COPYRIGHT AND USE OF THIS THESIS This thesis must be used in accordance with the provisions of the Copyright Act 1968. Reproduction of material protected by copyright may be an infringement of copyright and copyright owners may be entitled to take legal action against persons who infringe their copyright. Section 51 (2) of the Copyright Act permits an authorized officer of a university library or archives to provide a copy (by communication or otherwise) of an unpublished thesis kept in the library or archives, to a person who satisfies the authorized officer that he or she requires the reproduction for the purposes of research or study. The Copyright Act grants the creator of a work a number of moral rights, specifically the right of attribution, the right against false attribution and the right of integrity. You may infringe the author’s moral rights if you: - fail to acknowledge the author of this thesis if you quote sections from the work - attribute this thesis to another author - subject this thesis to derogatory treatment which may prejudice the author’s reputation For further information contact the University’s Copyright Service. sydney.edu.au/copyright Stereopharmacological research in anaesthesiology A thesis based on selected published works submitted in fulfilment of the requirements for the degree of Doctor of Medical Science of Sydney Medical School The University of Sydney by Laurence Edward Mather PhD, MSc, BSc ( NSW ), DipApplChem ( SydTechColl ), FANZCA, FRCA, FFPMANZCA (Hon) July 2015 “It will be at once admitted that the medical practitioner ought to be acquainted with the strength of the various compounds which he applies as remedial agents, and that he ought, if possible, to be able to regulate their potency.” (John Snow, On the inhalation of the vapour of ether. Lond Med Gaz IV: 498, 1847) “Dans les champs de l'observation le hasard ne favorise que les esprits préparés.” (Louis Pasteur, Lecture, University of Lille, December 7 1854) ii Summary This thesis is based on a theme of 63 selected publications taken from three of the author’s research programs (on local anaesthetic agents, intravenous anaesthetic agents, and nonsteroidal anti-inflammatory agents) and three individual drug projects (on halothane, ketamine and thalidomide). The publications have been selected to highlight the bearing of the chemistry of the drug, especially the stereochemistry, on some or other aspect of its pharmacology, of which 42 are designated Study (new experimental observations and knowledge), 18 are designated Review (syntheses of ideas from existent knowledge), and 3 are designated Patent (aspects of invention). The thesis is shaped by the narrative relating to the development of anaesthesia as first established by John Snow (1813-1858), and the contemporaneous stereochemical basis of pharmacology first described by Louis Pasteur (1822-1895). It presents each topic as a personal commentary identifying the historical context, rationale and outcome, and recognizing the work of collaborators and others at the institutions where the research was performed. The selected publications originate from research performed at the University of Sydney, the Flinders University of South Australia, and the University of Sheffield (UK), although prior work performed at the University of Washington (USA) also contributed. The ‘concepts and tools’ supporting the research are described in an historical context, corresponding to their evolution. In particular, the dual concepts of pharmacokinetics and pharmacodynamics underpin much of modern pharmacology and run throughout the theme of this thesis. Their investigation required reliable practical tools for physiological data acquisition, numerical data analysis, and drug assay methods, especially for the analytical resolution of chiral drugs used as racemates; however these tools evolved more slowly than the demands of the concepts. Eventually, when they were combined in appropriate ‘whole body’ pharmacological preparations, such as the author’s ‘multicannulated sheep preparation’ that allowed anatomically-correct and physiologically-sound assessment of drug disposition in association with quantitative measures of systemic drug effects, as well as with the ‘Stanford’ quantitative electroencephalography rodent model used for pharmacokinetics- pharmacodynamics, then real progress became possible, allowing greater insights into drugs used as racemates, such as those described in this thesis. The local anaesthetic program was instrumental in the registration and clinical introduction of two enantiopure substances, ropivacaine and levobupivacaine. The selections from the intravenous anaesthetic program focus on thiopentone and provide evidence of a greater margin of safety of R-thiopentone over the more potent S-thiopentone or the clinically-used racemate. Although the program led to a provisional patent for enantiopure thiopentone, clinical anaesthesia has since largely moved to using propofol which has a preferred pharmacokinetic profile to thiopentone. The selections from the nonsteroidal anti- inflammatory drug program focus on ketorolac, but produced inconclusive pharmacokinetic data; given the limited resources available, the program was changed to focus on (achiral) diclofenac. The ketamine project provided useful pharmacokinetic and pharmacodynamic data supporting the current clinical investigations of the pharmacologically preferred esketamine. The halothane project was designed to consider that differential enantiomeric disposition might influence halothane toxicity, but an enantiomeric difference was not found, and the project was terminated due to lack of funds. Finally, the thalidomide project documented that racemization precluded the use of a preferred enantiomer, but also produced a rationale for its use in future combined cancer chemotherapy developments. iii Table of Contents Stereopharmacological research in anaesthesiology Summary ii Table of Contents iii Foreword v Acknowledgements vi Thesis structure vii Declarations vii Selected publications viii 1. Concepts and tools 1 1.1 Basic medicinal chemical concepts 1 1.2 A glossary of basic stereochemical concepts 3 1.3 Essentials of experimental concepts and tools 7 1.4 Quantitation in anaesthesia – John Snow 8 1.5 Stereochemistry in pharmacology – Louis Pasteur 10 1.6 Stereopharmacology and anaesthesiology 12 1.7 Pharmacokinetics 15 1.8 Pharmacodynamics 17 1.9 Applied pharmacokinetics and pharmacodynamics 18 1.10 The ‘elephant in the laboratory’ 21 2. Translating chemistry into anaesthesiology – in stages 25 2.1 The University of Sydney (1966-1972) 25 2.2 The University of Washington (1972-1976) 27 2.3 Flinders Medical Centre (1976-1981) 31 2.4 The University of Massachusetts Medical Center (1981-1983) 37 2.5 Flinders Medical Centre (1983-1990) 37 2.6 The University of Sheffield (1988) 40 2.7 The University of Sydney (1991-2007) 41 3. Stereopharmacological research in anaesthesiology: the main theme 45 3.1 Introduction 45 3.11 Growing recognition of stereopharmacological issues 47 3.12 The main theme: stereopharmacological programs and projects 49 3.13 Basic concepts and experimental tools 49 Review 01. The rationale of our experimental techniques [110] 49 Reviews 02, 03 and 04. Early reviews of stereopharmacology [198,214,215] 50 3.2 Local anaesthetic agent program 51 3.21 Introduction 51 Reviews 05 and 06. Achiral aspects of local anaesthetic pharmacology [48,49] 51 3.22 Prilocaine 55 Study 01. Pharmacokinetics of prilocaine enantiomers in humans [197] 55 3.23 Bupivacaine 56 Study 02. Pharmacokinetics of mepivacaine and bupivacaine enantiomers in sheep [170] 57 Study 03. Bupivacaine pharmacokinetics with steady state infusion in sheep [177] 57 Study 04. Post-surgical plasma binding of bupivacaine enantiomers in sheep [188] 58 Study 05. Enantioselectivity of bupivacaine tissue distribution in sheep [190] 58 Study 06. Influence of i.v. infusion on bupivacaine pharmacokinetics in sheep [217] 58 Study 07. Bupivacaine pharmacokinetics with intercostal block in humans [218] 58 Study 08. Bupivacaine pharmacokinetics with epidural block in humans [246] 59 3.24 Levobupivacaine 59 Study 09. A new enantiospecific HPLC assay for the levobupivacaine program [250] 64 Study 10. Cardiovascular effects of i.v. bupivacaine and levobupivacaine [244] 64 Study 11. PK-PD analysis of bupivacaine and levobupivacaine [245] 64 Reviews 07 and 08. Levobupivacaine: critical data [243,273] 65 Patents 01 and 02. Use patents on levobupivacaine 65 Study 12. Analysis of fatal toxicity from levobupivacaine [276] 65 Study 13. Chaos model of the CNS toxicity of local anaesthetics [285] 65 Study 14. Direct cardiovascular toxicity of local anaesthetics [280] 66 Study 15. Direct CNS toxicity of local anaesthetics [299] 66 Review 09. Cardiotoxicity of local anaesthetics – rationale [282] 67 iv Study 16. Acid-base changes on PD and PK of bupivacaine (and thiopentone) [306] 67 Review 10. PK and PD concepts and the acute toxicity of local anesthetics [314] 68 Study 17. Effects of general anaesthesia on local anaesthetic toxicity [323] 68 Study 18. Effects of general anaesthesia on local anaesthetic pharmacokinetics [324] 68 3.25 Broader aspects of local anaesthetic pharmacology involving chirality 69 Review 11, 12 and 13. Translational pharmacology of local anaesthetics [325,327,328] 69 Postscript 1 69 3.3 Intravenous anaesthetic agent program 71 Patent 03. Synthesis and uses of thiopentone enantiomers 76 Study 19. Thiopentone: a chiral assay for future studies [221]