. 1997. Tissue-specific expression of unique mRNAs that encode proglucagon-derived peptides or exendin 4 in the lizard. J Biol Chem, 272, 4108-15. CHRISTENSEN, R., KRISTENSEN, P. K., BARTELS, E. M., BLIDDAL, H. & ASTRUP, A. 2007. Efficacy and safety of the weight-loss drug rimonabant: a meta-analysis of randomised trials. Lancet, 370, 1706-13. COHEN, D. M., JASPAN, J. B., POLONSKY, K. S., LEVER, E. G. & MOOSSA, A. R. 1984. Pancreatic hormone profiles and metabolism posthepatectomy in the dog. Evidence for a hepatotrophic role of insulin, glucagon, and pancreatic polypeptide. Gastroenterology, 87, 679-87. COHEN, M. A., ELLIS, S. M., LE ROUX, C. W., BATTERHAM, R. L., PARK, A., PATTERSON, M., FROST, G. S., GHATEI, M. A. & BLOOM, S. R. 2003. Oxyntomodulin suppresses appetite and reduces food intake in humans. J Clin Endocrinol Metab, 88, 4696-701. COLMERS, W. F. & EL BAHH, B. 2003. Neuropeptide Y and Epilepsy. Epilepsy Curr, 3, 53-58. CONNOLLY, H. M., CRARY, J. L., MCGOON, M. D., HENSRUD, D. D., EDWARDS, B. S., EDWARDS, W. D. & SCHAFF, H. V. 1997. Valvular heart disease associated with fenfluramine-phentermine. N Engl J Med, 337, 581-8. CONSIDINE, R. V., SINHA, M. K., HEIMAN, M. L., KRIAUCIUNAS, A., STEPHENS, T. W., NYCE, M. R., OHANNESIAN, J. P., MARCO, C. C., MCKEE, L. J., BAUER, T. L. & ET AL. 1996. Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med, 334, 292-5. COOKE, D. & BLOOM, S. 2006. The obesity pipeline: current strategies in the development of anti- obesity drugs. Nat Rev Drug Discov, 5, 919-31. COWLEY, M. A., PRONCHUK, N., FAN, W., DINULESCU, D. M., COLMERS, W. F. & CONE, R. D. 1999. Integration of NPY, AGRP, and melanocortin signals in the hypothalamic paraventricular nucleus: evidence of a cellular basis for the adipostat. Neuron, 24, 155-63. COWLEY, M. A., SMART, J. L., RUBINSTEIN, M., CERDAN, M. G., DIANO, S., HORVATH, T. L., CONE, R. D. & LOW, M. J. 2001. Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus. Nature, 411, 480-4. CRAWLEY, J. N. & BEINFELD, M. C. 1983. Rapid development of tolerance to the behavioural actions of cholecystokinin. Nature, 302, 703-6.

283

CUMMINGS, D. E., PURNELL, J. Q., FRAYO, R. S., SCHMIDOVA, K., WISSE, B. E. & WEIGLE, D. S. 2001. A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans. Diabetes, 50, 1714-9. CUMMINGS, D. E., WEIGLE, D. S., FRAYO, R. S., BREEN, P. A., MA, M. K., DELLINGER, E. P. & PURNELL, J. Q. 2002. Plasma ghrelin levels after diet-induced weight loss or gastric bypass surgery. N Engl J Med, 346, 1623-30. DAKIN, C. L., GUNN, I., SMALL, C. J., EDWARDS, C. M., HAY, D. L., SMITH, D. M., GHATEI, M. A. & BLOOM, S. R. 2001. Oxyntomodulin inhibits food intake in the rat. Endocrinology, 142, 4244- 50. DAKIN, C. L., SMALL, C. J., BATTERHAM, R. L., NEARY, N. M., COHEN, M. A., PATTERSON, M., GHATEI, M. A. & BLOOM, S. R. 2004. Peripheral oxyntomodulin reduces food intake and body weight gain in rats. Endocrinology, 145, 2687-95. DAKIN, C. L., SMALL, C. J., PARK, A. J., SETH, A., GHATEI, M. A. & BLOOM, S. R. 2002. Repeated ICV administration of oxyntomodulin causes a greater reduction in body weight gain than in pair-fed rats. Am J Physiol Endocrinol Metab, 283, E1173-7. DALE, G. E. A. O., C. 2006. Neprilysin. In: MESSERSCHMIDT, A. (ed.) Handbook of Metalloproteins. 3rd edition ed. Basel, Switzerland: Wiley-Blackwell. DALTON, L. D., CARPENTER, R. G. & GROSSMAN, S. P. 1981. Ingestive behavior in adult rats with dorsomedial hypothalamic lesions. Physiol Behav, 26, 117-23. DATE, Y. 2012. Ghrelin and the vagus nerve. Methods Enzymol, 514, 261-9. DATE, Y., KOJIMA, M., HOSODA, H., SAWAGUCHI, A., MONDAL, M. S., SUGANUMA, T., MATSUKURA, S., KANGAWA, K. & NAKAZATO, M. 2000. Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology, 141, 4255-61. DATE, Y., MURAKAMI, N., TOSHINAI, K., MATSUKURA, S., NIIJIMA, A., MATSUO, H., KANGAWA, K. & NAKAZATO, M. 2002. The role of the gastric afferent vagal nerve in ghrelin-induced feeding and growth hormone secretion in rats. Gastroenterology, 123, 1120-8. DAVIDSON, J. A., PARENTE, E. B. & GROSS, J. L. 2008. Incretin mimetics and dipeptidyl peptidase-4 inhibitors: innovative treatment therapies for type 2 diabetes. Arq Bras Endocrinol Metabol, 52, 1039-49. DAVIDSON, M. H., HAUPTMAN, J., DIGIROLAMO, M., FOREYT, J. P., HALSTED, C. H., HEBER, D., HEIMBURGER, D. C., LUCAS, C. P., ROBBINS, D. C., CHUNG, J. & HEYMSFIELD, S. B. 1999. Weight control and risk factor reduction in obese subjects treated for 2 years with orlistat: a randomized controlled trial. Jama, 281, 235-42. DAY, J. W., GELFANOV, V., SMILEY, D., CARRINGTON, P. E., EIERMANN, G., CHICCHI, G., ERION, M. D., GIDDA, J., THORNBERRY, N. A., TSCHOP, M. H., MARSH, D. J., SINHAROY, R., DIMARCHI, R. & POCAI, A. 2012. Optimization of co-agonism at GLP-1 and glucagon receptors to safely maximize weight reduction in DIO-rodents. Biopolymers, 98, 443-50. DAY, J. W., OTTAWAY, N., PATTERSON, J. T., GELFANOV, V., SMILEY, D., GIDDA, J., FINDEISEN, H., BRUEMMER, D., DRUCKER, D. J., CHAUDHARY, N., HOLLAND, J., HEMBREE, J., ABPLANALP, W., GRANT, E., RUEHL, J., WILSON, H., KIRCHNER, H., LOCKIE, S. H., HOFMANN, S., WOODS, S. C., NOGUEIRAS, R., PFLUGER, P. T., PEREZ-TILVE, D., DIMARCHI, R. & TSCHOP, M. H. 2009. A new glucagon and GLP-1 co-agonist eliminates obesity in rodents. Nat Chem Biol, 5, 749- 57. DE MEESTER, I., KOROM, S., VAN DAMME, J. & SCHARPE, S. 1999. CD26, let it cut or cut it down. Immunol Today, 20, 367-75. DEACON, C. F., NAUCK, M. A., TOFT-NIELSEN, M., PRIDAL, L., WILLMS, B. & HOLST, J. J. 1995. Both subcutaneously and intravenously administered glucagon-like peptide I are rapidly degraded from the NH2-terminus in type II diabetic patients and in healthy subjects. Diabetes, 44, 1126-31.

284

DECARR, L. B., BUCKHOLZ, T. M., COISH, P. D., FATHI, Z., FISK, S. E., MAYS, M. R., O'CONNOR, S. J. & LUMB, K. J. 2007. Identification of selective neuropeptide Y2 peptide agonists. Bioorg Med Chem Lett, 17, 538-41. DEEN, C., CLAASSEN, E., GERRITSE, K., ZEGERS, N. D. & BOERSMA, W. J. 1990. A novel carbodiimide coupling method for synthetic peptides. Enhanced anti-peptide antibody responses. J Immunol Methods, 129, 119-25. DEGEN, L., OESCH, S., CASANOVA, M., GRAF, S., KETTERER, S., DREWE, J. & BEGLINGER, C. 2005. Effect of peptide YY3-36 on food intake in humans. Gastroenterology, 129, 1430-1436. DEGN, K. B., JUHL, C. B., STURIS, J., JAKOBSEN, G., BROCK, B., CHANDRAMOULI, V., RUNGBY, J., LANDAU, B. R. & SCHMITZ, O. 2004. One week's treatment with the long-acting glucagon-like peptide 1 derivative liraglutide (NN2211) markedly improves 24-h glycemia and alpha- and beta-cell function and reduces endogenous glucose release in patients with type 2 diabetes. Diabetes, 53, 1187-94. DELGADO, J. M. & ANAND, B. K. 1953. Increase of food intake induced by electrical stimulation of the lateral hypothalamus. Am J Physiol, 172, 162-8. DENG, X., WOOD, P. G., SVED, A. F. & WHITCOMB, D. C. 2001. The area postrema lesions alter the inhibitory effects of peripherally infused pancreatic polypeptide on pancreatic secretion. Brain Res, 902, 18-29. DIDANGELOS, A., YIN, X., MANDAL, K., BAUMERT, M., JAHANGIRI, M. & MAYR, M. 2010. Proteomics characterization of extracellular space components in the human aorta. Mol Cell Proteomics, 9, 2048-62. DOANE, D. F., LAWSON, M. A., MEADE, J. R., KOTZ, C. M. & BEVERLY, J. L. 2007. Orexin-induced feeding requires NMDA receptor activation in the perifornical region of the lateral hypothalamus. Am J Physiol Regul Integr Comp Physiol, 293, R1022-6. DOH. 2011. Department of Health - Obesity General Information [Online]. Available: http://www.dh.gov.uk/en/Publichealth/Obesity/DH_078098 [Accessed]. DOMBROWSKI, S. U., KNITTLE, K., AVENELL, A., ARAUJO-SOARES, V. & SNIEHOTTA, F. F. 2014. Long term maintenance of weight loss with non-surgical interventions in obese adults: systematic review and meta-analyses of randomised controlled trials. BMJ, 348, g2646. DRUCE, M. R., MINNION, J. S., FIELD, B. C., PATEL, S. R., SHILLITO, J. C., TILBY, M., BEALE, K. E., MURPHY, K. G., GHATEI, M. A. & BLOOM, S. R. 2009. Investigation of structure-activity relationships of Oxyntomodulin (Oxm) using Oxm analogs. Endocrinology, 150, 1712-22. DRUCKER, D. J. 2001. Minireview: the glucagon-like peptides. Endocrinology, 142, 521-7. DRUCKER, D. J., SHI, Q., CRIVICI, A., SUMNER-SMITH, M., TAVARES, W., HILL, M., DEFOREST, L., COOPER, S. & BRUBAKER, P. L. 1997. Regulation of the biological activity of glucagon-like peptide 2 in vivo by dipeptidyl peptidase IV. Nat Biotechnol, 15, 673-7. DU, X., KOSINSKI, J. R., LAO, J., SHEN, X., PETROV, A., CHICCHI, G. G., EIERMANN, G. J. & POCAI, A. 2012. Differential effects of oxyntomodulin and GLP-1 on glucose metabolism. Am J Physiol Endocrinol Metab, 303, E265-71. DUAN, J., ZHOU, J., REN, F., TAN, C., WANG, S. & YUAN, L. 2014. Mid to distal small bowel resection with the preservation of the terminal ileum improves glucose homeostasis in diabetic rats by activating the hindgut-dependent mechanism. J Gastrointest Surg, 18, 1186-93. DUBE, M. G., KALRA, S. P. & KALRA, P. S. 1999. Food intake elicited by central administration of orexins/hypocretins: identification of hypothalamic sites of action. Brain Res, 842, 473-7. DUMONT, Y., JACQUES, D., BOUCHARD, P. & QUIRION, R. 1998. Species differences in the expression and distribution of the neuropeptide Y Y1, Y2, Y4, and Y5 receptors in rodents, guinea pig, and primates brains. J Comp Neurol, 402, 372-84. EGAWA, M., YOSHIMATSU, H. & BRAY, G. A. 1991. Neuropeptide Y suppresses sympathetic activity to interscapular brown adipose tissue in rats. Am J Physiol, 260, R328-34. EKBLAD, E. & SUNDLER, F. 2002. Distribution of pancreatic polypeptide and peptide YY. Peptides, 23, 251-61.

285

ELIAS, C. F., LEE, C., KELLY, J., ASCHKENASI, C., AHIMA, R. S., COUCEYRO, P. R., KUHAR, M. J., SAPER, C. B. & ELMQUIST, J. K. 1998. Leptin activates hypothalamic CART neurons projecting to the spinal cord. Neuron, 21, 1375-85. ELMQUIST, J. K., AHIMA, R. S., ELIAS, C. F., FLIER, J. S. & SAPER, C. B. 1998. Leptin activates distinct projections from the dorsomedial and ventromedial hypothalamic nuclei. Proc Natl Acad Sci U S A, 95, 741-6. EMA. 2011. European Medicines Agency starts review of orlistat-containing medicines [Online]. Available: http://www.ema.europa.eu/docs/en_GB/document_library/Press_release/2011/09/WC500 112798.pdf [Accessed]. EMA. 2014. Guideline on clinical evaluation of medicinal products used in weight control [Online]. Available: http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2014/07/W C500170278.pdf [Accessed]. ENGLISH, P. J., GHATEI, M. A., MALIK, I. A., BLOOM, S. R. & WILDING, J. P. 2002. Food fails to suppress ghrelin levels in obese humans. J Clin Endocrinol Metab, 87, 2984. ERDMANN, J., LIPPL, F., WAGENPFEIL, S. & SCHUSDZIARRA, V. 2005. Differential association of basal and postprandial plasma ghrelin with leptin, insulin, and type 2 diabetes. Diabetes, 54, 1371- 8. ERDOS, E. G. & SKIDGEL, R. A. 1989. Neutral endopeptidase 24.11 (enkephalinase) and related regulators of peptide hormones. FASEB J, 3, 145-51. FARIA, S. L., FARIA, O. P., CARDEAL MDE, A., DE GOUVEA, H. R. & BUFFINGTON, C. 2012. Diet- induced thermogenesis and respiratory quotient after Roux-en-Y gastric bypass. Surg Obes Relat Dis, 8, 797-802. FARIA, S. L., FARIA, O. P., CARDEAL MDE, A., ITO, M. K. & BUFFINGTON, C. 2014. Diet-induced thermogenesis and respiratory quotient after Roux-en-Y gastric bypass surgery: a prospective study. Surg Obes Relat Dis, 10, 138-43. FAROOQI, I. S., JEBB, S. A., LANGMACK, G., LAWRENCE, E., CHEETHAM, C. H., PRENTICE, A. M., HUGHES, I. A., MCCAMISH, M. A. & O'RAHILLY, S. 1999. Effects of recombinant leptin therapy in a child with congenital leptin deficiency. N Engl J Med, 341, 879-84. FAROOQI, I. S., MATARESE, G., LORD, G. M., KEOGH, J. M., LAWRENCE, E., AGWU, C., SANNA, V., JEBB, S. A., PERNA, F., FONTANA, S., LECHLER, R. I., DEPAOLI, A. M. & O'RAHILLY, S. 2002. Beneficial effects of leptin on obesity, T cell hyporesponsiveness, and neuroendocrine/metabolic dysfunction of human congenital leptin deficiency. J Clin Invest, 110, 1093-103. FDA. 2007. Guidance for Industry: Developing Products for Weight Management [Online]. Available: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidanc es/UCM071612.pdf [Accessed]. FEHMANN, H. C., JIANG, J., SCHWEINFURTH, J., DORSCH, K., WHEELER, M. B., BOYD, A. E., 3RD & GOKE, B. 1994. Ligand-specificity of the rat GLP-I receptor recombinantly expressed in Chinese hamster ovary (CHO-) cells. Z Gastroenterol, 32, 203-7. FEI, H., OKANO, H. J., LI, C., LEE, G. H., ZHAO, C., DARNELL, R. & FRIEDMAN, J. M. 1997. Anatomic localization of alternatively spliced leptin receptors (Ob-R) in mouse brain and other tissues. Proc Natl Acad Sci U S A, 94, 7001-5. FEINLE-BISSET, C., PATTERSON, M., GHATEI, M. A., BLOOM, S. R. & HOROWITZ, M. 2005. Fat digestion is required for suppression of ghrelin and stimulation of peptide YY and pancreatic polypeptide secretion by intraduodenal lipid. Am J Physiol Endocrinol Metab, 289, E948-53. FEKETE, C., LEGRADI, G., MIHALY, E., HUANG, Q. H., TATRO, J. B., RAND, W. M., EMERSON, C. H. & LECHAN, R. M. 2000. alpha-Melanocyte-stimulating hormone is contained in nerve terminals innervating thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic

286

paraventricular nucleus and prevents fasting-induced suppression of prothyrotropin- releasing hormone gene expression. J Neurosci, 20, 1550-8. FERRIER, L., SEGAIN, J. P., BONNET, C., CHERBUT, C., LEHUR, P. A., JARRY, A., GALMICHE, J. P. & BLOTTIERE, H. M. 2002. Functional mapping of NPY/PYY receptors in rat and human gastrointestinal tract. Peptides, 23, 1765-71. FEURLE, G. E., WIRTH, A., DIEHM, C., LORENZEN, M. & SCHLIERF, G. 1980. Exercise-induced release of pancreatic polypeptide and its inhibition by propranolol: evidence for adrenergic stimulation. Eur J Clin Invest, 10, 249-51. FISCH, A., CADILHAC, P., VIDOR, E., PRAZUCK, T., DUBLANCHET, A. & LAFAIX, C. 1996. Immunogenicity and safety of a new inactivated hepatitis A vaccine: a clinical trial with comparison of administration route. Vaccine, 14, 1132-6. FLEGAL, K. M., GRAUBARD, B. I., WILLIAMSON, D. F. & GAIL, M. H. 2007. Cause-specific excess deaths associated with underweight, overweight, and obesity. Jama, 298, 2028-37. FLUM, D. R., BELLE, S. H., KING, W. C., WAHED, A. S., BERK, P., CHAPMAN, W., PORIES, W., COURCOULAS, A., MCCLOSKEY, C., MITCHELL, J., PATTERSON, E., POMP, A., STATEN, M. A., YANOVSKI, S. Z., THIRLBY, R. & WOLFE, B. 2009. Perioperative safety in the longitudinal assessment of bariatric surgery. N Engl J Med, 361, 445-54. FOLTZ, I. N., HU, S., KING, C., WU, X., YANG, C., WANG, W., WEISZMANN, J., STEVENS, J., CHEN, J. S., NUANMANEE, N., GUPTE, J., KOMOROWSKI, R., SEKIROV, L., HAGER, T., ARORA, T., GE, H., BARIBAULT, H., WANG, F., SHENG, J., KAROW, M., WANG, M., LUO, Y., MCKEEHAN, W., WANG, Z., VENIANT, M. M. & LI, Y. 2012. Treating diabetes and obesity with an FGF21- mimetic antibody activating the betaKlotho/FGFR1c receptor complex. Sci Transl Med, 4, 162ra153. FOURNIER, A., GAGNON, D., QUIRION, R., CADIEUX, A., DUMONT, Y., PHENG, L. H. & ST-PIERRE, S. 1994. Conformational and biological studies of neuropeptide Y analogs containing structural alterations. Mol Pharmacol, 45, 93-101. FRANZ, M. J., VANWORMER, J. J., CRAIN, A. L., BOUCHER, J. L., HISTON, T., CAPLAN, W., BOWMAN, J. D. & PRONK, N. P. 2007. Weight-loss outcomes: a systematic review and meta-analysis of weight-loss clinical trials with a minimum 1-year follow-up. J Am Diet Assoc, 107, 1755-67. FRUEBIS, J., TSAO, T. S., JAVORSCHI, S., EBBETS-REED, D., ERICKSON, M. R., YEN, F. T., BIHAIN, B. E. & LODISH, H. F. 2001. Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice. Proc Natl Acad Sci U S A, 98, 2005-10. FU, L. Y. & VAN DEN POL, A. N. 2008. Agouti-related peptide and MC3/4 receptor agonists both inhibit excitatory hypothalamic ventromedial nucleus neurons. J Neurosci, 28, 5433-49. FUHLENDORFF, J., GETHER, U., AAKERLUND, L., LANGELAND-JOHANSEN, N., THOGERSEN, H., MELBERG, S. G., OLSEN, U. B., THASTRUP, O. & SCHWARTZ, T. W. 1990. [Leu31, Pro34]neuropeptide Y: a specific Y1 receptor agonist. Proc Natl Acad Sci U S A, 87, 182-6. FUJIMOTO, S., INUI, A., KIYOTA, N., SEKI, W., KOIDE, K., TAKAMIYA, S., UEMOTO, M., NAKAJIMA, Y., BABA, S. & KASUGA, M. 1997. Increased cholecystokinin and pancreatic polypeptide responses to a fat-rich meal in patients with restrictive but not bulimic anorexia nervosa. Biol Psychiatry, 41, 1068-70. GADDE, K. M., KOPPING, M. F., WAGNER, H. R., YONISH, G. M., ALLISON, D. B. & BRAY, G. A. 2012. Zonisamide for Weight Reduction in Obese Adults: A 1-Year Randomized Controlled Trial. Arch Intern Med, 1-8. GARBER, A. J. 2005. Pharmacologic modifications of hormones to improve their therapeutic potential for diabetes management. Diabetes Obes Metab, 7, 666-74. GEE, N. S., BOWES, M. A., BUCK, P. & KENNY, A. J. 1985. An immunoradiometric assay for endopeptidase-24.11 shows it to be a widely distributed enzyme in pig tissues. Biochem J, 228, 119-26.

287

GEHLERT, D. R., SCHOBER, D. A., BEAVERS, L., GADSKI, R., HOFFMAN, J. A., SMILEY, D. L., CHANCE, R. E., LUNDELL, I. & LARHAMMAR, D. 1996. Characterization of the peptide binding requirements for the cloned human pancreatic polypeptide-preferring receptor. Mol Pharmacol, 50, 112-8. GELLMAN, S. H. & WOOLFSON, D. N. 2002. Mini-proteins Trp the light fantastic. Nat Struct Biol, 9, 408-10. GERALD, C., WALKER, M. W., CRISCIONE, L., GUSTAFSON, E. L., BATZL-HARTMANN, C., SMITH, K. E., VAYSSE, P., DURKIN, M. M., LAZ, T. M., LINEMEYER, D. L., SCHAFFHAUSER, A. O., WHITEBREAD, S., HOFBAUER, K. G., TABER, R. I., BRANCHEK, T. A. & WEINSHANK, R. L. 1996. A receptor subtype involved in neuropeptide-Y-induced food intake. Nature, 382, 168-71. GERMAIN, N., MINNION, J. S., TAN, T., SHILLITO, J., GIBBARD, C., GHATEI, M. & BLOOM, S. 2013. Analogs of pancreatic polypeptide and peptide YY with a locked PP-fold structure are biologically active. Peptides, 39, 6-10. GHATEI, M. A., UTTENTHAL, L. O., CHRISTOFIDES, N. D., BRYANT, M. G. & BLOOM, S. R. 1983. Molecular forms of human enteroglucagon in tissue and plasma: plasma responses to nutrient stimuli in health and in disorders of the upper gastrointestinal tract. J Clin Endocrinol Metab, 57, 488-95. GIBBS, J., YOUNG, R. C. & SMITH, G. P. 1973. Cholecystokinin decreases food intake in rats. J Comp Physiol Psychol, 84, 488-95. GLOVER, I. D., BARLOW, D. J., PITTS, J. E., WOOD, S. P., TICKLE, I. J., BLUNDELL, T. L., TATEMOTO, K., KIMMEL, J. R., WOLLMER, A., STRASSBURGER, W. & ET AL. 1984. Conformational studies on the pancreatic polypeptide hormone family. Eur J Biochem, 142, 379-85. GOKE, R., FEHMANN, H. C., LINN, T., SCHMIDT, H., KRAUSE, M., ENG, J. & GOKE, B. 1993. Exendin-4 is a high potency agonist and truncated exendin-(9-39)-amide an antagonist at the glucagon- like peptide 1-(7-36)-amide receptor of insulin-secreting beta-cells. J Biol Chem, 268, 19650- 5. GOKE, R., LARSEN, P. J., MIKKELSEN, J. D. & SHEIKH, S. P. 1995. Distribution of GLP-1 binding sites in the rat brain: evidence that exendin-4 is a ligand of brain GLP-1 binding sites. Eur J Neurosci, 7, 2294-300. GOTOH, K., MASAKI, T., CHIBA, S., ANDO, H., FUJIWARA, K., SHIMASAKI, T., MITSUTOMI, K., KATSURAGI, I., KAKUMA, T., SAKATA, T. & YOSHIMATSU, H. 2013. Brain-derived neurotrophic factor, corticotropin-releasing factor, and hypothalamic neuronal histamine interact to regulate feeding behavior. J Neurochem, 125, 588-98. GRANDT, D., DAHMS, P., SCHIMICZEK, M., EYSSELEIN, V. E., REEVE, J. R., JR. & MENTLEIN, R. 1993. [Proteolytic processing by dipeptidyl aminopeptidase IV generates receptor selectivity for peptide YY (PYY)]. Med Klin (Munich), 88, 143-5. GRANDT, D., TEYSSEN, S., SCHIMICZEK, M., REEVE, J. R., JR., FETH, F., RASCHER, W., HIRCHE, H., SINGER, M. V., LAYER, P., GOEBELL, H. & ET AL. 1992. Novel generation of hormone receptor specificity by amino terminal processing of peptide YY. Biochem Biophys Res Commun, 186, 1299-306. GREEN, B. D., GAULT, V. A., MOONEY, M. H., IRWIN, N., BAILEY, C. J., HARRIOTT, P., GREER, B., FLATT, P. R. & O'HARTE, F. P. 2003. Novel dipeptidyl peptidase IV resistant analogues of glucagon- like peptide-1(7-36)amide have preserved biological activities in vitro conferring improved glucose-lowering action in vivo. J Mol Endocrinol, 31, 529-40. GREENWAY, F. L., FUJIOKA, K., PLODKOWSKI, R. A., MUDALIAR, S., GUTTADAURIA, M., ERICKSON, J., KIM, D. D. & DUNAYEVICH, E. 2010. Effect of naltrexone plus bupropion on weight loss in overweight and obese adults (COR-I): a multicentre, randomised, double-blind, placebo- controlled, phase 3 trial. Lancet, 376, 595-605. GRILL, H. J. & KAPLAN, J. M. 2002. The neuroanatomical axis for control of energy balance. Front Neuroendocrinol, 23, 2-40.

288

GRILL, H. J. & SMITH, G. P. 1988. Cholecystokinin decreases sucrose intake in chronic decerebrate rats. Am J Physiol, 254, R853-6. GROMADA, J., DING, W. G., BARG, S., RENSTROM, E. & RORSMAN, P. 1997. Multisite regulation of insulin secretion by cAMP-increasing agonists: evidence that glucagon-like peptide 1 and glucagon act via distinct receptors. Pflugers Arch, 434, 515-24. GROSSE, J., HEFFRON, H., BURLING, K., AKHTER HOSSAIN, M., HABIB, A. M., ROGERS, G. J., RICHARDS, P., LARDER, R., RIMMINGTON, D., ADRIAENSSENS, A. A., PARTON, L., POWELL, J., BINDA, M., COLLEDGE, W. H., DORAN, J., TOYODA, Y., WADE, J. D., APARICIO, S., CARLTON, M. B., COLL, A. P., REIMANN, F., O'RAHILLY, S. & GRIBBLE, F. M. 2014. Insulin-like peptide 5 is an orexigenic gastrointestinal hormone. Proc Natl Acad Sci U S A, 111, 11133-8. GRUNDEMAR, L., KRSTENANSKY, J. L. & HAKANSON, R. 1993. Activation of neuropeptide Y1 and neuropeptide Y2 receptors by substituted and truncated neuropeptide Y analogs: identification of signal epitopes. Eur J Pharmacol, 232, 271-8. GSK 2014. GSK receives US approval for once-weekly type 2 diabetes treatment, Tanzeum™ (albiglutide). GUERCIOLINI, R. 1997. Mode of action of orlistat. Int J Obes Relat Metab Disord, 21 Suppl 3, S12-23. GUO, Y., MA, L., ENRIORI, P. J., KOSKA, J., FRANKS, P. W., BROOKSHIRE, T., COWLEY, M. A., SALBE, A. D., DELPARIGI, A. & TATARANNI, P. A. 2006. Physiological evidence for the involvement of peptide YY in the regulation of energy homeostasis in humans. Obesity (Silver Spring), 14, 1562-70. GUSTAFSON, E. L., SMITH, K. E., DURKIN, M. M., WALKER, M. W., GERALD, C., WEINSHANK, R. & BRANCHEK, T. A. 1997. Distribution of the neuropeptide Y Y2 receptor mRNA in rat central nervous system. Brain Res Mol Brain Res, 46, 223-35. HAGOPIAN, W., LEVER, E. G., COHEN, D., EMMANOUEL, D., POLONSKY, K. S., PUGH, W., MOOSSA, A. & JASPAN, J. B. 1983. Predominance of renal and absence of hepatic metabolism of pancreatic polypeptide in the dog. Am J Physiol, 245, E171-7. HALATCHEV, I. G., ELLACOTT, K. L., FAN, W. & CONE, R. D. 2004. Peptide YY3-36 inhibits food intake in mice through a melanocortin-4 receptor-independent mechanism. Endocrinology, 145, 2585-90. HANAHAN, D. A. F. R. B. 1996. Mechanisms of DNA transformation. In: F. C. NEIDHARDT, R. C. C. I., J. L. INGRAHAM, E. C. C. LIN, K. B. LOW, B. MAGASANIK, W. S. REZNIKOFF, M. RILEY, M. SCHAECHTER, AND H. E. UMBARGER (ed.) Escherichia coli and Salmonella cellular and molecular biology. 2nd edition ed. Washington, DC: American Society for Microbiology. HANSEN, T. K., DALL, R., HOSODA, H., KOJIMA, M., KANGAWA, K., CHRISTIANSEN, J. S. & JORGENSEN, J. O. 2002. Weight loss increases circulating levels of ghrelin in human obesity. Clin Endocrinol (Oxf), 56, 203-6. HARDER, H., NIELSEN, L., TU, D. T. & ASTRUP, A. 2004. The effect of liraglutide, a long-acting glucagon-like peptide 1 derivative, on glycemic control, body composition, and 24-h energy expenditure in patients with type 2 diabetes. Diabetes Care, 27, 1915-21. HATOUM, I. J. & KAPLAN, L. M. 2013. Advantages of percent weight loss as a method of reporting weight loss after Roux-en-Y gastric bypass. Obesity (Silver Spring), 21, 1519-25. HELOU, N., OBEID, O., AZAR, S. T. & HWALLA, N. 2008. Variation of postprandial PYY 3-36 response following ingestion of differing macronutrient meals in obese females. Ann Nutr Metab, 52, 188-95. HERNANDEZ, E. J., WHITCOMB, D. C., VIGNA, S. R. & TAYLOR, I. L. 1994. Saturable binding of circulating peptide YY in the dorsal vagal complex of rats. Am J Physiol, 266, G511-6. HERRMANN, C., GOKE, R., RICHTER, G., FEHMANN, H. C., ARNOLD, R. & GOKE, B. 1995. Glucagon-like peptide-1 and glucose-dependent insulin-releasing polypeptide plasma levels in response to nutrients. Digestion, 56, 117-26. HERZOG, C. 2014. Influence of parenteral administration routes and additional factors on vaccine safety and immunogenicity: a review of recent literature. Expert Rev Vaccines, 13, 399-415.

289

HILL, J. O., HAUPTMAN, J., ANDERSON, J. W., FUJIOKA, K., O'NEIL, P. M., SMITH, D. K., ZAVORAL, J. H. & ARONNE, L. J. 1999. Orlistat, a lipase inhibitor, for weight maintenance after conventional dieting: a 1-y study. Am J Clin Nutr, 69, 1108-16. HINDLET, P., BADO, A., KAMENICKY, P., DELOMENIE, C., BOURASSET, F., NAZARET, C., FARINOTTI, R. & BUYSE, M. 2009. Reduced intestinal absorption of dipeptides via PepT1 in mice with diet- induced obesity is associated with leptin receptor down-regulation. J Biol Chem, 284, 6801-8. HODGES, G. M., LIVINGSTON, D. C. & FRANKS, L. M. 1973. The localization of trypsin in cultured mammalian cells. J Cell Sci, 12, 887-902. HOLST, J. J. 1994. Glucagonlike peptide 1: a newly discovered gastrointestinal hormone. Gastroenterology, 107, 1848-55. HOLZ, G. G. & HABENER, J. F. 1998. Black widow spider alpha-latrotoxin: a presynaptic neurotoxin that shares structural homology with the glucagon-like peptide-1 family of insulin secretagogic hormones. Comp Biochem Physiol B Biochem Mol Biol, 121, 177-84. HOSODA, H., KOJIMA, M., MATSUO, H. & KANGAWA, K. 2000. Ghrelin and des-acyl ghrelin: two major forms of rat ghrelin peptide in gastrointestinal tissue. Biochem Biophys Res Commun, 279, 909-13. HOTTA, K., FUNAHASHI, T., BODKIN, N. L., ORTMEYER, H. K., ARITA, Y., HANSEN, B. C. & MATSUZAWA, Y. 2001. Circulating concentrations of the adipocyte protein adiponectin are decreased in parallel with reduced insulin sensitivity during the progression to type 2 diabetes in rhesus monkeys. Diabetes, 50, 1126-33. HOUSTON, J. B. 1994. Relevance of in vitro kinetic parameters to in vivo metabolism of xenobiotics. Toxicol In Vitro, 8, 507-12. HSCIC 2014. Statistics on Obesity, Physical Activity and Diet in England. HU, E., LIANG, P. & SPIEGELMAN, B. M. 1996. AdipoQ is a novel adipose-specific gene dysregulated in obesity. J Biol Chem, 271, 10697-703. HUGHES, T. E., KIM, D. D., MARJASON, J., PROIETTO, J., WHITEHEAD, J. P. & VATH, J. E. 2013. Ascending dose-controlled trial of beloranib, a novel obesity treatment for safety, tolerability, and weight loss in obese women. Obesity (Silver Spring). HUI, H., NOURPARVAR, A., ZHAO, X. & PERFETTI, R. 2003. Glucagon-like peptide-1 inhibits apoptosis of insulin-secreting cells via a cyclic 5'-adenosine monophosphate-dependent protein kinase A- and a phosphatidylinositol 3-kinase-dependent pathway. Endocrinology, 144, 1444-55. HUPE-SODMANN, K., MCGREGOR, G. P., BRIDENBAUGH, R., GOKE, R., GOKE, B., THOLE, H., ZIMMERMANN, B. & VOIGT, K. 1995. Characterisation of the processing by human neutral endopeptidase 24.11 of GLP-1(7-36) amide and comparison of the substrate specificity of the enzyme for other glucagon-like peptides. Regul Pept, 58, 149-56. HUSZAR, D., LYNCH, C. A., FAIRCHILD-HUNTRESS, V., DUNMORE, J. H., FANG, Q., BERKEMEIER, L. R., GU, W., KESTERSON, R. A., BOSTON, B. A., CONE, R. D., SMITH, F. J., CAMPFIELD, L. A., BURN, P. & LEE, F. 1997. Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell, 88, 131-41. IHARA, T., TSUJIKAWA, T., FUJIYAMA, Y., UEYAMA, H., OHKUBO, I. & BAMBA, T. 2000. Enhancement of brush border membrane peptidase activity in rat jejunum induced by starvation. Pflugers Arch, 440, 75-83. IKEZAWA, Y., YAMATANI, K., OGAWA, A., OHNUMA, H., IGARASHI, M., DAIMON, M., MANAKA, H. & SASAKI, H. 1998. Effects of glucagon on glycogenolysis and gluconeogenesis are region- specific in periportal and perivenous hepatocytes. J Lab Clin Med, 132, 547-55. IRWIN, N., FRIZELLE, P., MONTGOMERY, I. A., MOFFETT, R. C., O'HARTE, F. P. & FLATT, P. R. 2012. Beneficial effects of the novel cholecystokinin agonist (pGlu-Gln)-CCK-8 in mouse models of obesity/diabetes. Diabetologia, 55, 2747-58. IRWIN, N., FRIZELLE, P., O'HARTE, F. P. & FLATT, P. R. 2013a. (pGlu-Gln)-CCK-8[mPEG]: a novel, long- acting, mini-PEGylated cholecystokinin (CCK) agonist that improves metabolic status in dietary-induced diabetes. Biochim Biophys Acta, 1830, 4009-16.

290

IRWIN, N., MONTGOMERY, I. A. & FLATT, P. R. 2013b. Comparison of the metabolic effects of sustained CCK1 receptor activation alone and in combination with upregulated leptin signalling in high-fat-fed mice. Diabetologia, 56, 1425-35. ITO, K., IWATSUBO, T., KANAMITSU, S., NAKAJIMA, Y. & SUGIYAMA, Y. 1998. Quantitative prediction of in vivo drug clearance and drug interactions from in vitro data on metabolism, together with binding and transport. Annu Rev Pharmacol Toxicol, 38, 461-99. JAHN, E.-M. & SCHNEIDER, C. K. 2009. How to systematically evaluate immunogenicity of therapeutic proteins – regulatory considerations. New Biotechnology, 25, 280-286. JARKOVSKA, Z., KRSEK, M., ROSICKA, M. & MAREK, J. 2004. Endocrine and metabolic activities of a recently isolated peptide hormone ghrelin, an endogenous ligand of the growth hormone secretagogue receptor. Endocr Regul, 38, 80-6. JELINEK, L. J., LOK, S., ROSENBERG, G. B., SMITH, R. A., GRANT, F. J., BIGGS, S., BENSCH, P. A., KUIJPER, J. L., SHEPPARD, P. O., SPRECHER, C. A. & ET AL. 1993. Expression cloning and signaling properties of the rat glucagon receptor. Science, 259, 1614-6. JESUDASON, D. R., MONTEIRO, M. P., MCGOWAN, B. M., NEARY, N. M., PARK, A. J., PHILIPPOU, E., SMALL, C. J., FROST, G. S., GHATEI, M. A. & BLOOM, S. R. 2007. Low-dose pancreatic polypeptide inhibits food intake in man. Br J Nutr, 97, 426-9. JILKOVA, Z. M., HENSLER, M., MEDRIKOVA, D., JANOVSKA, P., HORAKOVA, O., ROSSMEISL, M., FLACHS, P., SELL, H., ECKEL, J. & KOPECKY, J. 2013. Adipose tissue-related proteins locally associated with resolution of inflammation in obese mice. Int J Obes (Lond). JIN, S. L., HAN, V. K., SIMMONS, J. G., TOWLE, A. C., LAUDER, J. M. & LUND, P. K. 1988. Distribution of glucagonlike peptide I (GLP-I), glucagon, and glicentin in the rat brain: an immunocytochemical study. J Comp Neurol, 271, 519-32. JORDAN, J., GREENWAY, F. L., LEITER, L. A., LI, Z., JACOBSON, P., MURPHY, K., HILL, J., KLER, L. & AFTRING, R. P. 2008. Stimulation of cholecystokinin-A receptors with GI181771X does not cause weight loss in overweight or obese patients. Clin Pharmacol Ther, 83, 281-7. JUHL, C. B., HOLLINGDAL, M., STURIS, J., JAKOBSEN, G., AGERSO, H., VELDHUIS, J., PORKSEN, N. & SCHMITZ, O. 2002. Bedtime administration of NN2211, a long-acting GLP-1 derivative, substantially reduces fasting and postprandial glycemia in type 2 diabetes. Diabetes, 51, 424- 9. JUNG, C. H. & KIM, M. S. 2013. Molecular mechanisms of central leptin resistance in obesity. Arch Pharm Res, 36, 201-7. KENNY, A. J. 1986. Regulatory peptide metabolism at cell surfaces: the key role of endopeptidase- 24.11. Biomed Biochim Acta, 45, 1503-13. KENNY, A. J., FULCHER, I. S., RIDGWELL, K. & INGRAM, J. 1981. Microvillar membrane neutral endopeptidases. Acta Biol Med Ger, 40, 1465-71. KENNY, A. J. & STEPHENSON, S. L. 1988. Role of endopeptidase-24.11 in the inactivation of atrial natriuretic peptide. FEBS Lett, 232, 1-8. KESSLER, M., GOLDSMITH, D. & SCHELLEKENS, H. 2006. Immunogenicity of biopharmaceuticals. Nephrol Dial Transplant, 21 Suppl 5, v9-12. KIEFFER, T. J., MCINTOSH, C. H. & PEDERSON, R. A. 1995. Degradation of glucose-dependent insulinotropic polypeptide and truncated glucagon-like peptide 1 in vitro and in vivo by dipeptidyl peptidase IV. Endocrinology, 136, 3585-96. KIEVIT, P., HALEM, H., MARKS, D. L., DONG, J. Z., GLAVAS, M. M., SINNAYAH, P., PRANGER, L., COWLEY, M. A., GROVE, K. L. & CULLER, M. D. 2013. Chronic treatment with a melanocortin- 4 receptor agonist causes weight loss, reduces insulin resistance, and improves cardiovascular function in diet-induced obese rhesus macaques. Diabetes, 62, 490-7. KING, B. M. 2006. The rise, fall, and resurrection of the ventromedial hypothalamus in the regulation of feeding behavior and body weight. Physiol Behav, 87, 221-44.

291

KINZIG, K. P., COUGHLIN, J. W., REDGRAVE, G. W., MORAN, T. H. & GUARDA, A. S. 2007. Insulin, glucose, and pancreatic polypeptide responses to a test meal in restricting type anorexia nervosa before and after weight restoration. Am J Physiol Endocrinol Metab, 292, E1441-6. KISSILEFF, H. R., PI-SUNYER, F. X., THORNTON, J. & SMITH, G. P. 1981. C-terminal octapeptide of cholecystokinin decreases food intake in man. Am J Clin Nutr, 34, 154-60. KNUDSEN, L. B., NIELSEN, P. F., HUUSFELDT, P. O., JOHANSEN, N. L., MADSEN, K., PEDERSEN, F. Z., THOGERSEN, H., WILKEN, M. & AGERSO, H. 2000. Potent derivatives of glucagon-like peptide-1 with pharmacokinetic properties suitable for once daily administration. J Med Chem, 43, 1664-9. KNUDSEN, L. B. & PRIDAL, L. 1996. Glucagon-like peptide-1-(9-36) amide is a major metabolite of glucagon-like peptide-1-(7-36) amide after in vivo administration to dogs, and it acts as an antagonist on the pancreatic receptor. Eur J Pharmacol, 318, 429-35. KODA, S., DATE, Y., MURAKAMI, N., SHIMBARA, T., HANADA, T., TOSHINAI, K., NIIJIMA, A., FURUYA, M., INOMATA, N., OSUYE, K. & NAKAZATO, M. 2005. The role of the vagal nerve in peripheral PYY3-36-induced feeding reduction in rats. Endocrinology, 146, 2369-75. KOJIMA, M., HOSODA, H., DATE, Y., NAKAZATO, M., MATSUO, H. & KANGAWA, K. 1999. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature, 402, 656-60. KOJIMA, S., UENO, N., ASAKAWA, A., SAGIYAMA, K., NARUO, T., MIZUNO, S. & INUI, A. 2007. A role for pancreatic polypeptide in feeding and body weight regulation. Peptides, 28, 459-63. KOMADA, F., OKUMURA, K. & HORI, R. 1985. Fate of porcine and human insulin at the subcutaneous injection site. II. In vitro degradation of insulins in the subcutaneous tissue of the rat. J Pharmacobiodyn, 8, 33-40. KOOLE, C., PABREJA, K., SAVAGE, E. E., WOOTTEN, D., FURNESS, S. G., MILLER, L. J., CHRISTOPOULOS, A. & SEXTON, P. M. 2013. Recent advances in understanding GLP-1R (glucagon-like peptide-1 receptor) function. Biochem Soc Trans, 41, 172-9. KOOLE, C., WOOTTEN, D., SIMMS, J., MILLER, L. J., CHRISTOPOULOS, A. & SEXTON, P. M. 2012a. Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) has a critical role in GLP-1 peptide binding and receptor activation. J Biol Chem, 287, 3642-58. KOOLE, C., WOOTTEN, D., SIMMS, J., SAVAGE, E. E., MILLER, L. J., CHRISTOPOULOS, A. & SEXTON, P. M. 2012b. Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) differentially regulates orthosteric but not allosteric agonist binding and function. J Biol Chem, 287, 3659-73. KOOLE, C., WOOTTEN, D., SIMMS, J., VALANT, C., SRIDHAR, R., WOODMAN, O. L., MILLER, L. J., SUMMERS, R. J., CHRISTOPOULOS, A. & SEXTON, P. M. 2010. Allosteric ligands of the glucagon-like peptide 1 receptor (GLP-1R) differentially modulate endogenous and exogenous peptide responses in a pathway-selective manner: implications for drug screening. Mol Pharmacol, 78, 456-65. KOSINSKI, J. R., HUBERT, J., CARRINGTON, P. E., CHICCHI, G. G., MU, J., MILLER, C., CAO, J., BIANCHI, E., PESSI, A., SINHAROY, R., MARSH, D. J. & POCAI, A. 2012. The glucagon receptor is involved in mediating the body weight-lowering effects of oxyntomodulin. Obesity (Silver Spring), 20, 1566-71. KREYMANN, B., GHATEI, M. A., BURNET, P., WILLIAMS, G., KANSE, S., DIANI, A. R. & BLOOM, S. R. 1989. Characterization of glucagon-like peptide-1-(7-36)amide in the hypothalamus. Brain Res, 502, 325-31. KREYMANN, B., WILLIAMS, G., GHATEI, M. A. & BLOOM, S. R. 1987. Glucagon-like peptide-1 7-36: a physiological incretin in man. Lancet, 2, 1300-4. KUO, L. E., KITLINSKA, J. B., TILAN, J. U., LI, L., BAKER, S. B., JOHNSON, M. D., LEE, E. W., BURNETT, M. S., FRICKE, S. T., KVETNANSKY, R., HERZOG, H. & ZUKOWSKA, Z. 2007. Neuropeptide Y acts directly in the periphery on fat tissue and mediates stress-induced obesity and metabolic syndrome. Nat Med, 13, 803-11.

292

LAMBEIR, A. M., PROOST, P., SCHARPE, S. & DE MEESTER, I. 2002. A kinetic study of glucagon-like peptide-1 and glucagon-like peptide-2 truncation by dipeptidyl peptidase IV, in vitro. Biochem Pharmacol, 64, 1753-6. LAMERS, C. B., DIEMEL, C. M., VAN LEER, E., VAN LEUSEN, R. & PEETOOM, J. J. 1982. Mechanism of elevated serum pancreatic polypeptide concentrations in chronic renal failure. J Clin Endocrinol Metab, 55, 922-6. LARHAMMAR, D. 1996a. Evolution of neuropeptide Y, peptide YY and pancreatic polypeptide. Regul Pept, 62, 1-11. LARHAMMAR, D. 1996b. Structural diversity of receptors for neuropeptide Y, peptide YY and pancreatic polypeptide. Regul Pept, 65, 165-74. LARSEN, P. J., TANG-CHRISTENSEN, M., HOLST, J. J. & ORSKOV, C. 1997a. Distribution of glucagon-like peptide-1 and other preproglucagon-derived peptides in the rat hypothalamus and brainstem. Neuroscience, 77, 257-70. LARSEN, P. J., TANG-CHRISTENSEN, M. & JESSOP, D. S. 1997b. Central administration of glucagon-like peptide-1 activates hypothalamic neuroendocrine neurons in the rat. Endocrinology, 138, 4445-55. LASKIEWICZ, J., KROLCZYK, G., ZUROWSKI, G., SOBOCKI, J., MATYJA, A. & THOR, P. J. 2003. Effects of vagal neuromodulation and vagotomy on control of food intake and body weight in rats. J Physiol Pharmacol, 54, 603-10. LASSMANN, V., VAGUE, P., VIALETTES, B. & SIMON, M. C. 1980. Low plasma levels of pancreatic polypeptide in obesity. Diabetes, 29, 428-30. LE ROUX, C. W., BORG, C. M., MURPHY, K. G., VINCENT, R. P., GHATEI, M. A. & BLOOM, S. R. 2008a. Supraphysiological doses of intravenous PYY3-36 cause nausea, but no additional reduction in food intake. Ann Clin Biochem, 45, 93-5. LE ROUX, C. W., BORG, C. M., MURPHY, K. G., VINCENT, R. P., GHATEI, M. A. & BLOOM, S. R. 2008b. Supraphysiological doses of intravenous PYY3-36 cause nausea, but no additional reduction in food intake. Annals of Clinical Biochemistry, 45, 93-95. LEADBEATER, L. & DAVIES, D. R. 1964. The Stability of the Drug Metabolising Enzymes of Liver Microsomal Preparations. Biochem Pharmacol, 13, 1607-17. LEE, C. M., CHOI, Y., HUH, E. J., LEE, K. Y., SONG, H. C., SUN, M. J., JEONG, H. J., CHO, C. S. & BOM, H. S. 2005. Polyethylene glycol (PEG) modified 99mTc-HMPAO-liposome for improving blood circulation and biodistribution: the effect of the extent of PEGylation. Cancer Biother Radiopharm, 20, 620-8. LEE, S. W. & STANLEY, B. G. 2005. NMDA receptors mediate feeding elicited by neuropeptide Y in the lateral and perifornical hypothalamus. Brain Res, 1063, 1-8. LEIBOWITZ, S. F., HAMMER, N. J. & CHANG, K. 1981. Hypothalamic paraventricular nucleus lesions produce overeating and obesity in the rat. Physiol Behav, 27, 1031-40. LERCH, M., KAMIMORI, H., FOLKERS, G., AGUILAR, M. I., BECK-SICKINGER, A. G. & ZERBE, O. 2005. Strongly altered receptor binding properties in PP and NPY chimeras are accompanied by changes in structure and membrane binding. Biochemistry, 44, 9255-64. LERCH, M., MAYRHOFER, M. & ZERBE, O. 2004. Structural similarities of micelle-bound peptide YY (PYY) and neuropeptide Y (NPY) are related to their affinity profiles at the Y receptors. J Mol Biol, 339, 1153-68. LEUNG, W. Y., THOMAS, G. N., CHAN, J. C. & TOMLINSON, B. 2003. Weight management and current options in pharmacotherapy: orlistat and sibutramine. Clin Ther, 25, 58-80. LIANG, N. C., BELLO, N. T. & MORAN, T. H. 2013. Additive feeding inhibitory and aversive effects of naltrexone and exendin-4 combinations. Int J Obes (Lond), 37, 272-8. LIDDLE, R. A., GOLDFINE, I. D., ROSEN, M. S., TAPLITZ, R. A. & WILLIAMS, J. A. 1985. Cholecystokinin bioactivity in human plasma. Molecular forms, responses to feeding, and relationship to gallbladder contraction. J Clin Invest, 75, 1144-52.

293

LIJNEN, H. R., FREDERIX, L. & VAN HOEF, B. 2010. Fumagillin reduces adipose tissue formation in murine models of nutritionally induced obesity. Obesity (Silver Spring), 18, 2241-6. LIN, S., SHI, Y. C., YULYANINGSIH, E., ALJANOVA, A., ZHANG, L., MACIA, L., NGUYEN, A. D., LIN, E. J., DURING, M. J., HERZOG, H. & SAINSBURY, A. 2009. Critical role of arcuate Y4 receptors and the melanocortin system in pancreatic polypeptide-induced reduction in food intake in mice. PLoS One, 4, e8488. LIU, Y. L., FORD, H. E., DRUCE, M. R., MINNION, J. S., FIELD, B. C., SHILLITO, J. C., BAXTER, J., MURPHY, K. G., GHATEI, M. A. & BLOOM, S. R. 2010. Subcutaneous oxyntomodulin analogue administration reduces body weight in lean and obese rodents. Int J Obes (Lond), 34, 1715- 25. LLUIS, F., FUJIMURA, M., GOMEZ, G., SALVA, J. A., GREELEY, G. H., JR. & THOMPSON, J. C. 1989. [Cellular localization, half-life, and secretion of peptide YY]. Rev Esp Fisiol, 45, 377-84. LO, C. M., KING, A., SAMUELSON, L. C., KINDEL, T. L., RIDER, T., JANDACEK, R. J., RAYBOULD, H. E., WOODS, S. C. & TSO, P. 2010. Cholecystokinin knockout mice are resistant to high-fat diet- induced obesity. Gastroenterology, 138, 1997-2005. LO, K. M., ZHANG, J., SUN, Y., MORELLI, B., LAN, Y., LAUDER, S., BRUNKHORST, B., WEBSTER, G., HALLAKOU-BOZEC, S., DOARE, L. & GILLIES, S. D. 2005. Engineering a pharmacologically superior form of leptin for the treatment of obesity. Protein Eng Des Sel, 18, 1-10. LOCKIE, S. H., HEPPNER, K. M., CHAUDHARY, N., CHABENNE, J. R., MORGAN, D. A., VEYRAT-DUREBEX, C., ANANTHAKRISHNAN, G., ROHNER-JEANRENAUD, F., DRUCKER, D. J., DIMARCHI, R., RAHMOUNI, K., OLDFIELD, B. J., TSCHOP, M. H. & PEREZ-TILVE, D. 2012. Direct control of brown adipose tissue thermogenesis by central nervous system glucagon-like peptide-1 receptor signaling. Diabetes, 61, 2753-62. LONGUET, C., SINCLAIR, E. M., MAIDA, A., BAGGIO, L. L., MAZIARZ, M., CHARRON, M. J. & DRUCKER, D. J. 2008. The glucagon receptor is required for the adaptive metabolic response to fasting. Cell Metab, 8, 359-71. LUITEN, P. G., TER HORST, G. J. & STEFFENS, A. B. 1987. The hypothalamus, intrinsic connections and outflow pathways to the endocrine system in relation to the control of feeding and metabolism. Prog Neurobiol, 28, 1-54. MA, X., ZHANG, Y., GROMADA, J., SEWING, S., BERGGREN, P. O., BUSCHARD, K., SALEHI, A., VIKMAN, J., RORSMAN, P. & ELIASSON, L. 2005. Glucagon stimulates exocytosis in mouse and rat pancreatic alpha-cells by binding to glucagon receptors. Mol Endocrinol, 19, 198-212. MACDONALD, P. E., EL-KHOLY, W., RIEDEL, M. J., SALAPATEK, A. M., LIGHT, P. E. & WHEELER, M. B. 2002. The multiple actions of GLP-1 on the process of glucose-stimulated insulin secretion. Diabetes, 51 Suppl 3, S434-42. MADSBAD, S., KIELGAST, U., ASMAR, M., DEACON, C. F., TOREKOV, S. S. & HOLST, J. J. 2011. An overview of once-weekly glucagon-like peptide-1 receptor agonists-available efficacy and safety data and perspectives for the future. Diabetes Obes Metab, 13, 394-407. MAKRIS, A. & FOSTER, G. D. 2011. Dietary approaches to the treatment of obesity. Psychiatr Clin North Am, 34, 813-27. MANDEL, M. & HIGA, A. 1992. Calcium-dependent bacteriophage DNA infection. 1970. Biotechnology, 24, 198-201. MARTELLA, E. M., FERRARO, G., AZZONI, C., MARIGNANI, M. & BORDI, C. 1997. Pancreatic- polypeptide cell hyperplasia associated with pancreatic or duodenal gastrinomas. Hum Pathol, 28, 149-53. MATSON, C. A., REID, D. F., CANNON, T. A. & RITTER, R. C. 2000. Cholecystokinin and leptin act synergistically to reduce body weight. Am J Physiol Regul Integr Comp Physiol, 278, R882-90. MATSUDA, M. & SHIMOMURA, I. 2013. Roles of adiponectin and oxidative stress in obesity- associated metabolic and cardiovascular diseases. Rev Endocr Metab Disord. MATTHEWS, J. E., STEWART, M. W., DE BOEVER, E. H., DOBBINS, R. L., HODGE, R. J., WALKER, S. E., HOLLAND, M. C. & BUSH, M. A. 2008. Pharmacodynamics, pharmacokinetics, safety, and

294

tolerability of albiglutide, a long-acting glucagon-like peptide-1 mimetic, in patients with type 2 diabetes. J Clin Endocrinol Metab, 93, 4810-7. MCFADDEN, D. W., RUDNICKI, M., KUVSHINOFF, B. & FISCHER, J. E. 1992. Postprandial peptide YY release is mediated by cholecystokinin. Surg Gynecol Obstet, 175, 145-50. MCGOWAN, M. K., ANDREWS, K. M. & GROSSMAN, S. P. 1992. Chronic intrahypothalamic infusions of insulin or insulin antibodies alter body weight and food intake in the rat. Physiol Behav, 51, 753-66. MCLAUGHLIN, C. L., BAILE, C. A. & BUONOMO, F. C. 1985. Effect of CCK antibodies on food intake and weight gain in Zucker rats. Physiol Behav, 34, 277-82. MCMAHON, L. R. & WELLMAN, P. J. 1998. PVN infusion of GLP-1-(7-36) amide suppresses feeding but does not induce aversion or alter locomotion in rats. Am J Physiol, 274, R23-9. MCTIGUE, D. M., EDWARDS, N. K. & ROGERS, R. C. 1993. Pancreatic polypeptide in dorsal vagal complex stimulates gastric acid secretion and motility in rats. Am J Physiol, 265, G1169-76. MEDEIROS, M. D. & TURNER, A. J. 1994. Processing and metabolism of peptide-YY: pivotal roles of dipeptidylpeptidase-IV, aminopeptidase-P, and endopeptidase-24.11. Endocrinology, 134, 2088-94. MEDEIROS, M. D. & TURNER, A. J. 1995. Metabolic stability of some tachykinin analogues to cell- surface peptidases: roles for endopeptidase-24.11 and aminopeptidase N. Peptides, 16, 441- 7. MEDEIROS MDOS, S. & TURNER, A. J. 1994. Comparison of cholecystokinin metabolism by membrane preparations from the human astrocytoma clone D384 and the neuroblastoma line SH-SY5Y. Neurochem Int, 24, 369-77. MEDEIROS MDOS, S. & TURNER, A. J. 1996. Metabolism and functions of neuropeptide Y. Neurochem Res, 21, 1125-32. MEEREIS-SCHWANKE, K., KLONOWSKI-STUMPE, H., HERBERG, L. & NIEDERAU, C. 1998. Long-term effects of CCK-agonist and -antagonist on food intake and body weight in Zucker lean and obese rats. Peptides, 19, 291-9. MEISTER, B. 2007. Neurotransmitters in key neurons of the hypothalamus that regulate feeding behavior and body weight. Physiol Behav, 92, 263-71. MELIS, M. R., MASCIA, M. S., SUCCU, S., TORSELLO, A., MULLER, E. E., DEGHENGHI, R. & ARGIOLAS, A. 2002. Ghrelin injected into the paraventricular nucleus of the hypothalamus of male rats induces feeding but not penile erection. Neurosci Lett, 329, 339-43. MENENDEZ, J. A. & ATRENS, D. M. 1991. Insulin and the paraventricular hypothalamus: modulation of energy balance. Brain Res, 555, 193-201. MENTLEIN, R. 1999. Dipeptidyl-peptidase IV (CD26)--role in the inactivation of regulatory peptides. Regul Pept, 85, 9-24. MENTLEIN, R., DAHMS, P., GRANDT, D. & KRUGER, R. 1993a. Proteolytic processing of neuropeptide Y and peptide YY by dipeptidyl peptidase IV. Regul Pept, 49, 133-44. MENTLEIN, R., GALLWITZ, B. & SCHMIDT, W. E. 1993b. Dipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide, glucagon-like peptide-1(7-36)amide, peptide histidine methionine and is responsible for their degradation in human serum. Eur J Biochem, 214, 829-35. MERCER, J. G., MOAR, K. M. & HOGGARD, N. 1998. Localization of leptin receptor (Ob-R) messenger ribonucleic acid in the rodent hindbrain. Endocrinology, 139, 29-34. MERCHENTHALER, I., LANE, M. & SHUGHRUE, P. 1999. Distribution of pre-pro-glucagon and glucagon-like peptide-1 receptor messenger RNAs in the rat central nervous system. J Comp Neurol, 403, 261-80. MERTEN, N., LINDNER, D., RABE, N., ROMPLER, H., MORL, K., SCHONEBERG, T. & BECK-SICKINGER, A. G. 2007. Receptor subtype-specific docking of Asp6.59 with C-terminal arginine residues in Y receptor ligands. J Biol Chem, 282, 7543-51.

295

MISRA, M., MILLER, K. K., TSAI, P., GALLAGHER, K., LIN, A., LEE, N., HERZOG, D. B. & KLIBANSKI, A. 2006. Elevated peptide YY levels in adolescent girls with anorexia nervosa. J Clin Endocrinol Metab, 91, 1027-33. MOENS, K., HEIMBERG, H., FLAMEZ, D., HUYPENS, P., QUARTIER, E., LING, Z., PIPELEERS, D., GREMLICH, S., THORENS, B. & SCHUIT, F. 1996. Expression and functional activity of glucagon, glucagon-like peptide I, and glucose-dependent insulinotropic peptide receptors in rat pancreatic islet cells. Diabetes, 45, 257-61. MOJSOV, S., HEINRICH, G., WILSON, I. B., RAVAZZOLA, M., ORCI, L. & HABENER, J. F. 1986. Preproglucagon gene expression in pancreas and intestine diversifies at the level of post- translational processing. J Biol Chem, 261, 11880-9. MORAN, T. H., KATZ, L. F., PLATA-SALAMAN, C. R. & SCHWARTZ, G. J. 1998. Disordered food intake and obesity in rats lacking cholecystokinin A receptors. Am J Physiol, 274, R618-25. MORRIS, B. J. 1989. Neuronal localisation of neuropeptide Y gene expression in rat brain. J Comp Neurol, 290, 358-68. MORTON, G. J., CUMMINGS, D. E., BASKIN, D. G., BARSH, G. S. & SCHWARTZ, M. W. 2006. Central nervous system control of food intake and body weight. Nature, 443, 289-95. MOUNTJOY, K. G., MORTRUD, M. T., LOW, M. J., SIMERLY, R. B. & CONE, R. D. 1994. Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain. Mol Endocrinol, 8, 1298-308. MULLER, M. 2006. Biological therapies: concepts and challenges. Wien Klin Wochenschr, 118, 508- 12. MUNZBERG, H. 2010. Leptin-signaling pathways and leptin resistance. Forum Nutr, 63, 123-32. NANNIPIERI, M., BALDI, S., MARI, A., COLLIGIANI, D., GUARINO, D., CAMASTRA, S., BARSOTTI, E., BERTA, R., MORICONI, D., BELLINI, R., ANSELMINO, M. & FERRANNINI, E. 2013. Roux-en-Y Gastric Bypass and Sleeve Gastrectomy: Mechanisms of Diabetes Remission and Role of Gut Hormones. J Clin Endocrinol Metab. NASLUND, E., BARKELING, B., KING, N., GUTNIAK, M., BLUNDELL, J. E., HOLST, J. J., ROSSNER, S. & HELLSTROM, P. M. 1999. Energy intake and appetite are suppressed by glucagon-like peptide-1 (GLP-1) in obese men. Int J Obes Relat Metab Disord, 23, 304-11. NAUCK, M. A., NIEDEREICHHOLZ, U., ETTLER, R., HOLST, J. J., ORSKOV, C., RITZEL, R. & SCHMIEGEL, W. H. 1997. Glucagon-like peptide 1 inhibition of gastric emptying outweighs its insulinotropic effects in healthy humans. Am J Physiol, 273, E981-8. NEOVIUS, M. & NARBRO, K. 2008. Cost-effectiveness of pharmacological anti-obesity treatments: a systematic review. Int J Obes (Lond), 32, 1752-63. NEUMANN, T. A. & FOOTE, M. 2000. Megakaryocyte growth and development factor (MGDF): an Mpl ligand and cytokine that regulates thrombopoiesis. Cytokines Cell Mol Ther, 6, 47-56. NEUMILLER, J. J. 2009. Differential chemistry (structure), mechanism of action, and pharmacology of GLP-1 receptor agonists and DPP-4 inhibitors. J Am Pharm Assoc (2003), 49 Suppl 1, S16-29. NICOLAIDIS, S. & ROWLAND, N. 1976. Metering of intravenous versus oral nutrients and regulation of energy balance. Am J Physiol, 231, 661-8. NIELSEN, L. L., YOUNG, A. A. & PARKES, D. G. 2004. Pharmacology of exenatide (synthetic exendin-4): a potential therapeutic for improved glycemic control of type 2 diabetes. Regul Pept, 117, 77-88. NIEUWENHUIZEN, A. G., KARLSSON, S., FRIDOLF, T. & AHREN, B. 1994. Mechanisms underlying the insulinostatic effect of peptide YY in mouse pancreatic islets. Diabetologia, 37, 871-8. NIGRO, S. C., LUON, D. & BAKER, W. L. 2013. Lorcaserin: a novel serotonin 2C agonist for the treatment of obesity. Curr Med Res Opin, 29, 839-48. NYGAARD, E. B., MOLLER, C. L., KIEVIT, P., GROVE, K. L. & ANDERSEN, B. 2013. Increased fibroblast growth factor 21 expression in high-fat diet-sensitive non-human primates (Macaca mulatta). Int J Obes.

296

NYGAARD, R., NIELBO, S., SCHWARTZ, T. W. & POULSEN, F. M. 2006. The PP-fold solution structure of human polypeptide YY and human PYY3-36 as determined by NMR. Biochemistry, 45, 8350-7. OBICI, S., FENG, Z., KARKANIAS, G., BASKIN, D. G. & ROSSETTI, L. 2002. Decreasing hypothalamic insulin receptors causes hyperphagia and insulin resistance in rats. Nat Neurosci, 5, 566-72. OGISO, T., IWAKI, M., TANINO, T., NISHIOKA, S., HIGASHI, K. & KAMO, M. 1997. In vitro skin penetration and degradation of enkephalin, elcatonin and insulin. Biol Pharm Bull, 20, 54-60. OGISO, T., IWAKI, M., TANINO, T., YONO, A. & ITO, A. 2000. In vitro skin penetration and degradation of peptides and their analysis using a kinetic model. Biol Pharm Bull, 23, 1346-51. OHKUBO, I., HUANG, K., OCHIAI, Y., TAKAGAKI, M. & KANI, K. 1994. Dipeptidyl peptidase IV from porcine seminal plasma: purification, characterization, and N-terminal amino acid sequence. J Biochem, 116, 1182-6. OLSEN, H. L., THEANDER, S., BOKVIST, K., BUSCHARD, K., WOLLHEIM, C. B. & GROMADA, J. 2005. Glucose stimulates glucagon release in single rat alpha-cells by mechanisms that mirror the stimulus-secretion coupling in beta-cells. Endocrinology, 146, 4861-70. OLSZEWSKI, P. K., GRACE, M. K., BILLINGTON, C. J. & LEVINE, A. S. 2003. Hypothalamic paraventricular injections of ghrelin: effect on feeding and c-Fos immunoreactivity. Peptides, 24, 919-23. OREXIGEN. 2009. Phase 2b Trial for EmpaticTM Meets Primary Efficacy Endpoint Demonstrating Significantly Greater Weight Loss Versus Comparators in Obese Patients [Online]. Available: http://ir.orexigen.com/phoenix.zhtml?c=207034&p=irol- newsArticle&ID=1336796&highlight= [Accessed]. ORSKOV, C., WETTERGREN, A. & HOLST, J. J. 1993. Biological effects and metabolic rates of glucagonlike peptide-1 7-36 amide and glucagonlike peptide-1 7-37 in healthy subjects are indistinguishable. Diabetes, 42, 658-61. ORSKOV, C., WETTERGREN, A. & HOLST, J. J. 1996. Secretion of the incretin hormones glucagon-like peptide-1 and gastric inhibitory polypeptide correlates with insulin secretion in normal man throughout the day. Scand J Gastroenterol, 31, 665-70. ORTIZ, A. A., MILARDO, L. F., DECARR, L. B., BUCKHOLZ, T. M., MAYS, M. R., CLAUS, T. H., LIVINGSTON, J. N., MAHLE, C. D. & LUMB, K. J. 2007. A novel long-acting selective neuropeptide Y2 receptor polyethylene glycol-conjugated peptide agonist reduces food intake and body weight and improves glucose metabolism in rodents. J Pharmacol Exp Ther, 323, 692-700. OSAKA, T., ENDO, M., YAMAKAWA, M. & INOUE, S. 2005. Energy expenditure by intravenous administration of glucagon-like peptide-1 mediated by the lower brainstem and sympathoadrenal system. Peptides, 26, 1623-31. OTTO, B., CUNTZ, U., FRUEHAUF, E., WAWARTA, R., FOLWACZNY, C., RIEPL, R. L., HEIMAN, M. L., LEHNERT, P., FICHTER, M. & TSCHOP, M. 2001. Weight gain decreases elevated plasma ghrelin concentrations of patients with anorexia nervosa. Eur J Endocrinol, 145, 669-73. PADWAL, R. S. & MAJUMDAR, S. R. 2007. Drug treatments for obesity: orlistat, sibutramine, and rimonabant. Lancet, 369, 71-7. PANDOL, S. J., PERISKIC, S., GUKOVSKY, I., ZANINOVIC, V., JUNG, Y., ZONG, Y., SOLOMON, T. E., GUKOVSKAYA, A. S. & TSUKAMOTO, H. 1999. Ethanol diet increases the sensitivity of rats to pancreatitis induced by cholecystokinin octapeptide. Gastroenterology, 117, 706-16. PARDRIDGE, W. M., EISENBERG, J. & YANG, J. 1985. Human blood-brain barrier insulin receptor. J Neurochem, 44, 1771-8. PARKER, R. M. & HERZOG, H. 1999. Regional distribution of Y-receptor subtype mRNAs in rat brain. Eur J Neurosci, 11, 1431-48. PARKER, S. L., PARKER, M. S. & CROWLEY, W. R. 1999. Characterization of rabbit kidney and brain pancreatic polypeptide-binding neuropeptide Y receptors: differences with Y1 and Y2 sites in sensitivity to amiloride derivatives affecting sodium transport. Regul Pept, 82, 91-102.

297

PARKINSON, J. R., CHAUDHRI, O. B., KUO, Y. T., FIELD, B. C., HERLIHY, A. H., DHILLO, W. S., GHATEI, M. A., BLOOM, S. R. & BELL, J. D. 2009. Differential patterns of neuronal activation in the brainstem and hypothalamus following peripheral injection of GLP-1, oxyntomodulin and lithium chloride in mice detected by manganese-enhanced magnetic resonance imaging (MEMRI). Neuroimage, 44, 1022-31. PARLEVLIET, E. T., HEIJBOER, A. C., SCHRODER-VAN DER ELST, J. P., HAVEKES, L. M., ROMIJN, J. A., PIJL, H. & CORSSMIT, E. P. 2008. Oxyntomodulin ameliorates glucose intolerance in mice fed a high-fat diet. Am J Physiol Endocrinol Metab, 294, E142-7. PATZELT, C. & SCHILTZ, E. 1984. Conversion of proglucagon in pancreatic alpha cells: the major endproducts are glucagon and a single peptide, the major proglucagon fragment, that contains two glucagon-like sequences. Proc Natl Acad Sci U S A, 81, 5007-11. PEARSON, N. & BIDDLE, S. J. 2011. Sedentary behavior and dietary intake in children, adolescents, and adults a systematic review. Am J Prev Med, 41, 178-88. PEDERSEN, S. L., SASIKUMAR, P. G., VRANG, N. & JENSEN, K. J. 2010. Peptide architecture: adding an alpha-helix to the PYY lysine side chain provides nanomolar binding and body-weight- lowering effects. ChemMedChem, 5, 545-51. PEGORIER, J. P., GARCIA-GARCIA, M. V., PRIP-BUUS, C., DUEE, P. H., KOHL, C. & GIRARD, J. 1989. Induction of ketogenesis and fatty acid oxidation by glucagon and cyclic AMP in cultured hepatocytes from rabbit fetuses. Evidence for a decreased sensitivity of carnitine palmitoyltransferase I to malonyl-CoA inhibition after glucagon or cyclic AMP treatment. Biochem J, 264, 93-100. PFLUGER, P. T., KAMPE, J., CASTANEDA, T. R., VAHL, T., D'ALESSIO, D. A., KRUTHAUPT, T., BENOIT, S. C., CUNTZ, U., ROCHLITZ, H. J., MOEHLIG, M., PFEIFFER, A. F., KOEBNICK, C., WEICKERT, M. O., OTTO, B., SPRANGER, J. & TSCHOP, M. H. 2007. Effect of human body weight changes on circulating levels of peptide YY and peptide YY3-36. J Clin Endocrinol Metab, 92, 583-8. PIRNIK, Z., BUNDZIKOVA, J., HOLUBOVA, M., PYCHOVA, M., FEHRENTZ, J. A., MARTINEZ, J., ZELEZNA, B., MALETINSKA, L. & KISS, A. 2011. Ghrelin agonists impact on Fos protein expression in brain areas related to food intake regulation in male C57BL/6 mice. Neurochem Int, 59, 889- 95. PITTNER, R. A., MOORE, C. X., BHAVSAR, S. P., GEDULIN, B. R., SMITH, P. A., JODKA, C. M., PARKES, D. G., PATERNITI, J. R., SRIVASTAVA, V. P. & YOUNG, A. A. 2004. Effects of PYY[3-36] in rodent models of diabetes and obesity. Int J Obes Relat Metab Disord, 28, 963-71. PLUSCZYK, T., WESTERMANN, S., RATHGEB, D. & FEIFEL, G. 1997. Acute pancreatitis in rats: effects of sodium taurocholate, CCK-8, and Sec on pancreatic microcirculation. Am J Physiol, 272, G310-20. POLAK, J. M., BLOOM, S. R., RAYFORD, P. L., PEARSE, A. G., BUCHAN, A. M. & THOMPSON, J. C. 1975. Identification of cholecystokinin-secreting cells. Lancet, 2, 1016-8. POLONSKY, K. S., GIVEN, B. D., HIRSCH, L., SHAPIRO, E. T., TILLIL, H., BEEBE, C., GALLOWAY, J. A., FRANK, B. H., KARRISON, T. & VAN CAUTER, E. 1988a. Quantitative study of insulin secretion and clearance in normal and obese subjects. J Clin Invest, 81, 435-41. POLONSKY, K. S., GIVEN, B. D. & VAN CAUTER, E. 1988b. Twenty-four-hour profiles and pulsatile patterns of insulin secretion in normal and obese subjects. J Clin Invest, 81, 442-8. PORTE, D., JR., BASKIN, D. G. & SCHWARTZ, M. W. 2002. Leptin and insulin action in the central nervous system. Nutr Rev, 60, S20-9; discussion S68-84, 85-7. PRENTICE, A. & JEBB, S. 2004. Energy intake/physical activity interactions in the homeostasis of body weight regulation. Nutr Rev, 62, S98-104. PROOST, P., STRUYF, S., SCHOLS, D., OPDENAKKER, G., SOZZANI, S., ALLAVENA, P., MANTOVANI, A., AUGUSTYNS, K., BAL, G., HAEMERS, A., LAMBEIR, A. M., SCHARPE, S., VAN DAMME, J. & DE MEESTER, I. 1999. Truncation of macrophage-derived chemokine by CD26/ dipeptidylpeptidase IV beyond its predicted cleavage site affects chemotactic activity and CC chemokine receptor 4 interaction. J Biol Chem, 274, 3988-93.

298

QI, Y., TAKAHASHI, N., HILEMAN, S. M., PATEL, H. R., BERG, A. H., PAJVANI, U. B., SCHERER, P. E. & AHIMA, R. S. 2004. Adiponectin acts in the brain to decrease body weight. Nat Med, 10, 524- 9. RANE, A., WILKINSON, G. R. & SHAND, D. G. 1977. Prediction of hepatic extraction ratio from in vitro measurement of intrinsic clearance. J Pharmacol Exp Ther, 200, 420-4. REIDELBERGER, R. D., HAVER, A. C., CHELIKANI, P. K. & BUESCHER, J. L. 2008. Effects of different intermittent peptide YY (3-36) dosing strategies on food intake, body weight, and adiposity in diet-induced obese rats. Am J Physiol Regul Integr Comp Physiol, 295, R449-58. RICHTER, G., GOKE, R., GOKE, B. & ARNOLD, R. 1990. Characterization of receptors for glucagon-like peptide-1(7-36)amide on rat lung membranes. FEBS Lett, 267, 78-80. RODRIGUEZ, J., GUPTA, N., SMITH, R. D. & PEVZNER, P. A. 2008. Does trypsin cut before proline? J Proteome Res, 7, 300-5. ROLAND, B. L., SUTTON, S. W., WILSON, S. J., LUO, L., PYATI, J., HUVAR, R., ERLANDER, M. G. & LOVENBERG, T. W. 1999. Anatomical distribution of prolactin-releasing peptide and its receptor suggests additional functions in the central nervous system and periphery. Endocrinology, 140, 5736-45. ROQUES, B. P., NOBLE, F., DAUGE, V., FOURNIE-ZALUSKI, M. C. & BEAUMONT, A. 1993. Neutral endopeptidase 24.11: structure, inhibition, and experimental and clinical pharmacology. Pharmacol Rev, 45, 87-146. RORSMAN, P., BERGGREN, P. O., BOKVIST, K., ERICSON, H., MOHLER, H., OSTENSON, C. G. & SMITH, P. A. 1989. Glucose-inhibition of glucagon secretion involves activation of GABAA-receptor chloride channels. Nature, 341, 233-6. ROSES, A. D. 2009. Stimulation of cholecystokinin-A receptors with Gl181771X: a failed clinical trial that did not test the pharmacogenetic hypothesis for reduction of food intake. Clin Pharmacol Ther, 85, 362-5. ROSSNER, S., SJOSTROM, L., NOACK, R., MEINDERS, A. E. & NOSEDA, G. 2000. Weight loss, weight maintenance, and improved cardiovascular risk factors after 2 years treatment with orlistat for obesity. European Orlistat Obesity Study Group. Obes Res, 8, 49-61. ROTH, J. D., ROLAND, B. L., COLE, R. L., TREVASKIS, J. L., WEYER, C., KODA, J. E., ANDERSON, C. M., PARKES, D. G. & BARON, A. D. 2008. Leptin responsiveness restored by amylin agonism in diet-induced obesity: evidence from nonclinical and clinical studies. Proc Natl Acad Sci U S A, 105, 7257-62. ROUILLE, Y., WESTERMARK, G., MARTIN, S. K. & STEINER, D. F. 1994. Proglucagon is processed to glucagon by prohormone convertase PC2 in alpha TC1-6 cells. Proc Natl Acad Sci U S A, 91, 3242-6. RUCKER, D., PADWAL, R., LI, S. K., CURIONI, C. & LAU, D. C. 2007. Long term pharmacotherapy for obesity and overweight: updated meta-analysis. BMJ, 335, 1194-9. RUIZ-GRANDE, C., PINTADO, J., ALARCON, C., CASTILLA, C., VALVERDE, I. & LOPEZ-NOVOA, J. M. 1990. Renal catabolism of human glucagon-like peptides 1 and 2. Can J Physiol Pharmacol, 68, 1568-73. RUNGE, S., SCHIMMER, S., OSCHMANN, J., SCHIODT, C. B., KNUDSEN, S. M., JEPPESEN, C. B., MADSEN, K., LAU, J., THOGERSEN, H. & RUDOLPH, R. 2007. Differential structural properties of GLP-1 and exendin-4 determine their relative affinity for the GLP-1 receptor N-terminal extracellular domain. Biochemistry, 46, 5830-40. RUNGE, S., THOGERSEN, H., MADSEN, K., LAU, J. & RUDOLPH, R. 2008. Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain. J Biol Chem, 283, 11340-7. SACKS, F. M., BRAY, G. A., CAREY, V. J., SMITH, S. R., RYAN, D. H., ANTON, S. D., MCMANUS, K., CHAMPAGNE, C. M., BISHOP, L. M., LARANJO, N., LEBOFF, M. S., ROOD, J. C., DE JONGE, L., GREENWAY, F. L., LORIA, C. M., OBARZANEK, E. & WILLIAMSON, D. A. 2009. Comparison of

299

weight-loss diets with different compositions of fat, protein, and carbohydrates. N Engl J Med, 360, 859-73. SAINSBURY, A., SHI, Y. C., ZHANG, L., ALJANOVA, A., LIN, Z., NGUYEN, A. D., HERZOG, H. & LIN, S. 2010. Y4 receptors and pancreatic polypeptide regulate food intake via hypothalamic orexin and brain-derived neurotropic factor dependent pathways. Neuropeptides, 44, 261-8. SAKATA, I., NAKAMURA, K., YAMAZAKI, M., MATSUBARA, M., HAYASHI, Y., KANGAWA, K. & SAKAI, T. 2002. Ghrelin-producing cells exist as two types of cells, closed- and opened-type cells, in the rat gastrointestinal tract. Peptides, 23, 531-6. SALEHI, A., VIEIRA, E. & GYLFE, E. 2006. Paradoxical stimulation of glucagon secretion by high glucose concentrations. Diabetes, 55, 2318-23. SANTOPRETE, A., CAPITO, E., CARRINGTON, P. E., POCAI, A., FINOTTO, M., LANGELLA, A., INGALLINELLA, P., ZYTKO, K., BUFALI, S., CIANETTI, S., VENEZIANO, M., BONELLI, F., ZHU, L., MONTEAGUDO, E., MARSH, D. J., SINHAROY, R., BIANCHI, E. & PESSI, A. 2011. DPP-IV- resistant, long-acting oxyntomodulin derivatives. J Pept Sci, 17, 270-80. SARGENT, D. F. & SCHWYZER, R. 1986. Membrane lipid phase as catalyst for peptide-receptor interactions. Proc Natl Acad Sci U S A, 83, 5774-8. SAVAGE, A. P., ADRIAN, T. E., CAROLAN, G., CHATTERJEE, V. K. & BLOOM, S. R. 1987. Effects of peptide YY (PYY) on mouth to caecum intestinal transit time and on the rate of gastric emptying in healthy volunteers. Gut, 28, 166-70. SAVINI, E. C. 1964. [On tachyphylaxis]. Actual Pharmacol (Paris), 17, 117-42. SAWCHENKO, P. E. & SWANSON, L. W. 1983a. The organization and biochemical specificity of afferent projections to the paraventricular and supraoptic nuclei. Prog Brain Res, 60, 19-29. SAWCHENKO, P. E. & SWANSON, L. W. 1983b. The organization of forebrain afferents to the paraventricular and supraoptic nuclei of the rat. J Comp Neurol, 218, 121-44. SCHECHTER, I. & BERGER, A. 2012. On the size of the active site in proteases. I. Papain. 1967. Biochem Biophys Res Commun, 425, 497-502. SCHELLEKENS, H. 2002. Immunogenicity of therapeutic proteins: clinical implications and future prospects. Clin Ther, 24, 1720-40; discussion 1719. SCHEPP, W., SCHMIDTLER, J., RIEDEL, T., DEHNE, K., SCHUSDZIARRA, V., HOLST, J. J., ENG, J., RAUFMAN, J. P. & CLASSEN, M. 1994. Exendin-4 and exendin-(9-39)NH2: agonist and antagonist, respectively, at the rat parietal cell receptor for glucagon-like peptide-1-(7- 36)NH2. Eur J Pharmacol, 269, 183-91. SCHIRRA, J., STURM, K., LEICHT, P., ARNOLD, R., GOKE, B. & KATSCHINSKI, M. 1998. Exendin(9- 39)amide is an antagonist of glucagon-like peptide-1(7-36)amide in humans. J Clin Invest, 101, 1421-30. SCHJOLDAGER, B., MORTENSEN, P. E., MYHRE, J., CHRISTIANSEN, J. & HOLST, J. J. 1989. Oxyntomodulin from distal gut. Role in regulation of gastric and pancreatic functions. Dig Dis Sci, 34, 1411-9. SCHJOLDAGER, B. T., BALDISSERA, F. G., MORTENSEN, P. E., HOLST, J. J. & CHRISTIANSEN, J. 1988. Oxyntomodulin: a potential hormone from the distal gut. Pharmacokinetics and effects on gastric acid and insulin secretion in man. Eur J Clin Invest, 18, 499-503. SCHMIDTLER, J., DEHNE, K., ALLESCHER, H. D., SCHUSDZIARRA, V., CLASSEN, M., HOLST, J. J., POLACK, A. & SCHEPP, W. 1994. Rat parietal cell receptors for GLP-1-(7-36) amide: northern blot, cross-linking, and radioligand binding. Am J Physiol, 267, G423-32. SCHOLTZ, S., MIRAS, A. D., CHHINA, N., PRECHTL, C. G., SLEETH, M. L., DAUD, N. M., ISMAIL, N. A., DURIGHEL, G., AHMED, A. R., OLBERS, T., VINCENT, R. P., ALAGHBAND-ZADEH, J., GHATEI, M. A., WALDMAN, A. D., FROST, G. S., BELL, J. D., LE ROUX, C. W. & GOLDSTONE, A. P. 2014. Obese patients after gastric bypass surgery have lower brain-hedonic responses to food than after gastric banding. Gut, 63, 891-902.

300

SCHWARTZ, G. J., WHITNEY, A., SKOGLUND, C., CASTONGUAY, T. W. & MORAN, T. H. 1999. Decreased responsiveness to dietary fat in Otsuka Long-Evans Tokushima fatty rats lacking CCK-A receptors. Am J Physiol, 277, R1144-51. SCHWARTZ, M. W. & NISWENDER, K. D. 2004. Adiposity signaling and biological defense against weight gain: absence of protection or central hormone resistance? J Clin Endocrinol Metab, 89, 5889-97. SCHWARTZ, M. W., SIPOLS, A. J., MARKS, J. L., SANACORA, G., WHITE, J. D., SCHEURINK, A., KAHN, S. E., BASKIN, D. G., WOODS, S. C., FIGLEWICZ, D. P. & ET AL. 1992. Inhibition of hypothalamic neuropeptide Y gene expression by insulin. Endocrinology, 130, 3608-16. SCHWARTZ, T. W., FUHLENDORFF, J., KJEMS, L. L., KRISTENSEN, M. S., VERVELDE, M., O'HARE, M., KRSTENANSKY, J. L. & BJORNHOLM, B. 1990. Signal epitopes in the three-dimensional structure of neuropeptide Y. Interaction with Y1, Y2, and pancreatic polypeptide receptors. Ann N Y Acad Sci, 611, 35-47. SCHWARTZ, T. W., HOLST, J. J., FAHRENKRUG, J., JENSEN, S. L., NIELSEN, O. V., REHFELD, J. F., DE MUCKADELL, O. B. & STADIL, F. 1978. Vagal, cholinergic regulation of pancreatic polypeptide secretion. J Clin Invest, 61, 781-9. SCHWYZER, R. 1995. In search of the 'bio-active conformation'--is it induced by the target cell membrane? J Mol Recognit, 8, 3-8. SCOZZAFAVA, A., SUPURAN, C. T. & CARTA, F. 2013. Antiobesity carbonic anhydrase inhibitors: a literature and patent review. Expert Opin Ther Pat, 23, 725-35. SCROCCHI, L. A., BROWN, T. J., MACLUSKY, N., BRUBAKER, P. L., AUERBACH, A. B., JOYNER, A. L. & DRUCKER, D. J. 1996. Glucose intolerance but normal satiety in mice with a null mutation in the glucagon-like peptide 1 receptor gene. Nat Med, 2, 1254-8. SEAL, L. J., SMALL, C. J., DHILLO, W. S., STANLEY, S. A., ABBOTT, C. R., GHATEI, M. A. & BLOOM, S. R. 2001. PRL-releasing peptide inhibits food intake in male rats via the dorsomedial hypothalamic nucleus and not the paraventricular hypothalamic nucleus. Endocrinology, 142, 4236-43. SEELEY, R. J., BLAKE, K., RUSHING, P. A., BENOIT, S., ENG, J., WOODS, S. C. & D'ALESSIO, D. 2000. The role of CNS glucagon-like peptide-1 (7-36) amide receptors in mediating the visceral illness effects of lithium chloride. J Neurosci, 20, 1616-21. SHECHTER, Y., TSUBERY, H., MIRONCHIK, M., RUBINSTEIN, M. & FRIDKIN, M. 2005. Reversible PEGylation of peptide YY3-36 prolongs its inhibition of food intake in mice. FEBS Lett, 579, 2439-44. SHI, Y. C., LIN, Z., LAU, J., ZHANG, H., YAGI, M., KANZLER, I., SAINSBURY, A., HERZOG, H. & LIN, S. 2013. PYY3-36 and pancreatic polypeptide reduce food intake in an additive manner via distinct hypothalamic dependent pathways in mice. Obesity (Silver Spring). SHIIYA, T., NAKAZATO, M., MIZUTA, M., DATE, Y., MONDAL, M. S., TANAKA, M., NOZOE, S., HOSODA, H., KANGAWA, K. & MATSUKURA, S. 2002. Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion. J Clin Endocrinol Metab, 87, 240-4. SHIMIZU, N., OOMURA, Y., PLATA-SALAMAN, C. R. & MORIMOTO, M. 1987. Hyperphagia and obesity in rats with bilateral ibotenic acid-induced lesions of the ventromedial hypothalamic nucleus. Brain Res, 416, 153-6. SHOUSHA, S., GADIR, F., PESTON, D., BANSI, D., THILLAINAYGAM, A. V. & MURRAY-LYON, I. M. 2004. CD10 immunostaining of bile canaliculi in liver biopsies: change of staining pattern with the development of cirrhosis. Histopathology, 45, 335-42. SHUGHRUE, P. J., LANE, M. V. & MERCHENTHALER, I. 1996. Glucagon-like peptide-1 receptor (GLP1- R) mRNA in the rat hypothalamus. Endocrinology, 137, 5159-62. SILJEE, J. E., UNMEHOPA, U. A., KALSBEEK, A., SWAAB, D. F., FLIERS, E. & ALKEMADE, A. 2013. Melanocortin 4 receptor distribution in the human hypothalamus. Eur J Endocrinol, 168, 361-9.

301

SJOSTROM, L. 2013. Review of the key results from the Swedish Obese Subjects (SOS) trial - a prospective controlled intervention study of bariatric surgery. J Intern Med, 273, 219-34. SJOSTROM, L., NARBRO, K., SJOSTROM, C. D., KARASON, K., LARSSON, B., WEDEL, H., LYSTIG, T., SULLIVAN, M., BOUCHARD, C., CARLSSON, B., BENGTSSON, C., DAHLGREN, S., GUMMESSON, A., JACOBSON, P., KARLSSON, J., LINDROOS, A. K., LONROTH, H., NASLUND, I., OLBERS, T., STENLOF, K., TORGERSON, J., AGREN, G. & CARLSSON, L. M. 2007. Effects of bariatric surgery on mortality in Swedish obese subjects. N Engl J Med, 357, 741-52. SJOSTROM, L., RISSANEN, A., ANDERSEN, T., BOLDRIN, M., GOLAY, A., KOPPESCHAAR, H. P. & KREMPF, M. 1998. Randomised placebo-controlled trial of orlistat for weight loss and prevention of weight regain in obese patients. European Multicentre Orlistat Study Group. Lancet, 352, 167-72. SLENTZ, C. A., DUSCHA, B. D., JOHNSON, J. L., KETCHUM, K., AIKEN, L. B., SAMSA, G. P., HOUMARD, J. A., BALES, C. W. & KRAUS, W. E. 2004. Effects of the amount of exercise on body weight, body composition, and measures of central obesity: STRRIDE--a randomized controlled study. Arch Intern Med, 164, 31-9. SLOTH, B., DAVIDSEN, L., HOLST, J. J., FLINT, A. & ASTRUP, A. 2007. Effect of subcutaneous injections of PYY1-36 and PYY3-36 on appetite, ad libitum energy intake, and plasma free fatty acid concentration in obese males. Am J Physiol Endocrinol Metab, 293, E604-9. SMALL, C. J. & BLOOM, S. R. 2005. The therapeutic potential of gut hormone peptide YY3-36 in the treatment of obesity. Expert Opin Investig Drugs, 14, 647-53. SMITH, S. R., WEISSMAN, N. J., ANDERSON, C. M., SANCHEZ, M., CHUANG, E., STUBBE, S., BAYS, H. & SHANAHAN, W. R. 2010. Multicenter, placebo-controlled trial of lorcaserin for weight management. N Engl J Med, 363, 245-56. SOWDEN, G. L., DRUCKER, D. J., WEINSHENKER, D. & SWOAP, S. J. 2007. Oxyntomodulin increases intrinsic heart rate in mice independent of the glucagon-like peptide-1 receptor. Am J Physiol Regul Integr Comp Physiol, 292, R962-70. SPEAKMAN, J. R., LEVITSKY, D. A., ALLISON, D. B., BRAY, M. S., DE CASTRO, J. M., CLEGG, D. J., CLAPHAM, J. C., DULLOO, A. G., GRUER, L., HAW, S., HEBEBRAND, J., HETHERINGTON, M. M., HIGGS, S., JEBB, S. A., LOOS, R. J., LUCKMAN, S., LUKE, A., MOHAMMED-ALI, V., O'RAHILLY, S., PEREIRA, M., PERUSSE, L., ROBINSON, T. N., ROLLS, B., SYMONDS, M. E. & WESTERTERP- PLANTENGA, M. S. 2011. Set points, settling points and some alternative models: theoretical options to understand how genes and environments combine to regulate body adiposity. Dis Model Mech, 4, 733-45. STANLEY, B. G., HA, L. H., SPEARS, L. C. & DEE, M. G., 2ND 1993. Lateral hypothalamic injections of glutamate, kainic acid, D,L-alpha-amino-3-hydroxy-5-methyl-isoxazole propionic acid or N- methyl-D-aspartic acid rapidly elicit intense transient eating in rats. Brain Res, 613, 88-95. STANLEY, B. G. & LEIBOWITZ, S. F. 1985. Neuropeptide Y injected in the paraventricular hypothalamus: a powerful stimulant of feeding behavior. Proc Natl Acad Sci U S A, 82, 3940- 3. STANLEY, S., WYNNE, K. & BLOOM, S. 2004. Gastrointestinal satiety signals III. Glucagon-like peptide 1, oxyntomodulin, peptide YY, and pancreatic polypeptide. Am J Physiol Gastrointest Liver Physiol, 286, G693-7. STEPHENS, T. W., BASINSKI, M., BRISTOW, P. K., BUE-VALLESKEY, J. M., BURGETT, S. G., CRAFT, L., HALE, J., HOFFMANN, J., HSIUNG, H. M., KRIAUCIUNAS, A. & ET AL. 1995. The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature, 377, 530-2. STEPHENSON, S. L. & KENNY, A. J. 1987. Metabolism of neuropeptides. Hydrolysis of the angiotensins, bradykinin, substance P and oxytocin by pig kidney microvillar membranes. Biochem J, 241, 237-47. STEWART, J. R. & KENNY, A. J. 1984. Proteins of the kidney microvillar membrane. Biosynthesis of endopeptidase-24.11, dipeptidylpeptidase IV and aminopeptidases N and A in pig kidney slices. Biochem J, 224, 549-58.

302

STRODEL, W. E., VINIK, A. I., LLOYD, R. V., GLASER, B., ECKHAUSER, F. E., FIDDIAN-GREEN, R. G., TURCOTTE, J. G. & THOMPSON, N. W. 1984. Pancreatic polypeptide-producing tumors. Silent lesions of the pancreas? Arch Surg, 119, 508-14. SUMITHRAN, P., PRENDERGAST, L. A., DELBRIDGE, E., PURCELL, K., SHULKES, A., KRIKETOS, A. & PROIETTO, J. 2011. Long-term persistence of hormonal adaptations to weight loss. N Engl J Med, 365, 1597-604. SVENDSEN, M., RISSANEN, A., RICHELSEN, B., ROSSNER, S., HANSSON, F. & TONSTAD, S. 2008. Effect of orlistat on eating behavior among participants in a 3-year weight maintenance trial. Obesity (Silver Spring), 16, 327-33. TACHIBANA, T., OIKAWA, D., ADACHI, N., BOSWELL, T. & FURUSE, M. 2007. Intracerebroventricular injection of glucagon-like peptide-1 changes lipid metabolism in chicks. Comp Biochem Physiol A Mol Integr Physiol, 147, 1104-8. TAMURA, H., KAMEGAI, J., SHIMIZU, T., ISHII, S., SUGIHARA, H. & OIKAWA, S. 2002. Ghrelin stimulates GH but not food intake in arcuate nucleus ablated rats. Endocrinology, 143, 3268- 75. TAN, T. & BLOOM, S. 2013a. Gut hormones as therapeutic agents in treatment of diabetes and obesity. Curr Opin Pharmacol. TAN, T. & BLOOM, S. 2013b. Gut hormones as therapeutic agents in treatment of diabetes and obesity. Curr Opin Pharmacol, 13, 996-1001. TAN, T. M., FIELD, B. C., MCCULLOUGH, K. A., TROKE, R. C., CHAMBERS, E. S., SALEM, V., GONZALEZ MAFFE, J., BAYNES, K. C., DE SILVA, A., VIARDOT, A., ALSAFI, A., FROST, G. S., GHATEI, M. A. & BLOOM, S. R. 2013. Coadministration of glucagon-like peptide-1 during glucagon infusion in humans results in increased energy expenditure and amelioration of hyperglycemia. Diabetes, 62, 1131-8. TANG-CHRISTENSEN, M., LARSEN, P. J., GOKE, R., FINK-JENSEN, A., JESSOP, D. S., MOLLER, M. & SHEIKH, S. P. 1996. Central administration of GLP-1-(7-36) amide inhibits food and water intake in rats. Am J Physiol, 271, R848-56. TANG-CHRISTENSEN, M., LARSEN, P. J., THULESEN, J., NIELSEN, J. R. & VRANG, N. 2001a. [Glucagon- like peptide 2, a neurotransmitter with a newly discovered role in the regulation of food ingestion]. Ugeskr Laeger, 163, 287-91. TANG-CHRISTENSEN, M., VRANG, N. & LARSEN, P. J. 1998. Glucagon-like peptide 1(7-36) amide's central inhibition of feeding and peripheral inhibition of drinking are abolished by neonatal monosodium glutamate treatment. Diabetes, 47, 530-7. TANG-CHRISTENSEN, M., VRANG, N. & LARSEN, P. J. 2001b. Glucagon-like peptide containing pathways in the regulation of feeding behaviour. Int J Obes Relat Metab Disord, 25 Suppl 5, S42-7. TANNENBAUM, G. S., LAPOINTE, M., BEAUDET, A. & HOWARD, A. D. 1998. Expression of growth hormone secretagogue-receptors by growth hormone-releasing hormone neurons in the mediobasal hypothalamus. Endocrinology, 139, 4420-3. TARANTOLA, E., BERTONE, V., MILANESI, G., CAPELLI, E., FERRIGNO, A., NERI, D., VAIRETTI, M., BARNI, S. & FREITAS, I. 2012. Dipeptidylpeptidase--IV, a key enzyme for the degradation of incretins and neuropeptides: activity and expression in the liver of lean and obese rats. Eur J Histochem, 56, e41. TATEMOTO, K. 2004. Neuropeptide Y: History and Overview. In: MICHEL, M. C. (ed.) Neuropeptide Y and Related Peptides. Springer Berlin Heidelberg. TATEMOTO, K., CARLQUIST, M. & MUTT, V. 1982. Neuropeptide Y--a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature, 296, 659-60. TAYLOR, I. L., GARCIA, R. & ELASHOFF, J. 1985. Effects of vagotomy on satiety induced by gastrointestinal hormones in the rat. Physiol Behav, 34, 957-61. TAYLOR, I. L., IMPICCIATORE, M., CARTER, D. C. & WALSH, J. H. 1978. Effect of atropine and vagotomy on pancreatic polypeptide response to a meal in dogs. Am J Physiol, 235, E443-7.

303

TER HORST, G. J. & LUITEN, P. G. 1987. Phaseolus vulgaris leuco-agglutinin tracing of intrahypothalamic connections of the lateral, ventromedial, dorsomedial and paraventricular hypothalamic nuclei in the rat. Brain Res Bull, 18, 191-203. THOMPSON, J. C., FENDER, H. R., RAMUS, N. I., VILLAR, H. V. & RAYFORD, P. L. 1975. Cholecystokinin metabolism in man and dogs. Ann Surg, 182, 496-504. THOMPSON, N. M., GILL, D. A., DAVIES, R., LOVERIDGE, N., HOUSTON, P. A., ROBINSON, I. C. & WELLS, T. 2004. Ghrelin and des-octanoyl ghrelin promote adipogenesis directly in vivo by a mechanism independent of the type 1a growth hormone secretagogue receptor. Endocrinology, 145, 234-42. THORENS, B. 1992. Expression cloning of the pancreatic beta cell receptor for the gluco-incretin hormone glucagon-like peptide 1. Proc Natl Acad Sci U S A, 89, 8641-5. TICHANSKY, D. S., BOUGHTER, J. D., JR. & MADAN, A. K. 2006. Taste change after laparoscopic Roux- en-Y gastric bypass and laparoscopic adjustable gastric banding. Surg Obes Relat Dis, 2, 440- 4. TIMM, W., SCHERBART, A., BOCKER, S., KOHLBACHER, O. & NATTKEMPER, T. W. 2008. Peak intensity prediction in MALDI-TOF mass spectrometry: a machine learning study to support quantitative proteomics. BMC Bioinformatics, 9, 443. TIRUPPATHI, C., MIYAMOTO, Y., GANAPATHY, V., ROESEL, R. A., WHITFORD, G. M. & LEIBACH, F. H. 1990. Hydrolysis and transport of proline-containing peptides in renal brush-border membrane vesicles from dipeptidyl peptidase IV-positive and dipeptidyl peptidase IV- negative rat strains. J Biol Chem, 265, 1476-83. TOMAS, E. & HABENER, J. F. 2010. Insulin-like actions of glucagon-like peptide-1: a dual receptor hypothesis. Trends Endocrinol Metab, 21, 59-67. TRAEBERT, M., RIEDIGER, T., WHITEBREAD, S., SCHARRER, E. & SCHMID, H. A. 2002. Ghrelin acts on leptin-responsive neurones in the rat arcuate nucleus. J Neuroendocrinol, 14, 580-6. TREVASKIS, J. L., COFFEY, T., COLE, R., LEI, C., WITTMER, C., WALSH, B., WEYER, C., KODA, J., BARON, A. D., PARKES, D. G. & ROTH, J. D. 2008. Amylin-mediated restoration of leptin responsiveness in diet-induced obesity: magnitude and mechanisms. Endocrinology, 149, 5679-87. TSUBONE, T., MASAKI, T., KATSURAGI, I., TANAKA, K., KAKUMA, T. & YOSHIMATSU, H. 2005. Ghrelin regulates adiposity in white adipose tissue and UCP1 mRNA expression in brown adipose tissue in mice. Regul Pept, 130, 97-103. TURTON, M. D., O'SHEA, D., GUNN, I., BEAK, S. A., EDWARDS, C. M., MEERAN, K., CHOI, S. J., TAYLOR, G. M., HEATH, M. M., LAMBERT, P. D., WILDING, J. P., SMITH, D. M., GHATEI, M. A., HERBERT, J. & BLOOM, S. R. 1996. A role for glucagon-like peptide-1 in the central regulation of feeding. Nature, 379, 69-72. UEHARA, S., MUROYAMA, A., ECHIGO, N., MORIMOTO, R., OTSUKA, M., YATSUSHIRO, S. & MORIYAMA, Y. 2004. Metabotropic glutamate receptor type 4 is involved in autoinhibitory cascade for glucagon secretion by alpha-cells of islet of Langerhans. Diabetes, 53, 998-1006. UENO, H., YAMAGUCHI, H., MIZUTA, M. & NAKAZATO, M. 2008. The role of PYY in feeding regulation. Regul Pept, 145, 12-6. UENO, N., INUI, A., IWAMOTO, M., KAGA, T., ASAKAWA, A., OKITA, M., FUJIMIYA, M., NAKAJIMA, Y., OHMOTO, Y., OHNAKA, M., NAKAYA, Y., MIYAZAKI, J. I. & KASUGA, M. 1999. Decreased food intake and body weight in pancreatic polypeptide-overexpressing mice. Gastroenterology, 117, 1427-32. UHE, A. M., SZMUKLER, G. I., COLLIER, G. R., HANSKY, J., O'DEA, K. & YOUNG, G. P. 1992. Potential regulators of feeding behavior in anorexia nervosa. Am J Clin Nutr, 55, 28-32. UKMI. 2010. Taspoglutide development suspended [Online]. Available: http://www.ukmi.nhs.uk/applications/ndo/record_view_open.asp?newDrugID=4649 [Accessed].

304

VAN CAN, J., SLOTH, B., JENSEN, C. B., FLINT, A., BLAAK, E. E. & SARIS, W. H. 2013. Effects of the once-daily GLP-1 analog liraglutide on gastric emptying, glycemic parameters, appetite and energy metabolism in obese, non-diabetic adults. Int J Obes (Lond). VAN DEN HOEK, A. M., HEIJBOER, A. C., CORSSMIT, E. P., VOSHOL, P. J., ROMIJN, J. A., HAVEKES, L. M. & PIJL, H. 2004. PYY3-36 reinforces insulin action on glucose disposal in mice fed a high- fat diet. Diabetes, 53, 1949-52. VAN DEN TOP, M., LEE, K., WHYMENT, A. D., BLANKS, A. M. & SPANSWICK, D. 2004. Orexigen- sensitive NPY/AgRP pacemaker neurons in the hypothalamic arcuate nucleus. Nat Neurosci, 7, 493-4. VANNESTE, Y., MICHEL, A., DIMALINE, R., NAJDOVSKI, T. & DESCHODT-LANCKMAN, M. 1988. Hydrolysis of alpha-human atrial natriuretic peptide in vitro by human kidney membranes and purified endopeptidase-24.11. Evidence for a novel cleavage site. Biochem J, 254, 531-7. VERONESE, F. M. 2001. Peptide and protein PEGylation: a review of problems and solutions. Biomaterials, 22, 405-17. VINCENT, R. P. & LE ROUX, C. W. 2008. The satiety hormone peptide YY as a regulator of appetite. J Clin Pathol, 61, 548-52. VINES, L. & SCHIESSER, M. 2014. Gastric bypass: current results and different techniques. Dig Surg, 31, 33-9. VRANG, N., HANSEN, M., LARSEN, P. J. & TANG-CHRISTENSEN, M. 2007. Characterization of brainstem preproglucagon projections to the paraventricular and dorsomedial hypothalamic nuclei. Brain Res, 1149, 118-26. VRANG, N., MADSEN, A. N., TANG-CHRISTENSEN, M., HANSEN, G. & LARSEN, P. J. 2006. PYY(3-36) reduces food intake and body weight and improves insulin sensitivity in rodent models of diet-induced obesity. Am J Physiol Regul Integr Comp Physiol, 291, R367-75. WADDEN, T. A., HOLLANDER, P., KLEIN, S., NISWENDER, K., WOO, V., HALE, P. M. & ARONNE, L. 2013. Weight maintenance and additional weight loss with liraglutide after low-calorie-diet- induced weight loss: The Scale Maintenance randomized study. Int J Obes (Lond). WALTER, S., KUNST, A., MACKENBACH, J., HOFMAN, A. & TIEMEIER, H. 2009. Mortality and disability: the effect of overweight and obesity. Int J Obes (Lond), 33, 1410-8. WANG, L., SAINT-PIERRE, D. H. & TACHE, Y. 2002. Peripheral ghrelin selectively increases Fos expression in neuropeptide Y - synthesizing neurons in mouse hypothalamic arcuate nucleus. Neurosci Lett, 325, 47-51. WANG, Q. P., GUAN, J. L., PAN, W., KASTIN, A. J. & SHIODA, S. 2008. A diffusion barrier between the area postrema and nucleus tractus solitarius. Neurochem Res, 33, 2035-43. WEI, Y. & MOJSOV, S. 1995. Tissue-specific expression of the human receptor for glucagon-like peptide-I: brain, heart and pancreatic forms have the same deduced amino acid sequences. FEBS Lett, 358, 219-24. WENDT, A., BIRNIR, B., BUSCHARD, K., GROMADA, J., SALEHI, A., SEWING, S., RORSMAN, P. & BRAUN, M. 2004. Glucose inhibition of glucagon secretion from rat alpha-cells is mediated by GABA released from neighboring beta-cells. Diabetes, 53, 1038-45. WEST, D. B., FEY, D. & WOODS, S. C. 1984. Cholecystokinin persistently suppresses meal size but not food intake in free-feeding rats. Am J Physiol, 246, R776-87. WEST, D. B., GREENWOOD, M. R., SULLIVAN, A. C., PRESCOD, L., MARZULLO, L. R. & TRISCARI, J. 1987. Infusion of cholecystokinin between meals into free-feeding rats fails to prolong the intermeal interval. Physiol Behav, 39, 111-5. WETTERGREN, A., SCHJOLDAGER, B., MORTENSEN, P. E., MYHRE, J., CHRISTIANSEN, J. & HOLST, J. J. 1993. Truncated GLP-1 (proglucagon 78-107-amide) inhibits gastric and pancreatic functions in man. Dig Dis Sci, 38, 665-73. WHITCOMB, D. C., TAYLOR, I. L. & VIGNA, S. R. 1990. Characterization of saturable binding sites for circulating pancreatic polypeptide in rat brain. Am J Physiol, 259, G687-91.

305

WHO. 2011. World Health Organization Report Fact Sheet Number 311 [Online]. Available: http://www.who.int/mediacentre/factsheets/fs311/en/index.htm [Accessed]. WILLIAMS, D. L., GRILL, H. J., CUMMINGS, D. E. & KAPLAN, J. M. 2003. Vagotomy dissociates short- and long-term controls of circulating ghrelin. Endocrinology, 144, 5184-7. WILLIAMS, G. 2010. Withdrawal of sibutramine in Europe. BMJ, 340, c824. WILMS, B., ERNST, B., SCHMID, S. M., THURNHEER, M. & SCHULTES, B. 2013. Enhanced thermic effect of food after Roux-en-Y gastric bypass surgery. J Clin Endocrinol Metab, 98, 3776-84. WIRTH, M. M. & GIRAUDO, S. Q. 2000. Agouti-related protein in the hypothalamic paraventricular nucleus: effect on feeding. Peptides, 21, 1369-75. WITTEMAN, B. J., EDWARDS-TEUNISSEN, K., HOPMAN, W. P., JEBBINK, M. C., MASCLEE, A. A., LAMERS, C. B. & JANSEN, J. B. 1994. Nutrient-specific effects of modified sham feeding on pancreatic polypeptide release. Eur J Clin Nutr, 48, 556-60. WOLOSHIN, S. & SCHWARTZ, L. M. 2014. The new weight-loss drugs, lorcaserin and phentermine- topiramate: slim pickings? JAMA Intern Med, 174, 615-9. WOOD, E. J. 1983. Molecular cloning. A laboratory manual by T Maniatis, E F Fritsch and J Sambrook. pp 545. Cold Spring Harbor Laboratory, New York. 1982. $48 ISBN 0-87969-136-0. Biochemical Education, 11, 82-82. WREN, A. M., SMALL, C. J., WARD, H. L., MURPHY, K. G., DAKIN, C. L., TAHERI, S., KENNEDY, A. R., ROBERTS, G. H., MORGAN, D. G., GHATEI, M. A. & BLOOM, S. R. 2000. The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion. Endocrinology, 141, 4325-8. WWW.HARLAN.COM Growth curve of C57BL/6 mice. WYNNE, K., PARK, A. J., SMALL, C. J., MEERAN, K., GHATEI, M. A., FROST, G. S. & BLOOM, S. R. 2006. Oxyntomodulin increases energy expenditure in addition to decreasing energy intake in overweight and obese humans: a randomised controlled trial. Int J Obes (Lond), 30, 1729-36. WYNNE, K., PARK, A. J., SMALL, C. J., PATTERSON, M., ELLIS, S. M., MURPHY, K. G., WREN, A. M., FROST, G. S., MEERAN, K., GHATEI, M. A. & BLOOM, S. R. 2005. Subcutaneous oxyntomodulin reduces body weight in overweight and obese subjects: a double-blind, randomized, controlled trial. Diabetes, 54, 2390-5. XU, B., GOULDING, E. H., ZANG, K., CEPOI, D., CONE, R. D., JONES, K. R., TECOTT, L. H. & REICHARDT, L. F. 2003. Brain-derived neurotrophic factor regulates energy balance downstream of melanocortin-4 receptor. Nat Neurosci, 6, 736-42. XU, E., KUMAR, M., ZHANG, Y., JU, W., OBATA, T., ZHANG, N., LIU, S., WENDT, A., DENG, S., EBINA, Y., WHEELER, M. B., BRAUN, M. & WANG, Q. 2006. Intra-islet insulin suppresses glucagon release via GABA-GABAA receptor system. Cell Metab, 3, 47-58. YOSHIMOTO, S., HIROTA, M., OHBOSHI, C. & SHIMA, K. 1989. Identification of glucagon-like peptide- 1(7-36) amide in rat brain. Ann Clin Biochem, 26 ( Pt 2), 169-71. YOUNG, A. A. 2012. Brainstem sensing of meal-related signals in energy homeostasis. Neuropharmacology, 63, 31-45. YUAN, C. S., ATTELE, A. S., WU, J. A., ZHANG, L. & SHI, Z. Q. 1999. Peripheral gastric leptin modulates brain stem neuronal activity in neonates. Am J Physiol, 277, G626-30. ZERBE, O., NEUMOIN, A., MARES, J., WALSER, R., WALSER, R. & ZOU, C. 2006. Recognition of neurohormones of the NPY family by their receptors. J Recept Signal Transduct Res, 26, 487- 504. ZHANG, L., NGUYEN, A. D., LEE, I. C., YULYANINGSIH, E., RIEPLER, S. J., STEHRER, B., ENRIQUEZ, R. F., LIN, S., SHI, Y. C., BALDOCK, P. A., SAINSBURY, A. & HERZOG, H. 2012. NPY modulates PYY function in the regulation of energy balance and glucose homeostasis. Diabetes Obes Metab, 14, 727-36. ZHANG, T., UCHIDA, T., GOMEZ, G., LLUIS, F., THOMPSON, J. C. & GREELEY, G. H., JR. 1993. Neural regulation of peptide YY secretion. Regul Pept, 48, 321-8.

306

ZHANG, Y., PROENCA, R., MAFFEI, M., BARONE, M., LEOPOLD, L. & FRIEDMAN, J. M. 1994. Positional cloning of the mouse obese gene and its human homologue. Nature, 372, 425-32. ZHU, L., TAMVAKOPOULOS, C., XIE, D., DRAGOVIC, J., SHEN, X., FENYK-MELODY, J. E., SCHMIDT, K., BAGCHI, A., GRIFFIN, P. R., THORNBERRY, N. A. & SINHA ROY, R. 2003. The role of dipeptidyl peptidase IV in the cleavage of glucagon family peptides: in vivo metabolism of pituitary adenylate cyclase activating polypeptide-(1-38). J Biol Chem, 278, 22418-23. ZIPF, W. B., O'DORISIO, T. M., CATALAND, S. & DIXON, K. 1983. Pancreatic polypeptide responses to protein meal challenges in obese but otherwise normal children and obese children with Prader-Willi syndrome. J Clin Endocrinol Metab, 57, 1074-80. ZIPF, W. B., O'DORISIO, T. M., CATALAND, S. & SOTOS, J. 1981. Blunted pancreatic polypeptide responses in children with obesity of Prader-Willi syndrome. J Clin Endocrinol Metab, 52, 1264-6.

307

APPENDICES

308

APPENDIX A – AMINO ACID CODES

Name Abbreviation Single letter code

Alanine Ala A

Cystine Cys C

Aspartic acid Asp D

Glutamic acid Glu E

Phenylalanine Phe F

Glycine Gly G

Histidine His H

Isoleucine Ile I

Lysine Lys K

Leucine Leu L

Methionine Met M

Asparagine Asn N

Proline Pro P

Glutamine Gln Q

Arginine Arg R

Serine Ser S

Threonine Thr T

Valine Val V

Tryptophan Trp W

Tyrosine Tyr Y

309

APPENDIX B – ANALOGUE SEQUENCES

Figure B.1

Figure B.2

310

APPENDIX C – BUFFERS AND SOLUTIONS

LYSOGENY BROTH (LB)

1L distilled water, 10g Bactotryptone, 5g Yeast extract, 5g NaCl. Adjust pH to 7.5 with 10M NaOH. Sterilise by autoclaving.

TFB I (30MM KOAC/50MM MNCL2/100MM RBCL/10MM CACL2/15% GLYCEROL)

1.472g KoAc, 4.95g MnCl2, 6.05g RbCl, 0.735g CaCl2·2H2O, 75ml Glycerol. Adjust pH to 5.8 with 10% v/v acetic acid. Bring final volume up to 0.5L with distilled water (approximately 420ml). Sterile filter and store at 4°C.

TFB II (10MM NA MOPS PH 7.0/10MM RBCL/75MM CACL2/15% GLYCEROL)

1.046g MOPS, 5.513g CaCl2·2H2O, 0.605g RbCl, 75 ml Glycerol. Adjust pH to 7.0. Bring final volume up to 0.5L with distilled water (approximately 420ml). Sterile filter and store at 4°C.

ALKALINE SDS (0.2M NAOH/1% SDS) 93ml distilled water, 5ml 20% SDS, 2ml 10M NaOH

GTE (0.9% GLUCOSE/0.25M TRIS/0.01M EDTA) 182ml distilled water, 9ml 20% glucose, 5ml 1M Tris base pH8, 4ml 0.5M EDTA pH8

50X TAE (TRIS/ACETATE/EDTA) 242g Tris base, 57.1ml glacial acetic acid, 100ml 0.5M EDTA pH8. Bring final volume up to 1L with distilled water (approximately 840ml). This stock solution can be diluted 1:50 with water to make a 1x working solution (final concentrations: 0.04M Tris/0.02M acetic acid/ 0.001M EDTA)

GEL LOADING BUFFER (25% GLYCEROL/0.025M EDTA/1MG/ML ORANGE G) 3.125ml 80% glycerol, 50μl 0.5M EDTA, 6.325ml distilled water, 10 mg orange G

1X TE (0.01M TRIS/0.001M EDTA) 98.8ml distilled water, 1ml 1M Tris base pH8, 0.2ml 0.5M EDTA pH8

311

TES (0.06M TRIS/0.006M EDTA/0.06 NACL) 182.5ml distilled water, 12.5ml 1M Tris base pH8, 2.5ml 0.5M EDTA pH8, 2.5ml 5M NaCl

CAESIUM CHLORIDE SATURATED PROPAN-2-OL 100g CsCl vigorously mixed with 100ml distilled water and 1L propan-2-ol. Leave to settle. Three phases are produced: upper propan-2-ol layer saturated with CsCl, middle layer of water, lower phase of excess CsCl.

0.1M PEI (AV MW 25KDA)

450mg High MW PEI dissolved in 80ml GDW and allowed to solubilise by stirring for 10mins. The solution is brought to pH7 with concentrated HCl and the volume finally made up to 100ml using GDW. Sterile filter and store at room temperature for a maximum of two weeks.

10X PBS

Dissolve 80g Nacl, 2g KCl, 14.4g Na2HPO4, 2.4g KH2PO4 in 800ml of distilled water. Adjust to pH7.4 and bring final volume up to 1L with distilled water. This stock solution can be diluted 1:10 with distilled water to make a 1x working solution (final concentration: 0.01M PBS)

1MM HEPES BUFFER 1mM HEPES (pH7.4) with 10µg/ml soybean trypsin inhibitor (Sigma), 0.5µg/ml pepstatin (Sigma), 0.5µg/ml antipain (Sigma), 0.5µg/ml leupeptin (Sigma), 0.1mg/ml benzamidine (Sigma) and 1ml 10,000KIU/ml aprotinin (Trasylol, Bayer)) at 4°C.

HOMOGENISATION BUFFER 50mM HEPES (pH7.4) with the cocktail of protease inhibitors added as with 1mM HEPES buffer, at 4°C.

RBA BUFFER 20mM HEPES (pH 7.4) with 1mM MgCl2, 5mM CaCl2, 1% BSA, 0.05% Tween-20, 0.1mM diprotin A, 0.2mM PMSF and 0.5 mM phosphoramidon

312

APPENDIX D – CLONING MAP OF hY2 AND hY4 cDNA

Plasmids containing the cDNA sequence of the hY2 and hY4 receptors (Fig D.2) were purchased from www.cDNA.org (Missouri S&T cDNA Resource Center, University of Missouri-Rolla), and supplied in the pcDNA3.1+ vector (Fig D.1).

Overexpressing cells lines were produced using the protocol described in section 2 and further described below.

Figure D.1 Plasmid map of supplied clones (Invitrogen)

Figure D.2 Insert map of the hY2 and hY4 cDNA clones (www.cDNA.org)

313

Plasmids were transformed into competent E.coli (XL1-Blue) by heat-shock treatment and grown on agar supplemented with ampicillin to allow selection of only those bacteria which had successfully incorporated the plasmid. Single positive bacterial colonies were picked and small scale DNA preparations made from each one. DNA was extracted and a restriction endonuclease (RE) digest strategy used to ensure the correct plasmid DNA was present.

DNA was incubated with enzymes EcoR I (5’RE) and Xho I (3’RE) and the digest reactions size fractionated by electrophoresis on an agarose gel. The expected DNA fragment for the hY2 receptor coding sequence was 1146 base pairs (Fig D.3(A)) and a 1128 base pair fragment indicating the presence of an intact hY4 receptor coding sequence (Fig D.3(B)).

Colony 8 of the tested hY2 receptor containing bacteria and colony 4 of the hY4 receptor containing bacteria was selected for large scale DNA preparation and used to generate overexpressing cell lines.

Figure D.3 Restriction endonucleas digestions of plasmid DNA extracted from E.coli transformed with plasmid containing (A) Y2 receptor cDNA, (B) Y4 receptor cDNA. DNA incubated with enzymes EcoR I and Xho I for 60 minutes at 37°C and separated by gel electrophoresis. 1146 base pair fragment indicated presence of an intact Y2 receptor coding sequence. 1128 base pair fragment indicating presence of an intact Y4 receptor coding sequence.

314

APPENDIX E – GENERAL PRINCIPLE OF A RADIOIMMUNOASSAY

The principle of RIA is similar to that of RBA in that it is the competition between a radiolabelled and non-radiolabelled antigen for a constant number of antibody binding sites. When unlabelled antigen from standards or unknown samples, and a fixed amount of labelled antigen are allowed to react with a constant and limiting amount of antibody, decreasing amounts of labelled antigen are bound to the antibody as the amount of unlabelled antigen is increased. The radioimmunoassay is incubated and allowed to reach equilibrium, according to the equation:

*Ag + Ab + Ag ↔x *AgAb + AgAb

Ag = unlabelled antigen *Ag = radiolabelled antigen Ab = antibody

Separation of the bound from the free antigen is achieved by addition of either dextran- coated charcoal (free label is contained in the charcoal pellet following centrifugation) or using a primary-secondary antibody complex (free label is contained in the supernatant following centrifugation). The secondary antibody (2° antibody) is derived from an animal species different from that used to generate the primary antibody. After incubation and separation, the bound and free labels are counted in a γ-counter. The data is used to construct a standard curve from which the values of the unknowns can be obtained by interpolation.

Inter-assay variation can be calculated by assaying aliquots of the same sample in each assay performed and comparing the concentrations obtained in each. To measure and correct for baseline drift, tubes with no sample (‘zero’ tubes) are placed at regular intervals throughout the assay and standard curves (with known amounts of the peptide) are added at the beginning and end of each assay. The general structure of the RIA is outlined below, which shows the content of the tubes according to their designation:

315

Tube number Designation 1-2 Non-specific binding 3-4 ½x label 5-6 2x label 7-10 Zero 11-12 Standard 1

13-14 Standard 2 15-16 Standard 3

17-18 Standard 4 19-20 Standard 5 21-22 Standard 6 23-24 Standard 7 25-26 Standard 8 27-28 Standard 9 29-30 Standard 10 31-32 Zero 33-onwards Unknown samples Zeros Zero (in duplicate) approximately every 50 samples Standard curve (1-10) every 200 samples Final two tubes Excess antibody

Table E.1 Radioimmunoassay plan

The following tubes are important for the assessment of the performance of the assay:

 Non-specific binding: low binding indicates adequate label integrity.  ½x label: assesses if greater sensitivity could be achieved by adding half the volume of label.  2x label: assesses if greater sensitivity could be achieved by adding double the volume of label.  Zero tubes: allows assessment of assay drift.  Excess antibody: assesses the immunological integrity of the labelled peptide.  Quality Controls: includes previously aliquoted samples containing high and low levels of the antigen. These tubes allow the assays to be standardised.

316

ORIGINAL ARTICLES

1) Cegla J, Troke RC, Jones B, Tharakan G, Kenkre J, McCullough KA, Lim CT, Parvizi N, Hussein M, Chambers ES, Minnion J, Cuenco J, Ghatei MA, Meeran K, Tan TM, Bloom SR. 2014. Co-infusion of low-dose glucagon-like peptide-1 (GLP-1) and glucagon in man results in a reduction in food intake. Diabetes. Jun 17 pii: DB_140242

2) Christakis I, Georgiou P, Minnion J, Constantinides V, Cuenco J, Scott R, Tan T, Palazzo F, Murphy K, Bloom S. 2014. Learning curve of vessel cannulation in rats using cumulative sum analysis. J Surg Res. Jul 1. pii: S0022-4804(14)00626-X. doi: 10.1016/j.jss.2014.06.048

3) Germain N, Minnion JS, Tan T, Shillito J, Gibbard C, Ghatei M, Bloom S. 2013. Analogs of pancreatic polypeptide and peptide YY with a locked PP-fold structure are biologically active. Peptides, 13: 6-10

4) Tan TM, Field BC, Minnion JS, Cuenco-Shillito J, Chambers ES, Zac-Varghese S, Brindley CJ, Mt-Isa S, Fiorentino F, Ashby D, Ward I, Ghatei MA, Bloom SR. 2012. Pharmacokinetics, adverse effects and tolerability of a novel analogue of human pancreatic polypeptide, PP 1420. Br J Clin Pharmacol. 73(2): 232-9

5) Addison ML, Minnion JS, Shillito JC, Suzuki K, Tan TM, Field BC, Germain-Zito N, Becker-Pauly C, Ghatei MA, Bloom SR, Murphy KG. 2011. A role for metalloendopeptidases in the breakdown of the gut hormone, PYY3-36. Endocrinology. 152(12): 4630-40

6) Liu YL, Ford H, Druce M, Minnion J, Field B, Shillito JC, Baxter J, Murphy K, Ghatei M, Bloom S. 2010. Subcutaneous oxyntomodulin analogue administration reduces body weight in lean and obese rodents. Int J Obes (Lond). 34(12): 1715-25

7) Suzuki K, Simpson K, Minnion J, Shillito JC, Bloom S. 2010. The Role of Gut Hormones and the Hypothalamus in Appetite Regulation. Endocr J. 57(5): 359-72

8) Druce M, Minnion J, Field B, Patel S, Shillito JC, Tilby M, Beale K, Murphy K, Ghatei M, Bloom S. 2009. Investigation of structure-activity relationships of Oxyntomodulin (Oxm) using Oxm analogs. Endocrinology. 150(4): 1712-22

317