Assessment of novel drugs for treating preterm labour using a translational model A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2020 Ammar Ahmed Mohammed School of Health Sciences Division of Pharmacy and Optometry 1 Table of contents Table of contents .............................................................................................. 2 List of Figures .................................................................................................................. 9 List of Tables ................................................................................................................. 20 List of abbreviations ..................................................................................................... 22 Publications .................................................................................................................... 27 Abstract .. ....................................................................................................................... 28 Declaration ..................................................................................................................... 29 Acknowledgements ........................................................................................................ 30 Dedication ...................................................................................................................... 32 1 Chapter 1: ............................................................................................ 33 Introduction ......................................................................................... 33 1.1 The female reproductive system ......................................................................... 34 1.2 The human female reproductive system .............................................................. 34 1.2.1 The human uterus ...................................................................................... 34 1.3 The mouse female reproductive system .............................................................. 36 1.3.1 The mouse uterus ...................................................................................... 36 1.3.2 The mouse placenta ................................................................................... 38 1.4 The role of mouse uterus during oestrous cycle .................................................. 40 1.5 The role of the uterus during pregnancy ............................................................. 41 2 1.6 Normal parturition: The common pathways of labour ........................................ 41 1.7 Preterm Labour (PTL) ......................................................................................... 42 1.7.1 Risk factors ............................................................................................... 44 1.7.2 Pathophysiology of PTL ........................................................................... 45 1.7.3 Role of steroid hormones in the induction of labour................................. 45 1.7.4 Prostanoids ................................................................................................ 50 1.8 The ROCK enzymes ............................................................................................ 54 1.8.1 Role of ROCK pathway in uterine contraction ......................................... 55 1.9 Feasibility and translation of mouse model of PTL in humans ........................... 56 1.10 Treatments for preterm labour ............................................................................. 59 1.10.1 Drugs used in the treatment of PTL .......................................................... 60 1.10.2 Prophylactic therapy of premature rupture of the membranes in PTL ..... 71 1.11 Nitric Oxide (NO) and Nitric Oxide Synthase (NOS) isoforms ......................... 71 1.11.1 The role of NO in the uterus ..................................................................... 72 1.11.2 The potential use of NO-donor compounds during pregnancy and labour …………………………………………………………………………...74 1.11.3 The interaction between the NO and lipid mediators................................ 75 1.11.4 SE175 (NO-donor), structure and pharmacology ..................................... 75 1.12 Drug targeting and liposomes ............................................................................. 76 1.12.1 Liposomes as a drug delivery system ....................................................... 77 1.12.2 Liposomes composition ............................................................................ 77 1.13 Aims and objectives ............................................................................................ 79 3 2 Chapter 2: ............................................................................................ 80 Materials and Methods ....................................................................... 80 2.1 Source of the tissues ............................................................................................ 81 2.1.1 Non-pregnant mice .................................................................................... 81 2.2 Methods for investigating myometrial contractility ............................................ 83 2.2.1 Preparation of isolated tissue for immersion ............................................. 83 2.2.2 Solutions and drug compounds ................................................................. 85 2.2.3 Immersion ................................................................................................. 87 2.2.4 Administration of drugs ............................................................................ 89 2.2.5 Preliminary experiments to determine dosing regimens ........................... 91 2.2.6 Measuring contractile force relative to spontaneous activity .................... 94 2.3 Investigation of contractile proteins in mouse myometrium ............................... 95 2.3.1 Western Blot Analysis............................................................................... 95 2.3.2 Isolation of protein .................................................................................... 97 2.3.3 Sample preparation for Western Blotting ................................................. 99 2.3.4 Immunoblotting to investigate protein expression .................................. 103 2.4 Immunocytochemistry (ICC) ............................................................................ 106 2.4.1 Isolation of uterine myometrial cells for immunofluorescence studies .. 106 2.4.2 Cell plating and treatment ....................................................................... 109 2.4.3 Cell fixation ............................................................................................. 109 2.4.4 Permeabilisation ...................................................................................... 109 4 2.4.5 Blocking .................................................................................................. 110 2.4.6 Immunofluorescence (IF) ........................................................................ 110 2.4.7 Cell immunostaining ............................................................................... 111 2.4.8 Imaging analysis...................................................................................... 114 2.5 Enzyme-linked Immunosorbent Assay (ELISA) .............................................. 116 2.5.1 Overview of ELISA ................................................................................ 116 2.5.2 Collection of plasma samples.................................................................. 116 2.5.3 ELISA measurement of 17β-oestradiol ................................................... 117 2.6 Mass spectrometry analysis of total fatty acid composition ............................. 120 2.6.1 Overview of mass spectrometry .............................................................. 120 2.6.2 LC/ESI-MS/MS protocol ........................................................................ 121 2.6.3 Materials for LC/ESI-MS/MS ................................................................. 123 2.6.4 Preparation of internal standards ............................................................. 124 2.6.5 LC/ESI-MS/MS ...................................................................................... 126 2.7 Statistical analysis ............................................................................................. 137 3 Chapter 3: .......................................................................................... 138 Effect of Ripasudil, a ROCK inhibitor on U46619-, PGF2α- and 5-HT-induced myometrial contraction in non-pregnant C57 WT mice. .................................................................................................... 138 3.1 Introduction ....................................................................................................... 139 5 3.2 Spontaneous activity during dioestrous stage ................................................... 140 3.3 Effect of PGF2α on spontaneous myometrial contraction in non-pregnant C57 WT mice ............................................................................................................ 142 3.4 Effect of 5-HT on spontaneous myometrial contraction in non-pregnant C57 WT mice ................................................................................................................... 143 3.5 Effect of ripasudil