Pharmacological modulation of the adenosine system as a novel strategy for the treatment of inflammatory bowel diseases

CONTENTS

GLOSSARY

GENERAL SECTION

Adenosine: synthesis and metabolism 4

Adenosine transport 6 Adenosine metabolism 10 Interaction between AMP-activated protein kinase 13

(AMPK) and adenosine system Adenosine receptors 19 Biological functions of adenosine 26 Effects of adenosine on gastrointestinal functions 40

Inflammatory bowel diseases 44

EXPERIMENTAL SECTION 49

Introduction 50

Aim of the research program 53

Materials and methods 54

- Animals 54

PART 1. STUDY OF THE ANTI-INFLAMMATORY ACTIVITY 54 OF THE NOVEL ADENOSINE DEAMINASE INHIBITOR APP IN AN EXPERIMENTAL MODEL OF COLITIS - Structural characteristics of APP 54

- In vitro adenosine deaminase inhibition assay 55

- Induction of colitis, drug treatments and experimental design 56

- Assessment of colitis 60

- Evaluation of tissue myeloperoxidase levels 63

- Evaluation of tissues cytokines levels 64

- Evaluation of tissue malondialdehyde levels 64

PART 2. STUDY OF THE PHARMACOLOGICAL 66

MODULATION OF AMPK SYSTEM BY ACADESINE IN THE TREATMENT OF EXPERIMENTAL COLITIS - AMPK activator acadesine 66

- Induction of colitis, drug treatments and experimental design 68

- Assessment of colitis 69

- Evaluation of tissues cytokines levels 69

- Evaluation of tissue malondialdehyde levels 70

- Detection of superoxide anion production 70

PART 3. STUDY OF THE ANTI-INFLAMMATORY 72 ACTIVITY OF THE NOVEL A2A RECEPTOR AGONIST PSB0777 IN AN EXPERIMENTAL MODEL OF COLITIS - Structural characteristics of PSB0777 72

- Induction of colitis, drug treatments and experimental design 72

- Assessment of colitis 74

- Measurement of systolic blood pressure 75

- Evaluation of tissue myeloperoxidase levels 75

DRUGS AND REAGENTS 75 STATISTICAL ANALYSIS 77

RESULTS

PART 1. STUDY OF THE ANTI-INFLAMMATORY 79 ACTIVITY OF THE NOVEL ADENOSINE DEAMINASE INHIBITOR APP IN AN EXPERIMENTAL MODEL OF COLITIS In vitro adenosine deaminase inhibition assay 79

Evaluation of the anti-inflammatory effects in the chronic 79 phase of colitis

- Expression of colonic adenosine deaminase 79

- Evaluation of colonic inflammation under control conditions 79

- Effects of test drugs on colonic inflammation 80

- Food intake, body weight and spleen weight 80

- Evaluation of macroscopic damage score 82

- Evaluation of microscopic damage score 83

- Evaluation of TNF- and IL-6 colonic tissue levels 83 - Evaluation of MDA tissue levels 84

PART 2. STUDY OF THE PHARMACOLOGICAL 92 MODULATION OF AMPK SYSTEM BY ACADESINE IN THE TRATMENT OF EXPERIMENTAL COLITIS - Body weight and spleen weight 92

- Evaluation of macroscopic damage score 93 - Evaluation of microscopic damage score 93 - Evaluation of TNF- and IL-10 colonic tissue levels 93 - Evaluation of MDA tissue levels 95

- DHE analysis of superoxide anion generation 95

PART 3. STUDY ON THE ANTI-INFLAMMATORY 104

ACTIVITY OF THE NOVEL A2A ADENOSINE RECEPTOR AGONIST PSB0777 IN AN EXPERIMENTAL MODEL OF COLITIS - Body weight 104

- Blood pressure 104

- Evaluation of microscopic damage score 105 - Evaluation of MPO tissue levels 105

 Pharmacological modulation of adenosine deaminase and 110 intestinal inflammation

 Pharmacological modulation of AMPK and intestinal 117 inflammation

 Activation of local A2A receptors and intestinal inflammation 123

CONCLUSIONS

REFERENCES