This item was submitted to Loughborough University as a PhD thesis by the author and is made available in the Institutional Repository (https://dspace.lboro.ac.uk/) under the following Creative Commons Licence conditions. For the full text of this licence, please go to: http://creativecommons.org/licenses/by-nc-nd/2.5/ LOUGHBOROUGH UNIVERSITY OF TECHNOLOGY LIBRARY AUTHOR/FILING TITLE MAIL11'" ---------- -----------;----------------------'J'"'I-. --.. ____________________________________________ .______ i ACCESSION/COPY NO. <J't~,?,+ Iq & -------------_._- ---- ----------------------- --- - -- VOl. NO. CLASS MARK 0400941988 IIIIII BIOCHEMICAL AND PHARMACOLOGICAL STUDIES OF MORPHINE-6- GLUCURONIDE AND RELATED COMPOUNDS by Jason Lewis Martin A Doctoral Thesis submitted in partial fulfilment of the requirements for the award of Doctor of Philosophy of the Loughborough University of Technology. June 1994 <Slby Jason Lewis Martin (1994) Loughborough University oi Tach:lv")9Y Library .~- .. ,-.-. ,-, Date cJ\. 'i <{ o'~-'" ,,-_._-. Class Ace. No. (!\to () ~ of I ii"1 ACKNOWLEOOEMENTS Primarily I would like to thank my supervisor. Or. John Traynor for his help. guidance and encouragement throughout this project. I would also like to thank British Technology GrouP. especially Dr. Terry Smith. for funding the project. I am also indebted to Mr. John Brennan. Miss Jill Thorley. Mrs. Helen Tidmas and Mrs. Wendy Jones for their technical assistance. DEDICATION To my mum and dad I would like to thank my mother and father for their continual support and understanding while I have been studying, and I therefore dedicate this thesis to them. Daring ideas are like chessman moved forward; they may be beaten, but they may start a winning game JOHANN WOLFGANG VON GOETHE ABSTRACT Biochemical and pharmacological studies of morphine-6- glucuronide and related compounds Jason L. Martin Keywords morphine. morphine-6-glucuronide. morphine-3- glucuronide. synthetic opiates. brain. spinal cord. ligand-binding assays. isolated tissue bioassays. analgesia. metabolism and distribution. Morphine-6-glucuronide is a minor metabolite. representing 5% of an administered dose of morphine. The metabolite has analgesic activity exceeding that of morphine and may contribute to analgesia following morphine administration. The aims of the study were to attempt to identify the reasons behind the improved activity of morphine-6-glucuronide over the parent compound and to examine a series of 6-substituted compounds. based on 6-substituted benzoate esters. as potential mimics of morphine-6-glucuronide. Morphine-6-glucuronide was seen to have similar affinity to morphine at l1-opioid receptors as assessed by ligand-binding assays in mouse brain homogenates. However a three-fold improved affinity at S-opioid receptor binding sites was observed and a ten-fold reduction in affinity at K-opioid sites. Using in vitro bioassay systems. the glucuronide showed a two-fold improved potency over morphine. in both the guinea-pig ileum and the mouse vas deferens preparations. Following in vivo (s.c.) administration in the mouse the glucuronide was seen to be equipotent with morphine in the tail-flick test. but was of much longer duration. lasting up to 9 hours. Ex­ vivo binding assays confirmed that morphine-like material was still present in the central nervous system six hours after administration of the glucuronide. but was not observed at a similar time after morphine administration. Activity was retained if the hydroxyl groups of the sugar moiety of the glucuronide were protected as esters. In contrast the more prevalent morphine metabolite. morphine-3-glucuronide. was inactive in all in vitro and in vivo tests used. and did not antagonise morphine in vitro or in vivo. A group of 3-substituted derivatives containing saturated and unsaturated substituents. did show affinity for opioid receptors. but no agonist activity of the compounds could be demonstrated in vitro. A series of synthetic 6-substituted compounds showed a variety of affinities for, and agonist potencies at, opioid receptors, though low affinity at K­ opioid receptors was a general finding. For example, morphine-6- nitrobenzoate was l1-opioid receptor preferring, while morphine-6- phthalate had improved O-opioid receptor affinity and acted via Il-opioid receptors in the mouse vas deferens and in vivo. However the compounds were weaker than morphine and the duration of action in vivo was shorter than morphine-6-glucuronide. The conclusions from these studies are that morphine-6-glucuronide and morphine have similar in vitro affinities at the l1-receptor, although morphine-6-glucuronide has somewhat improved binding affinity for Il receptor sites, it has less affinity for K receptor sites. Pharmacokinetic reasons are probably responsible for the improved activity and duration of action of morphine-6-glucuronide over morphine. None of the synthetic compounds examined are potentially useful as direct mimics of the glucuronide because morphine-6-glucuronide is more potent and has a longer duration of action than the synthetic derivatives, though alteration at the 6-position of the morphine nucleus can lead to dramatic changes in selectivity and potency of ligands for the differing opioid receptors. Abbreviations used in this thesis AC Adenylate cyclase ATP Adenosine triphosphate BBB Blood-brain barrier CMC Carboxy methyl cellulose CNS Central nervous system CSF Cerebrospinal fluid CPM Cyclopropylmethyl DADL [D-Ala2,D-LeuS] enkephalin DALCE [D-Ala2, Lys4,CysS] enkephalin DAMGO [D-Ala2,MePhe4,G1y-oIS] enkephalin DPDPE [D-Pen2,D-PenS] enkephalin DSLET [D-Ser2, LeuS] enkephalin-Thr6 J3-FNA J3- Funaltrexamine Gpp(NH)p S' guanylimidodiphosphate GTP Guanosine triphosphate GPI Guinea-pig ileum HEPES N -2-Hydrox yethy I piperazine-N -eth anesu I phonic acid HPLC High performance liquid chromatography i.c.v. In trac ere b rov en tri cui ar Lm. Intramuscular i.t. Intrathecal i.v. Intravenous M6G Morphine-6-gl ucuronide M3G Morphi ne-3-gl ucuronide MPLM Myenteric plexus longitudinal muscle MVD Mouse vas deferens NEM N -Ethylmaleimide PAG Periaquaductal grey PAPS 3' -phosphoadenosine-S' -phosphosulphate p.o. oral administration RVD Rat vas deferens sem Standard error of the mean s.c. Subcutaneous tris Tris (hydroxymethyl) aminomethane 1LC Thin layer chromatography CONfENTS CHAPTER I Introduction Ll Historical I U Opioid receptors and endogenous peptides 3 1.2.1 Endogenous opioids 6 1.2.2 Distribution of opiate receptor types in the CNS 8 1.2.4 Intracellular mechanism of opioid action IO U Morphine analogues and related drugs 13 lA Pharmacokinetics and metabolism of morphine 16 1.4.1 Species differences in morphine metabolism 19 1.5 Pharmacological properties of morphine metabolites 21 1.5.1 M6G 21 II Aims of the project 25 CHAPTER 2 Materials and Methods 2...1 Materials U Methods 29 2.2.1 Ligand binding assays 29 i)Brain homogenate 29 ii)Guinea-pig myenteric-plexus homogenate 30 2.2.2 Alkylation by j3-funaltrexamine 30 U I~Qlall:!! li~~l!1: ~Il!!!i!;~ 31 2.3.1 Tissue preparation 31 a) Guinea-pig myenteric plexus-longitudinal muscle (MPLM) bioassay 31 b) Mouse, rat and hamster vas deferens preparation 31 2.3.2 Experimental 31 a) Agonist potencies 31 b) Antagonist affinities 32 c) Alkylation by ~FNA in the MPLM 32 lA Metabolism studies 33 2.4.1 Preparation of liver microsomes 33 2.4.2 Morphine metabolism 33 U Separation of morphine and its metabolites 33 2.5.1 Methods available for studying morphine metabolites 33 2.5.2 Separation analysis using HPLC 34 Ljphophjlicities of morphine and relaled compounds 39 Stability of morphine and related compounds 39 II In Vivo experiments 40 2.8.1 Antinociceptive properties of morphine and related compounds 40 2.8.2 Distribution of morphine and M6G into the CNS 40 CHAPTER 3 Analysis of binding and isolated tissue data .3..1 Radioreceptor assays 41 3.1.1 Determination of inhibitor binding constants 43 II I a vitro bioassays 44 3.2.1 An tagonist potencies 44 3.2.1.1 Using the Schild plot 44 3.2.1.2 Antagonist potencies where partial agonism is shown in the rat vas deferens 49 3.2.2 Determination of agonist potencies using Furchgott's analysis 51 3.2.2.1 Efficacy and spare receptors 51 3.2.2.2 Furchgott 's method 52 CHAPTER 4 la vivo antinociceptive activity of morphine and its glucuronide II Introduction 54 :u Results 54 4.2.1 Antinociceptive properties as measured by the tail-flick test 54 ~ Distribution of morphine-like actiyity in the brain and spinal cord after S,C, administration of morphine and M6G 62 1...1 Metabolism of morphine 65 4.4.1 Metabolism in liver homogenates 65 1...5. Discussion 68 CHAPTER 5 Binding and isolated studies of morphine and its glucuronide II Characterisation of the binding of u,O, and K ollioid Hgands to homogenates of mouse brain 75 .l2 Receptor binding profile of M6G and morphine 77 5,2, I Binding of [3H1DAMGO at low concentrations of 11 Hgands 79 5,2,2 Binding to opioid K receptors in guinea-pig cerebellum 80 5,2,3 Binding studies in mouse spinal cord 81 5,2.4 Binding in the myenteric plexus longitudinal muscle (MPLM) 82 5,2,5 Binding at 11 sites in brain under different buffer conditions 82 5,2,6 Competition studies using [3 H1 CTOP in mouse brain 91 .u Isolated tissue studies 93 5.3, I Myenteric plexus longitudinal muscle 93 5.3,2 The rat vas deferens preparation 95 5.3,3 Furchgolt's analysis in the MPLM 97 5.3.4 The mouse vas deferens preparation 101 5,3,5 The hamster vas deferens preparation 104 5.3,6 Alkylation of 11 receptors 105 5A Discussion 107 5.4, I Binding 107 5.4,2 Isolated tissue bioassays 114 5.4,3 Overview 117 CHAPTER 6 Synthetic morphine-6-substituted compounds as analogues of morphine-6- glucuronide ti Introduction 118 6.2 Compounds under study 122 .6...1 Ligand binding assays 125 M Isolated tissue studies 129 6.5 Stability studies 138 M Lipophilicities of morphine and related compounds 139 6.6.1 Rates of onset and offset of morphine and derivatives as measured in the MPLM 139 6.6.2 Liphophilicities as determined using an HPLC system 139 fW.